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Abstract_reci3

 
 
 
 
 
       
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scientific  Committee  
Teresa  Giraldez  (Hospital  Universitario  Ntra.  Sra.  de  Candelaria,  Tenerife,  Spain)   Luis  A.  Pardo  (Max  Planck  Institute  for  Experimental  Medicine,  Göttingen,  Germany)   Mª  Teresa  Pérez  García  (IBGM,  Universidad  de  Valladolid  –  CSIC,  Valladolid,  Spain)   Antonio  Ferrer  Montiel  (IBMC,  Universidad  Miguel  Hernández,  Alicante,  Spain)   Miguel  A.  Valverde  (Universidad  Pompeu  Fabra,  Barcelona,  Spain)   Diego  Alvarez  de  la  Rosa  (Universidad  de  La  Laguna  and  ITB,  Tenerife,  Spain)  
 
 
 
 
Organizing  Committee  
Teresa  Giraldez  (Hospital  Universitario  Ntra.  Sra.  de  Candelaria,  Tenerife,  Spain)   Diego  Alvarez  de  la  Rosa  (Universidad  de  La  Laguna  and  ITB,  Tenerife,  Spain)   Ricardo  Borges  Jurado  (Universidad  de  La  Laguna  and  ITB,  Tenerife,  Spain)   Rosario  González  Muñiz  (Instituto  de  Química  Médica  –  CSIC,  Madrid,  Spain)   RECI 3: Trends and challenges in ion channel research
Wednesday, 2 February 2011
9:00 Welcome by the Organizing Committee and local Authorities:
Excmo. Sr. Rector Magnífico de la Universidad de La Laguna, Dr. D. Eduardo Doménech Martínez Ilmo. Sr. Dr. D. Antonio Alarcó Hernández, Senador y Consejero Insular del área de Sanidad y Relaciones con la Universidad Sra. Dra. Dª Mercedes Cueto Serrano, Gerente del Hospital Universitario Ntra Sra de Candelaria, Tenerife 9:30-11:00. Session 1. Structure-function of ion channels I
Chairs: Pilar de la Peña (U. Oviedo)/ Walter Stühmer (MPI Goettingen) 9:30. Brad Rothberg (Temple University Medical School, PA, USA)
Mechanisms of Ca2+-dependent gating in MthK channels
9:45. Crina Nimigean (Weill Cornell Medical College, NY, USA).
Mechanism for selectivity-inactivation coupling in KcsA potassium
channels
10:00. Mª Lourdes Renart (Universidad Miguel Hernández, Elche, Spain)
Specific ions, specific lipids: sources of protein stability in an ion
channel model
10:15. Joao Morais Cabral (Instituto de Biologia Molecular e Celular,
Porto, Portugal)
PAS domains in EAG channels
10:30. Francisco Barros (Universidad de Oviedo, Spain)
Detection of physical interactions between the initial segment of the
amino terminus and the S4-S5 linker of the h-ERG potassium channel
10:45. M. Luisa Campo (Universidad de Extremadura, Cáceres, Spain)
Structure-function relationships in the channel of the mitochondrial
inner membrane translocase TIM22 and molecular correlations with the
outer membrane
11:00-12:00. Posters & coffee 11:00 Meeting of the CIBITEN Advisory and Steering Committees (members only) 12:00-13:30. Session 2. Connexins

Chairs: Luis C. Barrio (H. Ramón y Cajal)/Jorge Contreras (UMDNJ)
12:00. Mohamed Kreir (Nanion Technologies GmbH, Munich, Germany)
Study of Gap junction hemichannels reconstituted into lipid bilayers
and artificial gap junction formation between bilayers and cells
12:15 Alberto Pereda (Albert Einstein College of Medicine, NY, USA)
Gap junction channels as electrical synapses
12:30. Carles Solsona (Universidad de Barcelona, Spain)
Connexinopathies: Are they related to ATP permeability?
12:45. Luis C. Barrio (Hospital Ramón y Cajal, Madrid)
Properties of connexin-47 mutant channels causing dysmyelination
13:00. Victoria Moreno Manzano (Centro de Investigación Príncipe
Felipe, Valencia, Spain)
Expression and biological function of connexins in adult ependymal
cells before and after spinal cord traumatic injury
13:15. David García-Dorado (Hospital Universitario Vall d'Hebron,
Barcelona, Spain)
Connexin-43: a key player in myocardial ischemia-reperfusion
13:30-15:30. Posters & lunch 13:30-15:30. Nanion ‘hands-on' session (optional) Live Patch Clamp Measurements on Bilayers and Cells with the Port-a-Patch. 15:30-17:00. Session 3. Membrane Pumps and transporters

Chairs: Pablo Martin-Vasallo (ULL)/ Miguel Holmgren (NINDS/NIH)
15:30. Miguel Holmgren (NINDS, NIH, MD, USA)
Na+ access kinetics to the Na+/K+- ATPase
15:45. Alessio Accardi (Weill Cornell Medical College, NY, USA)
Bases for ion binding and selectivity in the CLC family
16:00. Joseph A. Mindell (NINDS, NIH, MD, USA)
Transport-related conformational changes in an extracellular loop of a
glutamate transporter homolog
16:15. Susan G. Amara (University of Pittsburgh, PA, USA)
Large collective motions regulate the functional properties of
glutamate transporter trimers
16:30. Alfonso Martínez-Cruz (CIC bioGUNE, Derio, Spain)
Purification, Crystallization and Preliminary Crystallographic Analysis
of the CBS pair of the human Metal Transporter CNNM4
16:45 Ignacio Giménez (Universidad de Zaragoza, Spain)
Renal Na-K-Cl cotransporter (NKCC2) activity is modulated by
phosphorylation at residue Ser126
17:00. José M. Siverio (Universidad de La Laguna, Tenerife, Spain)
Role of Ssu2 and Nar1 in Nitrate and Nitrite Efflux in the Yeast
Hansenula polymorpha.
17:15-18:00. Posters & coffee
18:00-19:00. KEYNOTE LECTURE (Society of General Physiologists Traveling
Scholars Program Award)
William N. Zagotta (University of Washington, WA, USA)
Molecular mechanisms for the regulation of ion channels by cyclic
nucleotides
(Presented by Teresa Giraldez, HUNSC)
Thursday, 3 February 2011
9:00-10:30. Session 4. Structure-function of ion channels II
Chairs: Asia Fernández-Carvajal (UMH)/ Kenton Swartz (NIH/NINDS)
9:00. Kenton Swartz (NINDS, NIH, MD, USA)
Pore-opening mechanism in trimeric P2X receptor channels
9:15. Alan Neely (Universidad de Valparaíso, Chile)
Post-translational modifications in the regulation of inactivation of
CaV2.3 calcium channels
9:30. Baron Chanda (University of Wisconsin – Madison, WI, USA)
Electromechanical coupling in the sodium channels: Domain IV
voltage-sensor is uniquely involved in coupling to the outer pore
9:45. Chris Ahern (University of British Columbia, BC, Canada)
Investigating the role of counter charges in voltage-sensor function and
channel gating
10:00. Manuel Criado (Instituto de Neurociencias de Alicante, Spain)
A cytoplasmic domain of alpha7 acetylcholine nicotinic receptors is
involved in their transport to the membrane
10:15. Pablo Miranda (HUNSC, Tenerife, Spain)
Calcium induces structural rearrangements of the gating ring of the
human BK channel
10:30-11:30. Posters & coffee
11:30-13:00. Session 5. Transient Receptor Potential Channels (TRPs)
Chairs: Eva Delpón (U. Complutense)/ Ana Gomis (IN, Alicante)
11:30. Sebastian Brauchi (Universidad Austral de Chile, Valdivia, Chile)
Hop-diffusion on cold sensing channels, a kiss that's sure to linger
11:45. Felix Viana (Instituto de Neurociencias de Alicante, Spain)
Pharmacology and function of the cold-activated ion channel TRPM8
12:00 Rosa Planells (Centro de Investigación Príncipe Felipe, Valencia,
Spain)
Complex modulation of TRPV1
12:15. Gines Salido (Universidad de Extremadura, Cáceres, Spain)
TRPCs regulate agonist-induced Ca2+ mobilization
12:30. Anna Lucia Conte (Instituto de Neurociencias de Alicante, Spain)
Gq-coupled receptors potentiate the osmotic activation of TRPC5
12:45. Carole Jung (Universidad Pompeu Fabra, Barcelona, Spain)
Protective effect of a gain-of-function polymorphysm in TRPC4
channel against myocardial infarction
13:00-15:00. Posters & lunch
15:00-16:30. Session 6. Membrane Receptors and Signal Transduction

Chairs: Oscar Casis (UPV)/ Juan Martinez-Pinna (U. Alicante) 15:00. Barbara Ehrlich (Yale University School of Medicine, CT, USA)
Regulation of intracellular calcium signaling by calcium binding
proteins
15:15. José López-Barneo (Instituto de Biomedicina de Sevilla, Spain)
Metabotropic role of L-type Ca2+ channels in vascular smooth muscle
15:30. María Rodríguez Moyano (Instituto de Biomedicina de Sevilla,
Spain)
Essential role of store-operated calcium channels in urotensin-II
evoked vascular smooth muscle cell proliferation
15:45. Patricia González-Rodríguez (Instituto de Biomedicina de Sevilla,
Spain)
HIF-1α and Rho/ROCK participate in the hypoxic-induction of T-type
calcium channels in rat cardiac myocytes
16:00. Juan Lerma (Instituto de Neurociencias de Alicante, Spain)
P2X2/GluN2B: A new ionotropic receptor complex
16:15. Magdalena Torres (Universidad Complutense de Madrid, Spain)
cGKII phosphorylates GluR1 and promotes its incorporation into
plasma membrane of cerebellar granule cells
16:30-17:30. Posters & coffee
17:30-19:00. Session 7. Chemical Modulators: Drug Discovery and Pharmacology

Chairs: J. Antonio Lamas (U. de Vigo)/ Rosario González-Muñiz (IQM)
17:30. Angel Messeguer (IQAC, CSIC, Barcelona, Spain)
Combinatorial chemistry approaches to identify valuable
pharmacological tools and potential drug candidates
17:45. Andrea Brüggemann (Nanion Technologies GmbH, Munich,
Germany)
Chips go deeper: New possibilities of Ion channel Screening including
Organelles
18:00. Antonio Ferrer-Montiel (Universidad Miguel Hernández, Elche,
Spain)
TRPducins, a novel paradigm to modulate ion channel activity
18:15. Donato del Camino (Hydra Biosciences Inc., MA, USA)
TRPA1 is a Key Contributor to Cold Hypersensitivity
18:30 Núria Comes (Universitat de Barcelona, Spain)
The non-steroidal anti-inflammatory drug Diclofenac targets the
voltage-dependent potassium channel Kv1.3 in leukocytes
18:45. Jorge Marrero-Alonso (Universidad de La Laguna, Tenerife,
Spain)
Novel tamoxifen derivatives for the study of membrane targets
20:00. Bus departs from the Hotel Las Aguilas to Bodega El Lomo.
20:30 (approx.) Visit to the Bodega El Lomo and Gala Dinner.
www.bodegaellomo.com
Friday, 4 February 2011
9:00-10:15. Session 8. Epithelial sodium channel/degenerin (ENaC/DEG) family of ion
Chairs: Diego Alvarez de la Rosa (ULL)/ Wolfgang Clauss (U. Giessen)
9:00. Cecilia M. Canessa (Yale University School of Medicine, CT, USA)
Insights into ASIC1 gating: Open and Closed conformations of the ion
pore
9:15. Xavier Gasull (Universitat de Barcelona-IDIBAPS, Barcelona,
Spain)
Role of ASIC3 channel in inflammatory and post-operative pain
9:30. Diana Wesch (Universidad de La Laguna, Tenerife, Spain)
Differential plasma membrane abundance of epithelial sodium channel
δ subunit splice isoforms
9:45. Martin Fronius (Justus-Liebeg University, Giessen, Germany)
Proteolytic cleavage rather than membrane fluidity and the cytoskeleton
modulates the activity of epithelial Na+ channels in response to shear
force
10:00. Alexander Staruschenko (Medical College of Wisconsin, WI, USA)
Cortactin regulates ENaC via Arp2/3 complex
10:15-11:15. Posters & coffee
11:15-13:00. Session 9. Cell Physiology

Chairs: Carmen Valenzuela (IIB)/J. M. Fernandez (UPF)
11:15. Mark Shapiro (University of Texas Health Science Center, San
Antonio, TX, USA)
Multi-faceted regulation of neuronal ion channels by PIP2
11:30. Raul Estevez (Universidad de Barcelona, Spain)
Identification of a new beta subunit of the ClC-2 chloride channel
11:45. Sonia Gallego (IBGM, Universidad de Valladolid – CSIC, Spain)
CALHM1 reduces endoplasmic reticulum calcium levels in HEK293T
cells
12:00. Patricio Rojas (Universidad de Santiago de Chile)
GABAA receptors located at the axon initial segment control
excitability of dentate gyrus neurons
12:15. Antonio. R. Artalejo (Universidad Complutense de Madrid, Spain)
P2X7 receptors trigger ATP exocytosis and modify secretory vesicle
dynamics in neuroblastoma cells
12:30. Ricardo Borges (Universidad de La Laguna, Tenerife, Spain)
Chromogranins as regulators of exocytosis
12:45. Rafael Fernández-Chacón (Instituto de Biomedicina de Sevilla,
Spain)
Cysteine string protein-alpha maintains the number of synaptic release
sites and dynamin-dependent endocytosis at motor nerve terminals
13:00-15:00. Posters & lunch
15:00-16:45. Session 10. Pathophysiology of ion channels

Chairs: Raúl Estevez (UB)/ Mª Teresa Pérez (IBGM)
15:00. Cathy Proenza (University of Colorado, Aurora, CO, USA)
PKA anchoring via AKAP150 is required for sympathetic regulation of
cardiac HCN4 pacemaker channels and heart rate in mice
15:15. Juan Tamargo (Universidad Complutense de Madrid, Spain)
Functional effects of a missense mutation in HERG associated with
type 2 Long QT syndrome
15:30. Ramon Brugada
Channelopathies and sudden cardiac death
15:45. José Ramón López-López (IBGM, Universidad de Valladolid,
Spain)
Role of Kv1.3 in vascular smooth muscle proliferation
16:00. Felix Claverie-Martin (HUNSC, Tenerife, Spain)
Pre-mRNA splicing defects caused by exonic mutations in hereditary
renal diseases
16:15. Luis A. Pardo (Max Planck Institute for Experimental Medicine,
Göttingen, Germany)
Cancer therapies targeting KV10.1 channels
16:30. Ruben Vicente (Universitat Pompeu Fabra, Barcelona, Spain)
ORMDL3 expression levels affect lymphocyte physiology
17:00-18:00 KEYNOTE LECTURE (Sponsored by the University of La Laguna).
K. George Chandy (University of California – Irvine, CA, USA)
Targeting ion channels in lymphocytes as therapy for autoimmune
diseases: A journey from bench towards bedside
Presented by Luis A. Pardo (MPI, Göttingen, Germany)
18:30-20:30 Wine & Beer Round Table (sponsored by Nanion)
Chair: WN Zagotta
Ramón Latorre, Kenton Swartz, Alessio Accardi, Miguel Holmgren
Further musings on ion channel structure problems for perspicacious
biophysicists, chemists and physiologists
(After the initial remarks the discussion will be open to everyone) PLENARY LECTURES
Molecular mechanisms for the regulation of ion channels by cyclic nucleotides
William N. Zagotta
Department of Physiology and Biophysics, University of Washington School of Medicine,
Seattle, Washington, USA
Ion channels are exquisite molecular machines. By opening and closing an ion selective pore
across the cell membrane, these proteins ultimately control everything from our senses to our
thoughts. Our long term goal is to understand the precise molecular motions that underlie this
gating behavior of ion channels. Our work has focused on cyclic nucleotide-regulated channels,
channels that are gated by the direct binding of cAMP and cGMP. This family included the
cyclic nucleotide-gated (CNG) channels important in sensory transduction and the
hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels, important for
pacemaker activity of cardiac and neuronal cells. We have studied the molecular mechanisms
of gating in these channels using a combination of electrophysiology, X-ray crystallography, and
fluorescence. From these studies a picture is starting to emerge for how the binding of cyclic
nucleotide to the channel causes opening of the pore.
Targeting ion channels in lymphocytes as therapy for autoimmune diseases: A journey
from bench towards bedside
K. George Chandy
Department of Physiology and Biophysics, University of California, Irvine, California, USA
For more than 25 years, it has been widely appreciated that Ca2+ influx is essential to trigger
lymphocyte activation. Patch clamp analysis, molecular identification, and functional studies
using blockers and genetic manipulation have shown that a unique contingent of ion channels
orchestrates the initiation, intensity, and duration of the Ca2+ signal. Five distinct types of ion
channels – Kv1.3, KCa3.1, Orai1+ STIM1 [Ca2+-release activating Ca2+ (CRAC) channel],
TRPM7, and Clswell – comprise a network that performs functions vital for ongoing cellular
homeostasis and for T-cell activation, offering potential targets for immunomodulation. Kv1.3, KCa3.1, STIM1, and Orai1 cluster at the immunological synapse following contact with an antigen-presenting cell and might function to modulate local signaling. The expression pattern of ion channels and hence the functional network can adapt depending upon the state of differentiation and activation, and this allows for different stages of an immune response to be targeted specifically. Intravital imaging approaches are beginning to shed light on ion channel function in vivo. Highly selective inhibitors of the Kv1.3 and KCa3.1 channels have been developed and these have demonstrated efficacy in several models of immune disorders. Two of these blockers are scheduled for first-in-man phase 1 clinical trials in 2011.
ORAL PRESENTATIONS

O-1.1
Mechanisms of Ca2+-dependent gating in MthK channels
Brad Rothberg
Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140,
USA
MthK is a Ca2+-gated K+ channel whose principal Ca2+ binding site has been identified by x-
ray crystallography. Despite our know ledge of its structure, the energetic contributions of the
Ca2+-coordinating residues D184, E210, and E212 to MthK gating is unknown. We studied the
effects of charge-neutralizing mutations (D to N and E to Q) at these residues on Ca2+-
dependent gating by single-channel electrophysiology and subsequent kinetic analysis. We
observe that D184N and E210Q show decreased Ca2+-sensitivity, shifting the EC50 for Ca2+-
activation by 3-fold and 2-fold compared to wild-type MthK (ΔΔG of +0.6 and +0.4 kcal/mole),
respectively. Interestingly, the E212Q mutation did not decrease Ca2+ sensitivity, but instead
showed a slight increase in Ca2+-sensitivity (ΔΔG of -0.2 kcal/mole). These results suggest that
the carboxylate sidechains at D184 and E210 are key elements responsible for Ca2+
dehydration and binding it this site, which subsequently leads to the conformational change that
underlies gating.
This work is supported by the NIH and American Heart Association.



O-1.2
Mechanism for selectivity-inactivation coupling in KcsA potassium channels
Crina M. Nimigean
Departments of Physiology and Biophysics and Department of Biochemistry, Weill Cornell
Medical College, New York, USA
Structures of the prokaryotic K+ channel, KcsA, highlight the role of the selectivity filter carbonyls
from the GYG signature sequence in determining a highly selective pore, but channels
displaying this sequence vary widely in their cation selectivity. Furthermore, variable selectivity
can be found within the same channel during a process called C-type inactivation. We
investigated the mechanism for changes in selectivity associated with inactivation in a model K+
channel, KcsA. We found that E71A, a non-inactivating KcsA mutant in which a hydrogen-bond
behind the selectivity filter is disrupted, also displays decreased K+ selectivity. In E71A
channels, Na+ permeates at higher rates as seen with 86Rb+ and 22Na+ flux measurements and
analysis of intracellular Na+ block. Crystal structures of E71A reveal that the selectivity filter no
longer assumes the "collapsed", presumed inactivated, conformation in low K+, but a "flipped"
conformation, that is also observed in high-K+, high-Na+ and even Na+-only conditions. The data
reveal the importance of the E71-D80 interaction in both favoring inactivation and maintaining
high K+ selectivity. We propose a molecular mechanism by which inactivation and K+ selectivity
are linked, a mechanism that may also be at work in other channels containing the canonical
GYG signature sequence.


O-1.3
Specific ions, specific lipids: sources of protein stability in an ion channel model
M.L. Renart, I. Triano, E.Montoya, J.A. Poveda, J.L. Ayala, F.J. Gómez, J.A. Encinar, A.M.
Fernández, G. Fernández-Ballester, A.V. Ferrer-Montiel, J.M. González-Ros
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain
Ion channels are complex membrane proteins which recognize and selectively bind specific
ions, while allowing their permeation at near diffusion-limited rates. How to reconcile these two
apparently opposed phenomena, ion selectivity and rapid permeation, still remains a matter of
debate. The potassium channel KcsA from S.lividans is been used as a model system to
attempt answering some of these fundamental questions. Using spectroscopic approaches
(mainly fluorescence) with detergent-solubilized KcsA, we have studied the interaction of ions
and lipids with the channel, particularly regarding their effects on the structure and stability of
the
Our data indicate that K+ and Na+ antagonize each other in binding to the protein according to their relative affinity, potassium being more effective in stabilizing the protein against thermal denaturation. Titrating the protein with increasing concentrations of K+ or Na+ (no competition conditions) reveals two binding events for K+ (µM and low mM range) and seems consistent with the proposed equilibrium of the selectivity filter between conductive and non-conductive states and related to the ion permeation process. Conversely, Na+ mildly stabilizes the protein with an apparent Kd in the low mM range, in agreement with IC50 values for channel blockade. On the other hand, we have been able to characterize phospholipids and alkyl sulfates binding to KcsA. Anionic phospholipids and alkyl sulfates longer than 10 carbon atoms are the more effective compounds in increasing the thermal stability of the channel through their binding to intersubunit In summary, specific ions and lipids have an essential role in the stability, conformational equilibrium and function, acting as cofactors or allosteric effectors of ion channels. Partly supported by Spanish BFU2008-00602/BMC and CSD2-2008-00005 O-1.4
PAS domains in EAG channels
Joao H. Morais-Cabral
Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
PAS domains are sensor/regulatory domains present in many protein systems in eukaryotes
and prokaryotes. This sensory role is performed in many cases with the participation of a bound
co-factor, heme, FAD or other small molecules. The eukaryotic EAG potassium channels are 6
TM voltage -gated potassium channels with a role in cardiac repolarization, tumor growth and
neuronal activity. A distinct characteristic of EAG channels is the presence of an N-terminal
cytoplasmic PAS domain. The function of the PAS domains in the EAG channels is not known
but it has been speculated that they will have a regulatory role. I will present and discuss some
of our results on PAS domains from EAG channels.



O-1.5
Detection of physical interactions between the initial segment of the amino terminus and
the S4-S5 linker of the h-ERG potassium channel
Francisco Barros, Jorge Fernández-Trillo, Angeles Machín, Rosa Alvarez, Luis Carretero, Pedro
Domínguez
Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Oviedo, Spain The h-ERG channel amino terminus acts as a crucial determinant of gating. It is also known that the S4-S5 linker couples the voltage sensing machinery to the channel gate. This linker has been repeatedly proposed as an interaction site for the distal portion of the amino terminus controlling channel gating, but direct evidence for such interaction is still lacking. We used a combination of site-directed mutagenesis, functional reconstitution of gating-altered mutant channels, disulfide bond formation between pairs of engineered cysteines and TIRF-FRET spectroscopy, to demonstrate the physical interaction between the distal portion of the amino terminus and the S4-S5 linker. Expression of a recombinant h-ERG N-terminal fragment restored the gating properties modified by deletion of most of the amino terminus, but also by specific truncation of the eag/PAS domain or by introduction of single point mutations in the initial sequence of the protein. This effect is not observed with channels carrying single point mutations in the S4-S5 linker. The functional reconstitution correlates with a substantial level of FRET between truncated channels and recombinant fragments labelled with CFP and YFP. Furthermore, the currents from channels with pairs of cysteines introduced at the beginning of the amino terminus and in specific positions of the S4-S5 linker, were strongly attenuated by an oxidizing agent, an effect rapidly reversed by dithiothreitol. Thus, specific interactions between the initial segment of the amino terminus and the S4-S5 linker constitute an essential component of the gating machinery, contributing to the unusual gating properties of h-ERG. Supported by MICINN grant BFU2009-11262 and CSD-2008-00005 SICI Consolider- Ingenio from Spanish MEC. J. F-T., R.A. and L.C. hold predoctoral fellowships from MEC, MICINN and FICYT. A.M. holds a postdoctoral contract from SICI-Consolider 2008. O-1.6
Structure-function relationships in the channel of the mitochondrial inner membrane
translocase TIM22 and molecular correlations with the outer membrane
Lauro González-Fernández, Jorge Luis Bermejo, Patricia Rojo, María Luisa Campo
Department of Biochemistry and Molecular Biology, Universidad de Extremadura, Cáceres,
Spain
Several multisubunit complexes convey the precise relocation of cytoplasmic encoded proteins
to one of the four compartments enfolded by the two mitochondrial membranes. Aqueous
channels are at the core of these translocases and have proven essential for normal functioning
of TOM, in the outer membrane, and TIM23 in the inner membrane. Also, we have reported the
conditions to uncover, in organello, the channel associated to TIM22, the translocase mediating
the insertion of multispaning proteins into the inner membrane. We have performed the
molecular dissection of the complex present in mitochondria of eight yeast strains with different
expression levels of its defined components. A combination of these results with those of the
native complex present in the genetically modified cells, and those obtained directly applying
patch-clamp techniques to the inner membranes of their mitochondria, outline the biogenesis of
TIM22 and the distinct role played by each component. Remarkably, and despite their different
composition, the channels of the three translocases: TOM, TIM23 and TIM22, are noteworthy
similar. Most likely these results are in tenant with their common task as membrane conduits
transiently clapping the proteins in route to their final destination inside mitochondria. In
addition, and for the first time, we have detected a specific correlation between TIM22 in the
inner membrane and the Voltage Dependent Anionic Channel (VDAC) of the outer membrane,
suggesting a potential interaction and feasible cooperation between the two mitochondrial
membranes
Supported by MCIN grant BFU2008-00475; Junta de Extremadura and Fondo Social Europeo.




O-2.1
Study of gap junction hemichannels reconstituted into lipid bilayers and artificial gap
junction formation between bilayers and cells
Mohamed Kreir1, Christian Carnarius3, Christoph Methfessel2, Volker Moehrle2, Michael Habig2,
Andrea Brüggemann1, Claudia Steinem3, Niels Fertig1.
1Nanion Technologies GmbH, Erzgiessereistr. 4, D-80335 Munich, Germany; 2Bayer Technology Services, D-51368 Leverkusen, Germany; 3Institut für Organische und Biomolekulare Chemie, Georg August Universität Göttingen, D-37077 Göttingen, Germany Connexins (Cx) are members of a multigene family of membrane-spanning proteins that form gap junctions, which are composed of two hexameric hemichannels, called connexons. These gap junctions, organized in so-called gap junctional plaques, span the extracellular space/matrix of adjacent cells and thus allow a passive exchange of small molecules up to about 1 kDa. Connexins are widely distributed with various subtypes of connexin and are involved in different biological processes such transmission of information and propagation of action potential for e.g. Recent studies indicates that hemichannels do open under physiological and pathological conditions. In our study, we investigated the biophysical properties of hemichannels Cx26 and Cx43 which were isolated biochemically and reconstituted into synthetic lipid membranes. Both hemichannels are present in different tissues and involved in different pathologies. The results on a study of the Cx26 are presented. Reconstitutions of functional Cx26 and mutant hemichannels were performed. Secondly, Cx43 was purified and reconstituted into bilayers. The hemichannel Cx43 properties were compared to previous studies and showed similarities of conductance on single channel recordings of Cx43 in cells. Our focus was then to form artificial gap junctions, first between two unrelated cells and then between cells and bilayers containing functional hemichannels. This was done using Cx26 or Cx43. The bilayer-cell configuration allows to measure electrophysiological properties of the cells indirectly via gap junctions. Single channel recordings of gap junctions were recorded using a bilayer containing Cx43 and Cardiomyocytes expressing Cx43. Macroscopic currents were as well recorded between bilayers and cell lines expressing Cx26 or Cx43. O-2.2
Gap junction channels as electrical synapses
Alberto Pereda
Albert Einstein School of Medicine, New York, USA
Trafficking and turnover of synaptic receptors is known to be required to maintain and modify
the strength of chemical synapses. In contrast, little is known regarding the involvement of
trafficking in gap junction-mediated electrical synapses. By combining ultrastructural and in-vivo
physiological analysis at identifiable mixed, electrical and chemical, synapses on the goldfish
Mauthner cell, we report here that gap junction channels are continuously trafficked in and out
of the membrane of these contacts. Strikingly, despite that electrical transmission is supported
by only 1-2% of intercellular channels, electrical coupling was readily modified by peptides that
interfere with endo- and exocytosis, suggesting that the strength of the electrical synapses at
these terminals is sustained, at least in part, by a fast turnover of gap junction channels. We
also found that interactions involving conserved regions of the carboxy-terminus of connexin 36
and its teleost homologs are required for the surface expression of gap junction proteins at
these contacts. Thus, our data indicates that electrical synapses should be considered highly
dynamic structures and that an active turnover of gap junction channels significantly contributes
to maintaining their strength. The evidence raises the possibility that regulated trafficking of gap
junction channels could underlie plastic changes of junctional conductance.


O-2.3
Connexinopathies: Are they related to ATP permeability?
Ezequiel Mas del Molino, Xenia Grandes, Anna Nualart, Mireia Martín Satué, Luis C. Barrio*
and Carles Solsona
U. Barcelona - IDIBELL, F. Medicine, Dept. Pathology and Experimental Therapeutics,
Hospitalet de Llobregat, Spain; * Hospital Ramon y Cajal. Experimental Neurology. Madrid.
Spain.
Connexins (Cx), in addition to form gap junctions, are also incorporated to the cell plasma
membrane functioning as hemichannels. We have tested if human Cx32, Cx30 and Cx26 will be
permeable for ATP. We have expressed these proteins in Xenopus oocytes and recorded
simultaneously the membrane currents and the light emited by luciferine-luciferase in the
presence of small traces of ATP. The Cx32 is transiently permeable to ATP and the release is
associated to a tail inward current. The Cx30 is permeable to ATP and in most cases the inward
current is not inactivated. The Cx26 is not permeable to ATP. The Cx32 is located, among other
parts of the human body, on peripheral nerves and mutations of this protein have been related
to Charcot-Marie-Tooth disease linked to Chromosome X (CMTX). This is a inherited senso-
motor neurodegenerative disease in which more than 290 mutations have been so far. We have
tested de following mutations S26L, P87A, Δ111-16, D178Y, and R220X. In some mutations the
protein does not reach the plasma membrane, in others, the ATP release is decreased or the
speed of release is altered. Connexins Cx26 and Cx30 are located at organ of Corti of the
cochlea and point mutations of Cx26 and Cx30 have been reported to cause sensorineural,
non-syndromic, prelingual hearing impairment. He have studied the following mutations of
Cx26: G12R, S17F, D50N, D50Y, and the T5M of Cx30. Heteromeric combination of Cx26 and
Cx30 are permeable to ATP.
Supported by SAF 2008/732.




O-2.4
Properties of connexin-47 mutant channels causing dysmyelination
I Fasciani1, D González-Nieto2, J Molano3, P. Martínez-Montero3, C Sierra4, J Piccirilli5, N
Deconinck5, A Monier5, Y Sznajer5, T Sekhara5 and Luis C. Barrio1
1IRYCIS-Madrid, 2UPM-CTB-Madrid, 3IdIPAZ-Madrid, 4CH-Jaén, Spain; 5ULB-Brussels, Belgium Gap junction protein connexin-47 (Cx47) is expressed by oligodentrocytes, the myelin forming cells of CNS. Cx47 can form "reflexive" channels intramyelin, intercellular channels between oligodentrocytes themselves and heterologous channels between oligodentrocytes and astrocytes, forming a "glial syncytium". This syncytium has been implicated in the spatial buffering of K+ released during axonal activity. Recessive mutations in GJA12/GJC2, the gene that encodes Cx47, cause Pelizeaus Merzbacher-like disease (PMDL), an early onset dysmyelinating disorder of the CNS, characterized by nystagmus, psychomotor delay and progressive spasticity. Here we describe two novel recessively inherited mutations: H252D and Y47H. To develop an understanding of how Cx47 gap junction channels might function in central myelin and how PMDL mutations cause dysmyelination, a primary goal is to determine the properties of constitutive channels. To address this issue, we first identified the start site for Cx47 translation since this mRNA contains two putative AUG codons (Met1 and Met4). We found that the translation initiates by a linear scanning at the first Met, yielding a full-length protein of 440 aa. The first three residues (Met-Thr-Asn) at the 5´ extreme of NT domain are critical determinants of unitary conductance, permeability and voltage- and pH-gating of homotypic Cx47 channels, and also account for the rectifying behavior of heterotypic channels of Cx47 with astrocytic Cx43. The analysis of H252D and Y47H mutations showed that the two mutant proteins fail to form functional homotypic channels and heterotypic channels with astrocytic Cx43 in dual voltage-clamp and dye-coupling assays, suggesting that disruption of glial Funding: Consolider-2008_0005 & SAF2009-11614
O-2.5
Expression and biological function of connexins in adult ependymal stem cells before
and after spinal cord traumatic injury
1Francisco Javier Rodríguez-Jiménez, 1Fabrice Durupt, 2Ilaria Fasciani, 3Carlos Paino, 1Miodrag Stojkovic, 2Luis C. Barrio, 1Victoria Moreno-Manzano 1Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain, 2 Hospital Ramon y Cajal. Experimental Neurology. Madrid. Spain. The presence of ependymal stem/progenitor cells (epSPC) in the adult spinal cord suggests that endogenous stem cell-associated mechanisms might be exploited to repair spinal cord lesions. We recently demonstrated that after acute transplantation of undifferentiated epSPC obtained from injured rats (epSPCi) or the resulting oligodendrocyte precursor cells into a rat model of severe spinal cord contusion produced a significant recovery of locomotor activity. Interestingly, a better yield of neurosphere proliferation and of differentiated cells to oligodendrocytes and neurons was obtained from epSPCi than non-activated epSPC cultures. Connexins (Cx) belong to a multigene family of proteins that form cell-to-cell channels mediating direct intercellular communication. Cx have been implicated in proliferation and differentiation of neuronal progenitors. Here we explore the expression of Cx as well as the cell communication through ion channels in ependymal stem cells before and after activation by spinal cord injury in the undifferentiated stage or during an oligodendrocyte directed-differentiation process. We detected genes of the cx family that are downregulated in epSPCi. Protein expression of Cx40, Cx43 and Cx50 declined in epSPCi. The expression of Cx43 and Cx50 during directed differentiation form epSPCi to oligodendrocytes exhibited an inverse tendency relationship to its expression levels. The incidence of cell-to-cell communication was assessed by microinjecting the membrane impermeable tracer 5 (and-6)-carboxyfluorescein (CF) into single cells in the center of neurospheres. A rapid transfer of tracer was detected from the injected cells to at least 30 neighboring cells in both epSPCi and epSPC neurospheres. For dye uptake experiments, CF (100 µM) was added in the culture medium for 1 hour. Retention of dye into the cell interior after a treatment with cx channel/hemichannel blockers (carbenoxolone 200 µM, La3+ 100 µM or high extracellular Ca2+) was analyzed with confocal z-images. The images showed that a large number of cells in epSPCi and epSPCc neurospheres are CF-positive. These observations demonstrate an intense intercellular communication among ependymal stem/progenitor cells into neurospheres via cell-to-cell channels as well as open hemichannels and substantiate the hypothesis for an active role of Cx in proliferation and differentiation of ependymal progenitor cells.
O-2.6
Connexin-43: a key player in myocardial ischemia-reperfusion
David García-Dorado, Marisol Ruiz-Meana, Elisabet Miró, Esperanza Agulló, Jesús Vazquez,
Estefanía Nuñez, Antonio Rodríguez-Sinovas, Ignasi Barba, Celia Fernandez.
Vall d'Hebron University Hospital and Research Institute - Universitat Autònoma de Barcelona,
CBM-Universidad Autónoma de Madrid
Cx43 forms gap junction channels connecting the cytoplasm of adjacent cardiomyocytes. It is
well known that Gap juntions (GJ) are essential for electrical impulse propagation in the heart
and that ischemia rapidly reduces GJ conductivity and distribution which can cause arrhythmias.
In previous studies we demonstrated that GJ communication may allow cell-to-cell propagation
of cell death injury and during myocardial ischemia reperfusion, explain the continuity of
patches of contraction band necrosis, and contribute to final infarct size. More recently we
proposed that delayed recovery of GJ communication during reperfusion could be an effector of
the protection against cell death induced by ischemic preconditioning (brief ischemic episodes
immediately before prolonged ischemia and reperfusion). Other groups confirmed that
preconditioning protection was lost in hearts deficient in Cx43, but it was soon discovered that
this effect was independent of changes in JC communication, since it could also be observed in
isolated cardiomyocytes.
More recent studies have demonstrated that Cx43 is present at the inner membrane of
subsarcolemmal cardiomyocyte mitochondria. Total replacement of Cx43 by Cx32 in a viable
mice model has allowed us to show that Cx43 has important effects on K+ handling and
Complex I-driven mitochondrial respiration. Moreover, although this replacement allows normal
impulse propagation and ventricular function, it is associated to deeply altered energy
metabolism and abolition of ischemic preconditioning. We are now investigating the effects of
preconditioning and ischemia-reperfusion on respiration and function of subsarcolemmal and
interfibrillar mitochondria, and comparing them with changes in mitochondrial proteins by means
of quantitative differential proteomics. The results indicate that Cx43 localized not only in GJ but
also in mitochondrial membranes play essential, unsuspected roles in cardiac function and in
myocardial response to ischemia. This knowledge may help to understand the pathophysiology
of myocardial ischemia-reperfusion and its modification by conditions associated to reduced
Cx43, such as ageing or heart failure, and lead to the development on new therapies.




O-3.1
Na+ access kinetics to the Na+/K+-ATPase
Miguel Holmgren
Molecular Neurophysiology Section, Porter Neuroscience Research Center, National Institute of
Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA
In exchanging three intracellular Na+ for two extracellular K+ the Na+/K+ pump moves net charge
through the membrane's electric field. Most of this charge movement occurs among transitions
involving Na+ release to the extracellular environment. The electrical signals associated with
these ion translocation steps can be isolated from pump cycle by constraining the pumps to
operate in the absence of K+. Under these conditions, at a given membrane potential and
external [Na+], pumps distribute among conformations with zero, one, two, or three Na+ bound.
With a sudden voltage jump, as pumps redistribute to a new steady-state they generate
transient currents. By fast voltage clamp of the squid giant axon membrane, we have identified
three components in these current relaxations: fast (which follows the voltage-jump time
course), medium-speed (tm 0.2-0.5 ms), and slow (ts 1-10 ms). Here we tracked the kinetics
of these components with exquisite time precision. In a forward pump cycle, each Na+ is
released in a separate transition, rate limited by the first ion to leave the pump, followed by the
sequential release of the next two Na+.



O-3.2
Bases for ion binding and selectivity in the CLC family
Alessio Accardi
Department of Anesthesiology, Weill Cornell Medical College, 525 East 68th Street, A-1050,
New York, NY 10065-4897, USA
Ion binding to a secondary active transporter triggers a cascade of conformational
rearrangements which lead to substrate translocation across a membrane. We investigated ion
binding and selectivity in CLC-ec1, a H+/Cl– exchanger of the CLC family of channels and
transporters. We found that Cl– affinity depends on the conformational state of the protein: it is
highest with the extracellular gate removed, and progressively weakens as the transporter
adopts the occluded configuration and then with the intracellular gate removed. Furthermore,
we found that the central ion-binding site determines selectivity in CLC transporters and
channels, a serine to proline substitution at this site confers NO3– selectivity upon the otherwise
Cl– specific CLC-ec1 transporter and CLC-0 channel. We propose that CLC-ec1 operates
through an affinity-switch mechanism and that the bases of substrate specificity are conserved
in the CLC channels and transporters.



O-3.3
Transport-related conformational changes in an extracellular loop of a glutamate
transporter homolog
Joseph Mindell
Membrane Transport Biophysics Section, Porter Neuroscience Research Center, National
Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda,
Maryland, USA
Conformational changes are essential for transporter function. In the model system GltPh, an
archaeal EAAT homologue from Pyrococcus horikoshii, we used limited trypsin proteolysis
experiments to identify a site in the long extracellular loop that stretches between helices 3 and
4 that becomes protected from proteolysis in the presence of a substrate, L-aspartate, or an
inhibitor, DL-TBOA in the presence of Na+, the cotransported ion. Using a combination of site-
directed cysteine-scanning mutagenesis and fluorescein-5-maleimide labelling we found that
positions throughout the loop experience these ligand-induced conformational changes. By
selectively cleaving the 3-4 loop (via introduced Factor Xa sites) we demonstrate that it plays a
vital role in the transport process; though structurally intact, the cleaved proteins are unable to
transport aspartate. These results inculcate the 3-4 loop as an important player in the transport
process, a finding not predicted by any of the available crystal structures of GltPh.


O-3.4
Large collective motions regulate the functional properties of glutamate transporter
trimers
Jie Jiang1, Indira Shrivastava2, Spencer D Watts1, Delany Torres-Salazar1, Ivet Bahar2, Susan
G Amara1
Departments of Neurobiology1 and Computational & Systems Biology2, School of Medicine,
University of Pittsburgh, PA 15261, USA
Glutamate transporters present at the plasma membrane maintain extracellular glutamate
concentrations below toxic levels and limit the action of glutamate released during excitatory
neurotransmission. Although these carriers serve a primary role in glutamate clearance, they
also possess a ligand-gated chloride channel activity that can regulate neuronal excitability. Our
laboratory has used site-directed mutagenesis, sulfhydryl modification, and chemical
crosslinking approaches together with biochemical, and electrophysiological analyses of the
mammalian carriers to examine the structural domains required for substrate transport and ion
permeation. Using cysteine crosslinking studies and computational simulations we have
recently identified several large-scale collective motions that are intrinsic to glutamate
transporter trimers. Single cysteine residues introduced into the extracellular gate on each of
the three subunits form reversible intersubunit crosslinks spontaneously and/or catalyzed by
oxidizing reagents, such as copper phenanthroline. After crosslinking, substrate uptake, but not
the substrate-activated anion conductance, is completely inhibited in these mutants. The
formation of disulfide bridges between pairs of cysteines that are more than 40Å apart in the
static structure can be explained by the concerted subunit movements predicted by anisotropic
network model (ANM) analysis. Furthermore, we have observed that these collective motions
are functionally important for the sequential inward movement of core domains, a critical step in
the glutamate transport cycle. The ability of the individual subunits to undergo separate
transitions between outward-facing and inward-facing forms, rather than an all-or-none
transition of the three subunits, is a feature also supported by ANM-predicted intrinsic dynamics.
Overall, these results shed light on how the large collective motions contribute to the functional
dynamics of these carriers.


O-3.5
Purification, Crystallization and Preliminary Crystallographic Analysis of the CBS pair of
the human Metal Transporter CNNM4.
Inmaculada Gómez García, Iker Oyenarte and Luis Alfonso Martínez-Cruz
Structural Biology Unit, CIC bioGUNE, Derio, Spain
This work describes the purification and preliminary crystallographic analysis of the CBS pair
regulatory domain of the human Ancient Domain Protein 4 (ACDP4), also known as CNNM4.
ACDP proteins represent the least studied members of the eight different types of magnesium
transporters identified in mammals so far. In humans the ACDP family includes four members,
CNNM1 to 4. CNNM1 acts as a cytosolic copper chaperone and has been ligated to the
Urofacial syndrome, whereas CNNM2 and CNNM4 have been identified as magnesium
transporters.
Interestingly, mutations in the CNNM4 gene have clinical consequences that are limited to
retinal function and biomineralization and are considered the cause of the Jalili Syndrome
[MIM607805], consisting of autosomal-recessive Cone-rod dystrophy and Amelogenesis
imperfecta. The truncated protein was overexpressed, purified and crystallized in the
orthorhombic space group C222. Crystals diffracted X-rays to 3.6 Å resolution using
synchrotron radiation. Matthews volume calculations suggested the presence of two molecules
in the asymmetric unit that likely correspond to a CBS module of the CBS pair of CNNM4.


O-3.6
Renal Na-K-Cl cotransporter (NKCC2) activity is modulated by phosphorylation at
residue Ser126
Ignacio Giménez, Carmen Ibañez
Aragon Health Sciences Institute, and Department of Pharmacology and Physiology, University
of Zaragoza, Spain
Kidney specific Na-K-2Cl cotransporter (NKCC2) is activated by phosphorylation of N-terminal
threonine residues (Thr99 and Thr104). An additional downstream conserved residue, Ser126,
was initially identified as being phosphorylated in vitro by AMP-activated kinase (AMPK).
Recent phosphoproteomic studies have shown Ser126 is phosphorylated in vivo in a regulated
fashion, likely mediated by PKA. To determine the relative importance of Ser126
phosphorylation in NKCC2 regulation, we have used site-directed mutagenesis and
heterologous expression in Xenopus oocytes. Our results suggest NKCC2 activity might depend
on Ser126 phosphorylation when Thr99 and Thr104 are not phosphorylated. In addition, Ser126
phosphorylation appears to modulate the phosphorylation of Thr99 and Thr104.
Supported by MICINN BFU2007/62119



O-3.7
Role of Ssu2 and Nar1 in Nitrate and Nitrite Efflux in the Yeast Hansenula polymorpha.
Elisa Cabrera, Rafaela González-Montelongo, Celia Rodríguez and José M. Siverio.
Department of Biochemistry and Molecular Biology, University of La Laguna, Tenerife, Spain
In yeast and fungi, nitrate and nitrite efflux has been somewhat neglected, the participant
molecular entities are unknown, while its relationship with net nitrate uptake has been
underestimated. Here we show the role of HpSsu2 (sulfite efflux permease) in nitrate efflux and
HpNar1 (belonging to the formate nitrite transporter family) in both nitrate and nitrite excretion in
the yeast Hansenula polymorpha. Expression of the Saccharomyces cerevisiae sulfite efflux
permease SSU1 in Hp∆ssu2 revealed its capacity to efflux nitrate and nitrite, while HpSsu2
effluxes only nitrate. Nitrate accumulation in ∆ssu1∆ynr1, ∆nar1∆ynr1 and ∆ssu2∆nar1∆ynr1
increases progressively from the first to the latter. These results indicate that Ssu2 and Nar1 are
involved in nitrate efflux and nitrate reductase activity is not required for net nitrate uptake as
has been claimed. Further evidence on the capacity of Ssu2 to excrete nitrate was obtained in
Xenopus oocytes. In Dssu2, growth and cell viability in nitrate were clearly diminished while in
∆nar1 they were mainly affected by nitrite. We conclude that Ssu2 and Nar1 play a role in
avoiding high levels of toxic nitrite accumulation by excreting nitrate, and nitrate and nitrite,
respectively. Ssu2 and Nar1 are also clearly involved in regulating the nitrate assimilation gene
pathway, since they regulate intracellular nitrate and nitrite content.


O-4.1
Pore-opening mechanism in trimeric P2X receptor channels
Kenton J. Swartz
Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National
Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda,
Maryland, USA.
The opening of ion channels in response to ligand-binding, voltage or membrane-stretch,
underlies electrical and chemical signaling throughout biology. The recent X-ray structure of a
trimeric ATP-activated P2X receptor channel in a closed state reveals that the pore-lining
helices traverse the membrane at unusually steep angles and cross one another to form a
constriction within the outer half of the membrane. I will discuss our efforts to explore how the
structure of the pore changes during opening in P2X receptors by measuring the modification of
introduced Cys residues by thiol-reactive reagents, and by engineering metal bridges. Our
results are consistent with the X-ray structure of the closed state, and demonstrate that
expansion of the gate region in the external pore is accompanied by an unexpected narrowing
of the inner pore, suggesting that the pore-forming helices straighten to open the channel. This
unique pore-opening mechanism has implications for the role of subunit interfaces in the gating
mechanism of P2X receptors and points to a role of the internal pore in ion permeation.


O-4.2
Post-translational modifications in the regulation of inactivation of CaV2.3 calcium
channels
Alan Neely2, Vivian Gonzalez1,2, Erick Miranda-Laferte3, Gustavo Contreras1,2 and Patricia
Hidalgo3
1Programa de Doctorado en Ciencias Biológicas, Mención Neurociencias y 2Centro Interdisciplinario de Neurociencias de Valparaíso. Universidad de Valparaiso. 3Institut für Neurophysiologie, Medizinische Hochschule Hannover The Voltage-dependent calcium channels undergo two type of inactivation that are strongly influenced by accessory protein such as the calcium channel β-subunit (CaVβ) and calmodulin. While calmodulin is a determinant of calcium dependent-inactivation, CaVβ regulates voltage-dependent inactivation. In the neuronal type of calcium channel CaV2.3, inactivation is accelerated by all CaVβ except the palmitoylated splice variant CaVβ2a. The prevalent view is that this membrane-anchoring posttranslational modification keeps the inactivation domain from occluding the pore. However, inactivation of heterologously expressed CaV2.3 channels is slowed significantly by non-palmitoylated CaVβ when injected as purified recombinant proteins on Xenopus laevis oocytes already expressing the pore forming subunit. This phenotype was reverted if recombinant proteins were injected together with CaV2.3 cRNA as if CaVβ can switch the inactivation phenotype by some posttranslational modifications during channel biogenesis that is not accessible to the membrane anchored CaVβ2a. Here we investigate the time course of the phenotype switch and its sensitivity to some kinases and palmitoylation inhibitors and found that independent on the biogenesis of the pore-forming subunit, the inactivation-accelerating phenotype develops in about 24. FONDECYT 1980635 to AN and CONICYT-DFG to AN and PH
O-4.3
Electromechanical coupling in the sodium channels: Domain IV voltage-sensor is
uniquely involved in coupling to the outer pore
Baron Chanda
Department of Physiology, University of Wisconsin-Madison Medical School, Madison,
Wisconsin, USA
Voltage-dependent sodium channels are responsible for initiating the rising phase of an action
potential in the nerve, muscle and heart. Activation of their voltage-sensing domains in
response to depolarization leads to the opening and inactivation of the channel pore. To probe
the interaction between voltage-sensor and pore of the sodium channels, we monitored the
movement of individual voltage-sensors by measuring gating pore currents. These currents are
due to ion flux through the voltage-sensing domains at rest. We find that the binding of outer
pore blockers such as tetrodotoxin modulates the activation of the domain IV voltage-sensor but
does not have any effect on the movement of the other voltage-sensing domains. These
findings provide evidence that the activation of domain IV is anomalously coupled to
conformational changes in the outer pore of the sodium channel.



O-4.4
Investigating the role of counter charges in voltage-sensor function and channel gating
Chris Ahern
Department of Anesthesiology, Pharmacology and Therapeutics, The University of British
Columbia, Vancouver, British Columbia, Canada.
S4 segments are charged with the task of transporting their cationic Arg and Lys side-chains
through the low dielectric environment presented by the core of the bilayer during voltage-
sensing. How voltage-sensors achieve this task remains unresolved, however, co-evolved
acidic residues have been proposed to stabilize S4 charges by forming transient, state
dependent 'salt-bridges'. Moreover, these acidic residues have been suggested to support
proper voltage-sensor folding and trafficking. We used in vivo nonsense suppression in tandem
with voltage-clamp fluorimetry to investigate the contribution of these acidic amino acids to
voltage-sensor function and channel gating. The data show that incorporation of isosteric
neutral derivatives of Asp and Glu at three highly conserved negative charges in the voltage-
sensor of Shaker channels, E283, E393 and D316, has little impact on channel gating or S4
movement as determined by voltage-clamp fluorimetry of unnatural amino acid containing
channels. These results suggest, surprisingly, that the negative side-chains from these
residues do not engage electrostatically in salt-bridge formation with S4 charges. This result
may be explained by the possibility that salt-bridge formation is strongly reliant on the local
dielectric, and are weakest in a high-dielectric environment, a possibility supported here by ab
initio calculations utilizing crystallographic coordinates of the charges involved. We interpret
these results to suggest that these co-evolved negative charges promote the formation of
aqueous vestibules whose presence, in turn, diminish the ability of negative residues to interact
electrostatically with S4 charges.


O-4.5
A cytoplasmic domain of alpha7 acetylcholine nicotinic receptors is involved in their
transport to the membrane
Manuel Criado, José Mulet, Susana Gerber, Salvador Sala, Francisco Sala
Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Sant Joan
d'Alacant, Alicante, Spain
Certain domains of acetylcholine nicotinic receptor (nAChR) subunits seem to be involved in
different steps of receptor biosynthesis. Previously, we have shown that deletion of the
cytoplasmic region inmediately adjacent to the fourth transmembrane domain of alpha7
nAChRs (from Glu437 to Arg447) abolished receptor expression at the membrane of Xenopus
oocytes. Now we have analyzed this region in more detail. Alanine mutants of Trp438, Lys439,
Phe440 and Val445 showed moderate decreases in membrane expression (28-65% of wild-
type receptors) as measured by alpha-bungarotoxin binding, whereas only 10% of expression
was observed for V444A and D446A mutants. Interestingly, when two alanines at positions 441
and 442 were changed to prolines, membrane expression was totally abolished. Since high
resolution electron microscopy data have suggested that the fourth transmembrane domain and
the adjacent cytoplasmic region analyzed here, might form a continuous alpha-helix, our
hypothesis was that preservation of this helix was important for receptor biogenesis. This was
confirmed when introduction of prolines at other positions (mutants K439P, F440P, C443P and
V444P) also abolished membrane expression. On the other hand, in selected mutants with low
or null membrane expression, we discovered a significant proportion of mature receptors (30-
70% of wild-type receptors) inside oocytes, so that we postulate that elements in this
cytoplasmic region influence receptor transport to the cell membrane. This effect could be
direct, through interactions with the transport machinery, and/or indirect by the selection of only
properly
Supported by the Ministry of Science and Innovation of Spain O-4.6
Calcium induces structural rearrangements of the gating ring of the human BK channel
Pablo Miranda1, Jorge Contreras2, Diana Wesch1, Fred Sigworth3, Miguel Holmgren4, and
Teresa Giraldez1
1 Unidad de Investigación, Hospital Universitario Ntra Sra de Candelaria, 38010-Tenerife, Spain; 2 Department of Pharmacology & Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA; 3 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA; 4National Institute for Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. Large conductance voltage- and calcium-activated potassium channel (BK) activation is regulated by intracellular calcium and membrane voltage. Ca2+-sensitivity of this channel is conferred by a large, cytosolic domain forming a gating ring structure. Crystal structures of the gating ring have been solved in several prokaryotic channels and transporters such as the Ca2+-activated MthK channel, and recently in eukaryotic BK channels. Calcium binding to this region reduces the energy required to open the channel, but the exact mechanism underlying this process is still uncertain. Different structures of MthK have led to the theory that Ca2+ binding causes an increase in its diameter, physically inducing the opening of the channel gate. Given its sequence homology and structural similarity to MthK, it could be inferred that BK's gating mechanism by Ca2+ is similar to that of the prokaryotic channel. We have measured movements within the BK gating ring domain of whole functional BK channels expressed in oocyte membrane macropatches using Patch-Clamp Fluorometry. We used heterotetramers of YFP- and CFP-tagged subunits to measure simultaneously ionic currents and FRET signals between equivalent tagged-sites on adjacent subunits. We have detected relative movement at three different positions within the gating ring. Our results unequivocally demonstrate that upon Ca2+ binding there is a structural rearrangement of the gating ring. While this molecular rearrangement induced by Ca2+ is voltage independent, the opening and closing of pore remain voltage-dependent with higher sensitivity toward more negative potential. This suggests that rearrangements at the gating ring upon Ca2+ binding facilitate the opening of the pore, but do not directly gate the pore.
O-5.1
Hop-diffusion on cold sensing channels, a kiss that's sure to linger
Sebastian E. Brauchi
Instituto de Fisiologia, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile
TRPM channels have been described not exclusively at the plasma membrane (PM), but also at
intracellular membranes where its role is not understood. Knowledge regarding channel
intracellular localization, trafficking, and recruitment to the PM represent an important piece of
information needed to integrate channel activity with more complex cellular responses.
TRPM8 is a non-selective cation channel expressed on a subset of peripheral neurones, and is
the molecular machine that allow us to detect cold signals from our surroundings. There is
growing evidence linking TRPM8 to the development and maintenance of cold allodynia and
hyperalgesia in the somatosensory system. Additionally, TRPM8 has been also identified in
different tissues and associated with an important variety of tumors, where its role is not entirely
understood.
Some members of the TRP channel family changes their cellular distribution in response to
stimulation. Here, we will describe membrane/near-membrane dynamics of TRPM8-GFP
containing particles in both, HEK-293T and F-11 transfected cells. 2D and 3D trajectories
together with the velocity of individual protein containing vesicles were obtained by Total
Internal Reflection of Fluorescence Microscopy (TIRFM) and single particle tracking (SPT).
Trajectories were analysed by plotting the mean-square displacement against time. Four
characteristic types of motion were observed: (a) stationary; (b) simple Brownian diffusion; (c)
directed diffusion; and (d) confined diffusion, in which particles undergoing Brownian diffusion
are confined within a limited area. Our data suggests that TRPM8, when inserted into the
plasma membrane, is confined into small domains of about 3 um in diameter, in which receptor
molecules resides in the time scale of 2-8 s. In the absence of stimuli TRPM8 vesicles
constantly move along a network that cover the plasma membrane, periodically stopping, most
often just briefly. Stimulation halted this hop-diffusion probably by stabilizing TRPM8 channels,
as a result, release from plasma membrane became significantly slower. This slower release of
TRPM8 determined the overall increase of available receptors.
Support from: Fondecyt 11070190 and 1110906; Pew Programs in Biomedical Sciences;
SeedingLabs.


O-5.2
Pharmacology and function of the cold-activated ion channel TRPM8
Félix Viana, Cruz Morenilla, Annika Mälkiä, María Valero, Rodolfo Madrid, Elvira de la Peña,
María Pertusa, Víctor Meseguer, Gregorio Fernández, Antonio Ferrer-Montiel & Carlos
Belmonte
Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones
Científicas. San Juan de Alicante, 03550 Alicante, Spain
TRPM8 is a non-selective cation channel of the TRP family activated by voltage, mild cold
temperatures and cooling compounds (e.g. menthol). TRPM8 is selectively expressed in small
sensory neurons innervating the skin and in prostate cancer cells. In the peripheral nervous
system TRPM8 is clearly involved in the transduction of ambient cold temperatures. In contrast,
the function of TRPM8 in prostate tissue is currently unknown. Recently, we examined the
pharmacological and functional properties of TRPM8 channels in both tissues, and compared
them with the properties in various recombinant systems. A summary of findings follows:
In native and recombinant channels cooling and menthol shift, dose-dependently, the activation
curve of TRPM8 towards more negative potentials. The activation V1/2 of TRPM8 is
approximately 140 mV more negative in native channels compared to recombinant channels. Functionally, this large difference translates into a shift of >5ºC in the apparent temperature threshold. TRPM8 is glycosylated at position N934, a process that favours its accumulation within membrane lipid rafts. Removing plasma membrane cholesterol potentiates TRPM8 function. Several drugs, including BCTC, SKF96365 and clotrimazole inhibit the channel, acting as negative allosteric modulators of gating. The same blockers are less effective in prostate cancer cells where the channel is retained within intracellular compartments. Using site directed mutagenesis, we found that the aa Y745 at the menthol binding site, within the 2 TM region, is critical for the inhibition of cold- and voltage-activated TRPM8 currents produced by SKF96365. In contrast, the inhibition by BCTC was unaffected, suggesting a different binding site. Supported by MCINN (SAF2010-14990) and CONSOLIDER_INGENIO 2010 (CS2007-00023).
O-5.3
Complex modulation of TRPV1
Rosa Planells Cases
Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain.
The Transient Receptor Potential Vanilloid 1 (TRPV1), founding member of thermo-TRPs, is a
molecular integrator of noxious stimuli, considered to play a pivotal role in pain transduction. A
wide variety of regulating mechanisms control TRPV1 channel activity, leading to disease states
that involve nociceptor sensitization (and TRPV1 activity potentiation) or, alternatively, to a
refractory state (or TRPV1 desensitization). Specifically, regulation of TRPV1 activity can be
achieved at different levels encompassing from gene expression, to receptor post-translational
modification, formation of channel heteromers, association to intracellular regulatory proteins,
up to receptor compartmentalization. Nevertheless, an important principle that emerges from
signal transduction research considers that membrane receptors and ion channels assemble
into discrete macromolecular entities (or signalplexes) together with signalling molecules whose
physical contact would warrant an efficient and specific stimuli transduction. Several signalling,
trafficking and scaffolding proteins have been described to interact with TRPV1 and sharply
modulate its function, thus suggesting as being part of a TRPV1 signalplex. Here, we describe
that the cytosolic GABAA Receptor Associated Protein (GABARAP) interacts with TRPV1 in
heterologous expression systems and DRG neurons, and modulates TRPV1 biosynthesis,
trafficking and gating properties. Functionally, GABARAP co-expression attenuated voltage and
capsaicin sensitivity of TRPV1 in the presence of extracellular calcium. Furthermore, the
presence of GABARAP selectively increased the interaction of tubulin with the C-terminal
domain of TRPV1. Disruption of tubulin cytoskeleton suggested that GABARAP and
cytoskeleton interaction contributes to traffic and cluster TRPV1 on the plasma membrane,
modulating TRPV1 channel gating.



O-5.4
TRPCs regulate agonist-induced Ca2+ mobilization
Ginés M. Salido, Geoffrey E. Woodard, José J. López, Isaac Jardín and Juan A. Rosado
Department of Physiology, University of Extremadura, Ficell, Cáceres, Spain
Ca2+ signals are finely regulated by Ca2+ handling proteins that assemble into signaling
complexes. It has been reported that TRPC3 regulates IP3R function by mediating interaction
between IP3R and the scaffolding protein RACK1 (receptor for activated protein kinase C-1), a
protein that plays a key role in transduction of plasma membrane signals to downstream
effectors, thus regulating agonist-induced Ca2+ release (Bandyopadhyay et al., 2008). We have
found that stimulation with agonists results in association of Orai1 with types I and II IP3Rs, as
well as with TRPC3, RACK1, and STIM1 (stromal interaction molecule 1). TRPC3 expression
silencing was found to abolish the interaction of Orai1 with TRPC3 and the type I IP3R, but not
with the type II IP3R, thus suggesting that TRPC3 selectively mediates interaction between
Orai1 and type I IP3R. In addition, TRPC3 expression silencing attenuated agonist-stimulated
interaction between RACK1 and the type I IP3R, as well as Ca2+ release and entry (Woodard
et al., 2010). In conclusion, our results indicate that agonist stimulation results in the assembly
of the Orai1-STIM1-TRPC3-RACK1-type I IP3R complex, where TRPC3 plays a central role
playing
Supported by MICINN BFU2010-21043-C02-01 and Junta de Extremadura -FEDER (GR10010). References Bandyopadhyay, B. C., Ong, H. L., Lockwich, T. P., Liu, X., Paria, B. C., Singh, B. B., and Ambudkar, I. S. (2008) J. Biol. Chem. 283, 32821–32830. Woodard, G.E., López, J. J., Jardín, I., Salido, G. M., and Rosado, J. A. (2010) J. Biol. Chem. 285, 8045–8053. O-5.5
Gq-coupled receptors potentiate the osmotic activation of TRPC5
Anna Lucia Conte, Sergio Soriano, Félix Viana, Ana Gomis
Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones
Científicas. Sant Joan d'Alacant, 03550 Alicante.
The canonical transient receptor potential (TRPC) channels are a family of non-selective cation
channels that are activated by increases in intracellular Ca2+ and stimulation of
Gq/phospholipase C-coupled receptors. TRPC5, a member of this family is widely expressed in
the central and peripheral nervous system where it has an inhibitory impact on neurite extension. TRPC5 is activated by a range of stimuli, such as lanthanides and also lysophospholipids which suggests that this channel exhibits sensitivity to the structure of the lipid bilayer. However its activation mechanism and function is poorly understood. We have investigated the potential role of TRPC5 as an osmo-mechanical transducer channel. Here we show that extracellular hypoosmotic stimuli (210mOsm), leading to cell swelling and membrane stretch, produce an increase in the intracellular calcium concentration in HEK293 cells transfected with TRPC5. The activation is further potentiated by osmotic modulation of G-protein-coupled receptors via PLC-coupled and Ca2+-dependent signalling pathways. When TRPC5 is co-expressed with the Gq/11-coupled type1 histamine-receptor (H1), the osmotically-evoked [Ca2+]i response results from a dual mechanism: calcium entry from the extracellular medium and calcium release from intracellular stores. Recent work has indicated the possibility that regulated vesicular trafficking mechanisms might play a critical role in the activation of TRPC5. Using a biotinylation assay we found that the surface expression of TRPC5 channels was significantly increased after the treatment with hipoosmotic solution in cells transfected with TRPC5-H1 compared with cells transfected only with TRPC5. Our results suggest that osmotic activation of the G-protein-couple receptors could contribute to TRPC5 activation by increasing its surface expression. Supported by: MCINN (BFU2009-07853).CSIC (Intramural 2009-20I098) and CONSOLIDER_INGENIO 2010 (CS2007-00023). O-5.6
Protective effect of a gain-of-function polymorphysm in TRPC4 channel against
myocardial infarction
Carole Jung, Gemma G. Gené, Marta Tomas, Cristina Plata, Jana Selent, Manuel Pastor,
César Fandos, Sanela Mrkonic, Anna Garcia-Elias, Mariano Senti, Gavin Lucas, Roberto
Elosua and Miguel A. Valverde
Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona,
Spain.
The TRPC4 non-selective cation channel is widely expressed in the endothelium where
generates Ca2+ signals that participate in the endothelium-mediated vasodilatatory response.
This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC4 associated
to
METHODS AND RESULTS. Our candidate-gene association studies identified a missense SNP (TRPC4-I957V) associated with a reduced risk for myocardial infarction in diabetic patients (OR= 0.61; CI, 0.40-0.95, P=0.02). TRPC4 was also associated with myocardial infarction in the WTCCC genome-wide data, an intronic SNP (rs7319926) within the same LD block as TRPC4-I957V showed an OR of 0.86 (CI, 0.81-0.94; P=10-4). Functional studies of the missense SNP were carried out in heterologous expression systems. Patch-clamp studies and measurement of intracellular [Ca2+] in response to muscarinic agonists and direct G-protein activation showed increased channel activity in TRPC4-I957V transfected cells, compared to TRPC4-WT. Site directed mutagenesis and molecular modeling of TRPC4-I957V suggested that the gain of function was due to the presence of a less bulky Val-957. This permits a firmer interaction between the TRPC4 and the catalytic site of the tyrosine kinase that phosphorylates Tyr-959 and CONCLUSIONS. We provide evidence for the association of a TRPC4 SNP with myocardial infarction in population-based genetic studies. The higher Ca2+ signals generated by TRPC4-I957V may ultimately facilitate the generation of endothelium- and nitric oxide-dependent vasorelaxation, thereby explaining its protective effect at the vasculature. O-6.1
Regulation of intracellular calcium signaling by calcium binding proteins
Barbara Ehrlich
Deparment of Pharmacology, Yale University School of Medicine, New Haven, Connecticut,
USA
The inositol 1,4,5-trisphospate receptor (InsP3R), an intracellular calcium channel, is critical for
the initiation of intracellular calcium (Ca2+) signaling. To fulfill this function, the InsP3R depends
on interaction with accessory subunits and regulatory proteins. In this presentation I will
compare and contrast the effects of protein partners in the cytoplasm and in the lumen of the
endoplasmic reticulum (ER). In particular I will discuss the effects of two calcium binding
proteins, neuronal calcium sensor 1 (NCS-1) and chromogranin b (CgB) on InsP3R channel
function and intracellular calcium transients. NCS-1 is a high affinity, low capacity calcium
binding protein found in the cytoplasm. Binding of NCS-1 to the InsP3R increases intracellular
signaling and these signals are attenuated by pretreatment with the chemotherapueutic agent,
paclitaxel. CgB is a low affinity, high capacity calcium binding protein found in the lumen of the
ER. CgB is the most potent regulator of the InsP3R identified to date. Levels of CgB are
increased in heart failure. The pathophysiological relevance of InsP3R modulation becomes
apparent when the functional interaction of these proteins is enhanced or abolished by mutation
or overexpression.


O-6.2
Metabotropic role of L-type Ca2+ channels in vascular smooth muscle
José López-Barneo
Instituto de Biomedicina de Sevilla (Spain)




O-6.3
Essential role of store-operated calcium channels in urotensin-II evoked vascular smooth
muscle cell proliferation
María Rodríguez-Moyano, Antonio Ordóñez, Tarik Smani.
Instituto de Biomedicina de Sevilla, Spain
Urotensin-II (U-II) is a neuropeptide that acts through the de-orphanized G-protein-coupled
Urotensin receptor (UTS2R), which has been linked to several cardiovascular diseases. U-II
evokes vascular smooth muscle cell (SMC) proliferation, although the precise mechanism
activated downstream of UTS2R remains elusive. Recently, the store-operated Ca2+ entry
pathway (SOCE) has been demonstrated to play an important role in cell proliferation and
migration.
In this study, we aim to determine the implication of SOCE pathway in SMC proliferation evoked
by
The proliferation was assessed by 5-bromo-2'-deoxyuridine (BrdU) labeling of rat aortic SMC incubated with or without U-II. In addition, intracellular Ca2+ mobilization was performed in isolated SMC loaded with fura-2AM using a Ca2+ imaging system. First, we determined that U-II (100 nM) induces Ca2+ and Mn2+ entry that was significantly reduced by store-operated Ca2+ channels (SOCC) inhibitors, Gadolinium (10 µM) and 2-aminoethyl diphenylborinate (2APB, 75 µM). Furthermore, small interfering RNA (siRNA) of the new players of SOCE, STIM1 or Orai1, abolished the Ca2+ and Mn2+ entry induced by U-II. Then, we determined that U-II promotes dose dependent SMC proliferation, reaching maximum effect with 100 nM concentration, which was sensitive to SOCC inhibitors and by molecular knockdown of STIM1 and In summary, we demonstrate for the first time an essential role of Ca2+ entry through a STIM1 and Orai1 dependent SOCE pathway in U-II induced vascular SMC proliferation. Acknowledgements: This study was supported by MICINN (BFU-2010-21043-C02-02), ISCIII (PS09-02287 and Recava RD06-0014-0020) and from la Junta de Andalucía (P08-CVI-3913).
O-6.4
HIF-1α and Rho/ROCK participate in the hypoxic-induction of T-type calcium channels in
rat cardiac myocytes
Patricia González-Rodríguez, María J. Castro, José López-Barneo and Antonio Castellano
Instituto de Biomedicina de Sevilla, Spain
Voltage-gated T-type Ca2+ channels (T-channels) are expressed during embryonic
development in ventricular myocytes, but are undetectable in adult ventricular myocytes.
Interestingly, T-channels are reexpressed in pathological conditions associated with ischemic
episodes. We have data confirming that in a rat cardiac failure model, induced by coronary
artery ligation, T-channels are de novo expressed in ventricular myocytes. However, whether T-
channels play a role in the pathogenesis of cardiomyopathy and its underlying mechanisms is
unclear.
We report here that the Cav3.2 (α1H gene) T- channel is highly induced in hypoxia in dispersed
cardiomyocytes (1% O2) as well as in a hypoxic animal model (8-9% O2). The effect of hypoxia
on Cav3.2 gene expression is time- and dose-dependent, and is accompanied by an increase in
the density of T- type calcium channel currents recorded in patch-clamped cells. HIF-1α seems
to be involved in T- channel induction by hypoxia in cardiomyocytes, since cobalt chloride and
dimethyloxalylglycine, compounds that produce HIF stabilization, simulate the hypoxic effect.
Importantly, regulation by hypoxia is specific for the Cav3.2 T-type calcium channel, since it is
not observed in L-type channels. In addition, we have investigated whether the Rho/ROCK
signaling pathway is involved in the hypoxic HIF-1α stabilization in ventricular myocytes. We
show that hypoxia increases Rho-kinase protein level and RhoA activity. Fasudil, a ROCK
inhibitor, inhibits the hypoxia-induced HIF-1α protein expression, with no changes in HIF-1α
mRNA levels, and inhibits T-channel mRNA up-regulation in hypoxia. These results suggest
that the activation of the Rho/ROCK signaling participates in the hypoxia-induced HIF-1α
stabilization and T-channel induction.


O-6.5
P2X2/GluN2B: A new ionotropic receptor complex
Juan Lerma, Isabel Aller and Ricardo J. Rodrigues
Instituto de Neurociencias de Alicante, CSIC-UMH; San Juan de Alicante
In the nervous system, fast signalling is achieved by activation of ligand-gated ion channels.
Previous studies have reported a tight interaction and cross-talk between ionotropic receptors,
namely between P2XRs and other ionotropic receptors such as nAChRs, 5-HT3Rs and
GABAARs. These interactions are all characterized by a physical interaction that leads to an
activity-dependent cross-inhibition designed to negatively control the excess of function of the
other signalling systems by ATP. Since ATP and glutamate are co-released from nerve
terminals and P2XRs and NMDARs have an overlapping subcellular localization in the CNS, we
decided to study the existence of putative dimerization between P2X and NMDA receptors. P2X
and NMDA receptors are coincidence detectors of neuronal function and their interaction would
be of particular interest. We found the existence of hybrid P2X2/GluN2B receptors, ionotropic
receptors composed by subunits from dissimilar receptor families that are able to sense more
than one signalling molecule. Indeed, NMDA (300 µM) was able to elicit [Ca2+]i transients only
in HEK293 cells expressing P2X2 and GluN2B (formerly NR2B) subunits, but not in non-
transfected cells or cells solely transfected with GluN2B. Whole-cell patch clamp analysis in
combination with a fast perfusion system for agonist application revealed that the activation of
this putative P2X2/GluN2B receptor requires the co-application of ATP and NMDA. The "hybrid
current" was inhibited by APV, PPADS and ifenprodil, but not by MK801. The I/V relationship
and Mg2+-sensitivity observed for P2X2/ GluN2BRs were similar to those observed for P2X2Rs,
but they do not share P2X2Rs' ability to permeate NMDG+ ions. Co-immunoprecipitation of
GluN2B and P2X2 was also observed in brain tissue, and bimolecular fluoresce
complementation studies showed that P2X2 carries GluN2B to the plasma membrane.
Likewise, in P2X2-transfected hippocampal neurons the presence of functional P2X2/
GluN2BRs could also be demonstrated. The present study provides evidence for the formation
of a hybrid P2X2/ GluN2B ionotropic receptor, which may constitute a new concept of functional
neurotransmitter receptor.
Supported by CSD2007-0023 and BFU-2006-007138


O-6.6
cGKII phosphorylates GluR1 and promotes its incorporation into plasma membrane of
cerebellar granule cells
Salvatore Incontro, José Sánchez-Prieto and Magdalena Torres
Department of Biochemistry, Universidad Complutense de Madrid, Spain
Trafficking of AMPA receptors (AMPARs) is regulated by specific interactions with other proteins
and by posttranslational mechanisms like phosphorylation by different kinases. We have found
that cGMP-dependent protein kinase II (cGKII) phosphorylates the GluR1 at S845 and
increases the surface expression of AMPARs mostly at extrasynaptic sites. Activation of cGKII
by 8-Br-cGMP increases the surface expression of GluR1 whereas its inhibition or its
suppression effectively decreased the surface level of this protein. GluR1 is mainly incorporated
into calcium permeable AMPAR because the AMPA elicited calcium increase was clearly
abrogated in the presence of 1-naphthyl acetyl spermine (NASPM), a synthetic analogue of
Joro spider toxin (JSTX) that specifically blocks the Ca2+-permeable AMPA receptors. NMDA
receptor stimulation (NMDAR) effectively stimulates nitric oxide and cGMP production in
granule cells that in turns activates cGMP dependent protein kinases (cGKs), which stimulates
GluR1 accumulation in the plasma membrane. This work identifies a novel pathway
downstream from the NMDA receptor (NMDAR) involved in controlling GluR1 trafficking that
may play an important role in synaptic plasticity.


O-7.1
Combinatorial chemistry approaches to identify valuable pharmacological tools and
potential drug candidates
Angel Messeguer, Gloria Vendrell-Navarro, Federico Rúa.
Department of Chemical and Biomolecular Nanotechnology. IQAC.CSIC. Barcelona, Spain
It is widely admitted that Combinatorial Chemistry has become one of the most useful tools
developed by medicinal chemists for exploring the chemical diversity space in the drug
discovery area. In this communication we will report our recent work on the design, construction
and validation of combinatorial libraries of small organic molecules. In particular, libraries of
controlled mixtures of oligomers of N-alkylglycines (peptoids) and their application to identify
compounds active against different pharmaceutical targets will be presented.

Acknowledgements. Financial support from MICINN SAF2008-00048 Grant and Programme
SICI-CONSOLIDER INGENIO 2010 (CSD2008-00005).





O-7.2
Chips go deeper: New possibilities of Ion channel Screening including Organelles
Andrea Brüggemann
Nanion Technologies GmbH, Munich, Germany
Over the last several years ion channels have received more and more interest as potential
drug targets, because of their known involvement in chronic and acute disease. To meet the
ever-increasing demand for higher throughput in ion channel screening and safety testing the
development a highly parallel patch clamp platforms, like the SyncroPatch 96, was urgently
needed. The platform supports giga-seal recordings, continuous recording during compound
application and addition of multiple compounds at each of the 96 cells recorded from at a time.
The possible advantages of ion channel screening do not stop with the increase in throughput.
Moreover it also opens up a new field of experiments. The planar design allows continues
internal perfusion of whole cell measurements, which is very helpful for the study of internal
second messengers. By being able to manufacture variable hole sizes the planar patch clamp
technique allows the measurements of cell organelles like Lysosomes and Mitochondria. The
characterisation of the lysosmal Ca2+ release channels TPCN2 was made possible*. TPCN2 is
activated by nicotinic
acid adenine dinucleotide phosphate and is regulated by intralysosomal pH. Using site-directed
mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for
conferring high Ca2+ selectivity. Our glass chipbased method provides electrophysiological
access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion
channels.
References
* Characterization of two pore channel 2 (TPCN2)-mediated Ca2+ currents in isolated
lysosomes. The Journal of Biological Chemistry, 2010, 285 (28)
Planar Patch Clamp Approach to Characterize Ionic Currents from Intact Lysosomes
Science Signaling, 2010, 3 (151)


O-7.3
TRPducins, a novel paradigm to modulate ion channel activity
Antonio Ferrer-Montiel
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain
The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor
implicated in diverse physiological and pathological processes. The TRP domain, a highly
conserved region in the C-terminus adjacent to the internal channel gate, is critical for subunit
tetramerization and channel gating. We found that peptides patterned after this protein domain
block TRPV1 activity by binding to the intracellular side of the receptor, and presumably
interfering with protein-protein interactions at the level of the TRP domain that are essential for
the conformational change that leads to gate opening. Palmitoylation of active peptides reveals
that they are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking
receptor activity in intact rat primary sensory neurons and their peripheral axons. The most
potent lipidated peptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy
(IC50≤10 µM), without significantly affecting other thermoTRP channels. In contrast, its
retrosequence or the corresponding sequences of other thermoTRP channels did not alter TRPV1 channel activity (IC50>100 µM). TRP-p5 display anit-hyperalgesic and anti-prurito activity in a model of chronic hepatic failure. Therefore, these palmitoylated peptides, that we coined TRPducins, are non-competitive, voltage-independent, sequence-specific TRPV1 blockers with in vivo activity. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels, as well as other channel families. O-7.4
TRPA1 is a Key Contributor to Cold Hypersensitivity
Donato del Camino
Hydra Biosciences, Inc, Cambridge, Massachusetts 02139, USA
TRPA1 is a calcium permeable, non-selective cation channel expressed by nociceptors. It
serves as a broad irritancy receptor for a variety of reactive chemicals, both endogenous and
exogenous. Whether TRPA1 plays an additional role in cold sensation remains controversial.
Here, we demonstrate that even mild cooling markedly increases TRPA1 currents in a
recombinant system, but only following agonist activation. In the absence of an agonist, noxious
cold has a clear but very small effect on current amplitude. Extending this finding in acutely
isolated neurons from the rat dorsal root ganglia, cooling significantly potentiates agonist-
evoked TRPA1 currents. Cooling alone, however, is not sufficient to produce measurable
TRPA1 currents. We conclude that TRPA1 is likely a key mediator of cold hypersensitivity in
pathological conditions where pro-inflammatory activators of the channel are present, but plays
only a minor role in acute cold sensation. Supporting this, cold hypersensitivity can be induced
in wild-type but not Trpa1-/- mice by subcutaneous administration of a non-noxious
concentration of the TRPA1 agonist 4- HNE. Furthermore, the selective TRPA1 antagonist HC-
030031 reduces cold hypersensitivity in rodent models of inflammatory and neuropathic pain
without altering normal cold sensation in naïve animals.


O-7.5
The non-steroidal anti-inflammatory drug Diclofenac targets the voltage-dependent
potassium channel Kv1.3 in leukocytes
Núria Comes1, Núria Villalonga1, Miren David2, Joanna Bielańska1, Teresa González2, Mireia
Pérez-Verdaguer1, Concepció Soler3, Carmen Valenzuela2 and Antonio Felipe1
1Molecular Physiology laboratory, Departament de Bioquímica i Biología Molecular, Institut de
Biomedicina, Universitat de Barcelona. Avda Diagonal 645, E-08028 Barcelona, Spain.
2Instituto de Investigaciones Biomédicas "Alberto Sols", CSIC-UAM. C/Arturo Duperier 4, E-
28029 Madrid, Spain. 3Departament de Patologia i Terapèutica Experimental, Facultat de
Medicina, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, E-08907,
L'Hospitalet de Llobregat, Barcelona, Spain.
Kv1.3 plays a crucial role in the activation and proliferation of T-lymphocytes and macrophages.
While Kv1.3 is responsible for the voltage-dependent potassium current in T-cells, in
macrophages this K+ current is generated by the association of Kv1.3 and Kv1.5. Patients with
autoimmune diseases show a high number of effector memory T cells that are characterized by
a high expression of Kv1.3 and Kv1.3 antagonists ameliorate autoimmune disorders in vivo.
Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) used in patients who suffer from
painful autoimmune diseases such as rheumatoid arthritis. In this study, we show that
diclofenac impairs immune response via a mechanism that involves Kv1.3. While diclofenac
inhibited Kv1.3 expression in activated macrophages and T-lymphocytes, Kv1.5 remained
unaffected. Diclofenac also decreased iNOS levels in Raw 264.7 cells, impairing their activation
in response to lipopolysaccharide (LPS). LPS-induced macrophage migration and IL-2
production in stimulated Jurkat T-cells were also blocked by pharmacological doses of
diclofenac. These effects were mimicked by Margatoxin, a specific Kv1.3 inhibitor, and
Charybdotoxin, which blocks both Kv1.3 and Ca2+-activated K+ channels (KCa3.1). Because
Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of
high pharmacological relevance.


O-7.6
Novel tamoxifen derivatives for the study of membrane targets
Jorge Marrero-Alonso1,3,5, Araceli Morales1,3, Benito García Marrero4,5, Alicia Boto4, Raquel
Marín2,3,5, Tomás Gómez1,3,5, Mario Díaz1,3,5
Departments of Animal Biology(1) and Physiology(2), (3) Institute of Biomedical Technologies,
University of La Laguna, 38206 Tenerife, Spain. (4) Institute of Natural Products and
Agrobiology, CSIC, 38206 Tenerife, Spain. (5) Canarian Institute of Cancer Research, ICIC,
Tenerife, Spain.
It is widely recognized that tamoxifen, a Selective Estrogen Receptor Modulator (SERM)
extensively used on estrogen receptor-positive breast cancer treatment, leads to undesirable
side effects during chronic therapies. Numerous studies have shown that some of these effects
may be exerted through acute non-genomic mechanisms. In this sense, we have shown that
both, tamoxifen and novel tamoxifen salts derivatives synthesized in our laboratory, rapidly and
reversibly inhibited (in a dose-dependent manner) the contractile activity of mouse duodenal
and uterine smooth muscles likely through the interference with L-type calcium channels
activity.
The development of fluorescent conjugates on permeable and impermeable derivatives will help
to unravel the cellular targets for tamoxifen, both at the plasma membrane and intracellular
levels, raising the possibility of designing more specific and selective compounds to treat breast
cancer with less undesirable side-effects. In this sense, the first fluorescent tamoxifen derivative
has been synthesized by our group maintaining the triphenylethylene core of tamoxifen, which
is essential to interact with estrogen receptors. Efficiency and viability of this fluorescent
compound were assessed by confocal microscopy on estrogen receptor-positive cell lines
MCF7. Finally, competition studies showed that the fluorescent derivative binding at the plasma
membrane could be readily antagonized by unlabelled tamoxifen, indicating that the plasma
membrane binding sites are specific for triphenylethylene compounds.
Supported by grant SAF2007-66148-C02-02 from MEC and FICIC funds (Spain). J. Marrero-
Alonso is hired by FICIC with funds from ACIISI (Spain) and FSE (EU). A. Morales was a fellow
of the "Juan de la Cierva" programme from MEC (Spain).



O-8.1
Insights into ASIC1 gating: Open and Closed conformations of the ion pore
Tianbo Li, Youshan Yang and Cecilia M. Canessa
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut,
USA
ASIC1 is a proton-activated sodium channel found in the plasma membrane of most neurons in
the mammalian nervous system. ASIC1 modulates synaptic transmission, induction of fear and
memory, and nociception in peripheral neurons. Structurally, ASIC1 is a trimer of identical or
homologous subunits. Each subunit has two transmembrane segments, short intracellular
amino- and carboxytermini, and a large extracellular domain. This architecture is the hallmark of
all members of the ENaC/Degenerin family regardless of their particular function and mode of
activation. We screened TM1 and TM2 by SCAM and MTS reagents of lamprey ASIC1, a
channel with little intrinsic desensitization and with higher tolerance to cysteine mutagenesis
than the mammalian counterpart. The effect of MTS reagents applied to the external or internal
side of the channel (outside-out or inside-out patches) in either the open or closed
conformations revealed a pattern of reactivity consistent with an open architecture having the
narrowest segment at the level of residues G443A444S445 in TM2. In the closed conformation
MTSEA modifies outer residues of TM2 (429-to-432) when applied from the external side but when applied from the internal side it reaches position 440 indicating that the constriction made by G443A444S445 widens in the open conformation allowing the entrance of the reagent deeper into the ion pathway. In addition, substitutions of residues G443A444S445 either abolish current or change ion conductance consistent with forming the selectivity filter. We conclude that the open configuration has a constriction at the level of the selectivity filter (G443A444S445) whereas the closed configuration has a constriction at the level of G432-to-Q437. These findings imply: first that the selectivity filter is the narrowest segment of the pore in the open conformation but it widens and disassembles in the closed state. Second, that the constriction formed by G432-to-Q437 functions as the opening, closing and desensitization gates, and third, that the transition from
closed to open requires straightening of the 50o angle of TM2.



O-8.2
Role of ASIC3 channel in inflammatory and post-operative pain
Xavier Gasull1,2, Emmanuel Deval1, Jacques Noel1, Anne Delaunay1, Abdelkrim Alloui3,4, Sylvie
Diochot1, Miguel Salinas1, Anne Baron1, Valérie Friend1, Alain Eschalier3,4, Michel Lazdunski1,
Eric Lingueglia1
1Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS/Université de Nice-Sophia Antipolis, Sophia Antipolis, Valbonne, France; 2Facultat de Medicina, Universitat de Barcelona-IDIBAPS, Barcelona, Spain; 3Inserm U766, Clermont-Ferrand ; 4Clermont Université, Université d'Auvergne, Pharmacologie fondamentale et clinique de la douleur, Clermont-Ferrand, France Acid-sensing ion channels (ASICs) are voltage-independent cationic channels activated by extracellular protons expressed in both, peripheral and central nervous tissues. Members of the ASIC family of channels expressed in peripheral sensory neurons are well placed to sense changes in extracellular pH in acidic pain conditions (inflammation, ischemia, lesions or tumors). About ∼60% of rat cutaneous sensory neurons express ASIC3-like currents. Moderate changes in pH, alone or in combination with hypertonicity or arachidonic acid, increase nociceptor
excitability and produces pain, which is suppressed by the sea anemone toxin APETx2, a
specific blocker of ASIC3. In a rat model of primary inflammation-induced hyperalgesia, both,
APETx2 injection and in vivo knockdown of ASIC3 with a specific siRNA show potent analgesic
effects. In addition, in a rat model of postoperative pain (i.e., hindpaw skin/muscle incision), high
levels of ASIC-type currents ( 77%) in sensory neurons innervating the hindpaw muscles, with
a prevalence of ASIC3-like currents are found. The ASIC3 protein is largely expressed in
lumbar DRG neurons innervating the plantar muscle, and its mRNA and protein levels are
increased by plantar incision 24 h after surgery. Pharmacological inhibition of ASIC3 channels
with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by siRNA led to a
significant reduction of postoperative spontaneous and postural pain behaviors (spontaneous
flinching and weight bearing). The specific homomeric ASIC1a blocker PcTx1 has no effect on
spontaneous flinching, when applied peripherally. Together, these data demonstrate a
significant role for peripheral ASIC3-containing channels in inflammatory and postoperative
pain.



O-8.3
Differential plasma membrane abundance of epithelial sodium channel δ subunit splice
isoforms
Diana Wesch1,2, Mike Althaus3, Pablo Miranda2, Martin Fronius3, Wolfgang G Clauss3, Teresa
Giraldez2, Diego Alvarez de la Rosa1
1Department of Physiology and Institute of Biomedical Technologies, University of La Laguna,
La Laguna, Spain, 2Research Unit, Hospital Universitario Ntra. Sra. de Candelaria, Santa Cruz
de Tenerife, Spain, 3Institute of Animal Physiology, Justus-Liebig University, Giessen, Germany
Epithelial Na+ channel (ENaC) α, β and γ subunits form heterotrimeric Na+-selective ion
channels with a well-characterized role in Na+ reabsorption across tight epithelia, such as the
distal tubule of the kidney. However, channels formed by other subunit combinations may be
physiologically relevant. In humans and primates there is a fourth ENaC subunit, named δ,
which is expressed in various organs such as pancreas, nervous system and lung. The human
δ ENaC gene produces at least two splice isoforms (δ1 and δ2) that differ in their n-terminal
intracellular sequence. When expressed in Xenopus oocytes, δ1 Na+ transport activity is
approximately four-fold higher than δ2, regardless of the presence of accessory β and γ
subunits. Single channel analysis and plasma membrane abundance of δ1 and δ2 show that
the main difference between isoforms is their steady-state plasma membrane abundance. This
difference is independent of PY-motif mediated endocytosis or the presence of additional lysine
residues in the n-terminus of δ2. We have delineated a sequence motif in δ2 responsible for the
lower plasma membrane expression. Experiments are underway to determine whether this
sequence differentially controls channel insertion, endocytosis or both.
Supported by the Spanish Ministry of Science (grants FIS-PS09/00406, Consolider CSD-2008-
00005 and HD2008-0025) and the German Academic Exchange Service (DAAD).





O-8.4
Proteolytic cleavage rather than membrane fluidity and the cytoskeleton modulates the
activity of epithelial Na+ channels in response to shear force
Martin Fronius, Althaus M, Rößler D, Frenz S, Assmann M and Clauss W
Institute of Animal Physiology, Justus-Liebig University, Giessen, Germany
Epithelial Na+ channels (ENaCs) belong to a highly conserved protein family whose members
were originally identified to be part of a mechanosensor in C. elegans. From this perspective it
is not surprising that ENaCs have been found to be mechanosensitive as well. Nevertheless the
mechanisms how mechanical forces are transduced in conformal changes and thus ENaC
activation are unknown. ENaC activity of heterologously expressed human ENaCs (α,β,γ, δ1βγ,
δ2,β,γ) was recorded by the two-electrode-voltage-clamp technique in response to a fluid
stream that was used to produce shear force at the surface of the oocytes. Under basal
conditions, all three ENaC channels are activated by shear force. This mechanical activation
was mimicked by preincubation with compounds known to increase the open probability of the
channels. Alterations of the membrane fluidity achieved by preincubation of the oocytes with
ethanol did not affect the response to shear force. Pretreatment with cytochalasin D and
phalloidin to stabilize/disrupt the actin cytoskeleton also did not affect the response to shear
force. With trypsin, known to activate ENaCs by proteolytic cleavage the response to shear
force was potentiated in α,β,γ-ENaC but attenuated in δ1,β,γ and δ2,β,γ-ENaC. These data
indicate that mechanical activation of ENaC is independent of membrane composition as well
as interactions with the cytoskeleton. It seems that the extracellular loops, which are accessible
for proteases are primarily involved in mechanical gating and that proteolytic cleavage is
capable to modulate ENaC behaviour in response to shear force.




O-8.5
Cortactin regulates ENaC via Arp2/3 complex
Daria V. Ilatovskaya1,2, Tengis S. Pavlov1, Vladislav Levchenko1, Yuri A. Negulyaev2, and
Alexander Staruschenko1
1Department of Physiology and Kidney Disease Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 2Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russian Federation Epithelial Na+ Channel (ENaC) activity is regulated, in part, by the cortical cytoskeleton. Here we demonstrate that cortical actin binding protein cortactin is highly expressed in the kidney cortex, polarized epithelial cells, and is localized to the cortical collecting duct. Co-expression of cortactin with ENaC decreases ENaC activity, as measured in patch-clamp experiments. Biotinylation experiments and single channel analysis reveal that cortactin decreases ENaC activity via affecting channel open probability (Po). Knock down of cortactin in mpkCCDc14 principal cells results in an increase in ENaC activity and sodium reabsorption. Co-
immunoprecipitation analysis shows direct interactions between cortactin and all three ENaC
subunits in cultured and native cells. To address the question of what mechanism underlies the
action of cortactin on ENaC activity, we assayed the effects of various mutants of cortactin. The
data shows that only a cortactin mutant unable to bind Arp2/3 complex does not influence ENaC
activity. Furthermore, inhibitor of the Arp2/3 complex CK-0944666 precludes the effect of
cortactin. Depolymerization of the actin microfilaments and inhibition of the Arp2/3 complex
does not result in the loss of association between ENaC and cortactin. Thus, these results
indicate that cortactin is functionally important for ENaC activity and that Arp2/3 complex is
involved in this mechanism.




O-9.1
Multi-faceted regulation of neuronal ion channels by PIP2
Mark S. Shapiro
Department of Physiology, University of Texas Health Science Center, San Antonio, Texas,
USA
Many ion channels critical to neuronal function are regulated by phosphatidylinositol 4,5-
bisphosphate (PIP2) in the plasma membrane. In most cases, PIP2 acts as an "inverse" 2nd-
messenger, with its unbinding causing inhibition. We focus on "M-type" (KCNQ, Kv7) K+
channels and "N-type" Ca2+ channels, of which both play critical roles in regulation of neuronal
excitability and release of neurotransmitter. A number of different receptors coupled to Gq/11 G
proteins modulate these channels via lipid signals, either by consumption of membrane PIP2,
generation of calcified calmodulin, phosphorylation by protein kinase C, or a combination of all
three. We will explore the mechanistic bases of these modulatory signals, with focus on the
underlying mechanism(s) of receptor specificity, the current state of structural knowledge, and
the implications for physiological regulation of neurobiological function.





O-9.2
Identification of a new beta subunit of the ClC-2 chloride channel
Tania López-Hernández1, Elena Jeworutzki2, Luiza Bengtsson3, Xavier Capdevila-Nortes1,
Sonia Sirisi1, Marisol Montolio1,4, Albert Martínez5, Thomas Jentsch6, Michael Pusch2, Raul
Estevez1,4
1Seccion de Fisiologia. Departamento de Ciencias Fisiologicas II. Universidad de Barcelona; 2Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genoa, Italy; 3Leibniz-Institut für
Molekulare Pharmakologie and Max-Delbrück-Centrum für Molekulare Medizin, Berlin D-13125,
Germany; 4CIBERER U-750; 5IRBB-PCB. Universidad de Barcelona; 6Leibniz-Institut für
Molekulare Pharmakologie and Max-Delbrück-Centrum für Molekulare Medizin, Berlin D-13125,
Germany
We recently identified a second gene for megalencephalic leukoencephalopathy with subcortical
cysts (Lopez-Hernandez et al submitted), called MLC2. Using specific antibodies directed
against MLC2 we performed coIP experiments finding specifically to the ClC2 chloride channel.
MLC2 and ClC-2 interact directly. This interaction causes an increase in ClC-2 function and
changed its subcellular localization. Mutations found in MLC2 in human patients alter the
subcellular localization of the ClC-2/MLC2 complex. This work identified the first beta subunit of
the ClC-2 chloride channel and implies chloride channel function in the physiology of the
process of ion siphoning by glial cells.





O-9.3
CALHM1 reduces endoplasmic reticulum calcium levels in HEK293T cells
Sonia Gallego-Sandín, María Teresa Alonso and Javier García-Sancho
Instituto de Biología y Genética Molecular (IBGM). Universidad de Valladolid - Consejo Superior
de Investigaciones Científicas, Valladolid, Spain
A role for calcium disregulation in Alzheimer's disease was postulated 20 years ago. A novel
calcium-conducting channel called CALHM1 (Calcium Homeostasis Modulator 1) is associated
to
Expression of CALHM1 was found in different brain regions and cells of neuronal lineages. It is localized predominantly to the endoplasmic reticulum (ER) but also exist at the plasma membrane. We have investigated the properties of CALHM1 in transfected HEK293T cells. The effects of CALHM1 on cytosolic Ca2+ changes have been monitored by fura-2 or targeted aequorins. The cytosolic Ca2+ increase induced by Ca2+ release from the ER was reduced in cells expressing CALHM1. Using aequorins targeted to the ER, we have shown that CALHM1 decreases the calcium concentration in these intracellular stores. In permeabilized cells we observed a reduced Ca2+ uptake and also a faster Ca2+ leak from the ER. This Ca2+ depletion of the ER triggers In summary, we find that CALHM1 disturbs ER calcium homeostasis and induces ER stress in HEK293T This work was supported by a grant from The Spanish Ministerio de Ciencia e Innovación
(MICINN; BFI2007-60157 and BFU2010-17379). SGS holds a postdoctoral contract within the
JAE-doc program of the Consejo Superior de Investigaciones Científicas.



O-9.4
GABAA receptors located at the axon initial segment control excitability of dentate gyrus
neurons
Patricio Rojas
Departamento de Química y Biología, Universidad de Santiago de Chile. Centro de Estudios
Avanzados en Zonas Aridas, La Serena Chile.
The site of action potential (AP) initiation is located at the axon initial segment (AIS) where a
high density of voltage dependent channels is present, determining both threshold and the
shape and frequency of APs. In many principal cells GABAergic inputs to the AIS and soma
come from separate types of interneurons. Here we studied the effect of axonal versus somatic
GABA receptor activation by local application of an exogenous GABAA receptor agonist to
granule cells of the dentate gyrus, the gatekeepers of the hippocampal tri-synaptic circuit. Local
application of 5 µM muscimol to the AIS robustly depolarized the voltage threshold for AP
initiation (1.7 ± 0.2 mV, N=13) with relatively little change in input resistance (15%). Because
presynaptic inputs to dentate granule neurons produce small EPSPs (0.1-0.5 mV), this
threshold change is significant to granule cell function. Somatic application did not alter voltage
threshold but strongly decreased input resistance (58 %), resulting in a large increase in
rheobase. Interestingly, synaptic-like stimulus that were suprathreshold under basal conditions,
failed to reach threshold with axonal muscimol application. With sustained current injections,
axonal muscimol application decreased the number of APs generated at a fixed current
amplitude These results shows that activation of GABAA channels are not restricted to single
AP and can have effects on temporal summation and repetitive firing. In summary, our results
show that GABAA channels at the AIS have a role not only on the AP initiation, but can also
modulate the firing pattern in granule cells.




O-9.5
P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in
neuroblastoma cells
Rosa Gómez-Villafuertes,Yolanda Gutiérrez-Martín, Diego Bustillo Merino, Jesús Sánchez-
Nogueiro, Cristina Torregrosa-Hetland, Thomas Binz, Luis Miguel Gutiérrez, Mª Teresa Miras-
Portugal, Antonio R. Artalejo
Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad
Complutense de Madrid, Spain
Previously, we have reported that purinergic ionotropic P2X7 receptors negatively regulate
neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-
dependent kinase II-related mechanism. In the present study, we have used this cell line to
investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-
regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by
high-resolution membrane capacitance measurements. Using dual-wavelength total internal
reflection microscopy we have observed both the increase in near-membrane Ca2+
concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor
stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical
and horizontal directions, thus involving this receptor type in the control of early steps –docking
and priming– of the secretory pathway. Immunocytochemical and RT-PCR experiments
evidenced that N2a cells express the three neuronal SNAREs as well as nucleotide and
monoamine, VMAT-1, VMAT-2, vesicular transporters. Biochemical measurements indicated
that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor
stimulation and ionomycin increased the incidence of small transient inward currents (STICs),
reminiscent of postynaptic quantal events observed at synapses. STICs were dependent on
extracellular Ca2+ and were abolished by brilliant blue G, suggesting they were mediated by
P2X7 receptors. Altogether, these results suggest the existence of a positive feedback
mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on
P2X7 receptors on the same or neighbor cells to further stimulate its own release and
negatively control N2a cell differentiation.



O-9.6
Chromogranins as regulators of exocytosis
Dominguez, N.; Estévez, J.; Arnau, M.R.; Machado, J.D. and Borges, R
Unidad de Farmacología, Facultad de Medicina, Universidad de La Laguna. Tenerife. Spain
Chromogranins A and B are the major soluble proteins of dense-core secretory vesicles
(LDCV). We have reported that the absence of chromogranin A (CgA) caused important
changes in the accumulation and in the exocytosis of catecholamines (CA) using a CgA-KO
mouse. We have recently obtained a new results obtained in a CgB-KO mouse strain. These
mice have 36% reduction of adrenomedullary epinephrine when compared to WT whereas the
norepinephrine content was similar. The total CA evoked-release was 33% lower than WT. This
decrease was not due to a lower frequency of exocytotic events but to a lesser secretion per
quanta (≈30%) measured by amperometry, therefore amperometric spikes exhibited a slower
ascending phase. Cell incubation with L-DOPA increased the vesicle CA content of WT- but not
in CgB-KO cells. We have developed a CgA&B KO strain, which resulted viable and fertile.
Accordingly with the sorting function assigned to Cgs these mice should not contain LDCV.
However, single-cell amperometry recordings showed that although their amine content is
halved the secretory responses remained conserved. Electron microscopy revealed the
presence of giant and highly altered secretory vesicles. Proteomic analysis showed the
overexpression of fibrinogen and other proteins.
In summary, data from CgA-KO and CgB-KO cells indicate that the mechanisms for vesicular
accumulation of CA were saturated while there is ample capacity for further accumulation in WT
cells. We conclude that Cgs are highly efficient system directly implicated in monoamine
accumulation and in the kinetics of exocytosis of LDCV.
We thank the personnel of the animal house of the ULL for the maintenance of the mouse
strains. JDM had a CONSOLIDER contract (CSD2008-00005). ND is recipient of a FPU
fellowship from the Spanish Ministry of Science and Innovation (MICINN). Supported by
MICINN Grant BFU2007-64963 (RB) and Canary Agency for Research, Innovation and Society
of Information (ACIISI/FEDER) PI 2007/017 (JDM).




O-9.7
Cysteine string protein-alpha maintains the number of synaptic release sites and
dynamin-dependent endocytosis at motor nerve terminals
José L. Rozas1, Leonardo Gómez-Sánchez1, Pedro Linares-Clemente1, Eugenio Vázquez2,
Rafael Luján3 and Rafael Fernández-Chacón1
1Instituto de Biomedicina de Sevilla, IBiS, Hosp.Univ. Virgen del Rocío/CSIC/Universidad de
Sevilla y Dept. Fisiología Médica y Biofísica, y CIBERNED, Sevilla, España; 2Dpto. Química
Orgánica, Universidad de Santiago, Santiago, España; 3Dpto. de Ciencias Medicas, Facultad
de Medicina & Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La
Mancha, Albacete, España
Cysteine String Protein-alpha (CSP-alpha) is a synaptic vesicle protein that prevents
neurodegeneration of presynaptic terminals. CSP-alpha knock-out mice mice suffer from a
lethal neurological phenotype that is evident after the second postnatal week. We have found
striking changes in the synaptic vesicle cycle using electrophysiology and imaging at the
neuromuscular junction (NMJ). Exocytosis at the synaptic terminals is decreased in CSP-alpha
KO mice at P16-20. Nerve terminals fail to sustain prolonged release. At these synapses, the
SNARE protein SNAP25, is dramatically decreased. That observation could explain a lower
number of transmitter release sites observed in mutant synapses. In addition, we have studied
the synaptic vesicle cycle in mutant and wild-type synapses. To monitor synaptic exo- and
endocytosis in vivo we have generated transgenic mice that express in neurons
synaptopHluorin (SpH) but lack CSP-alpha. Combining imaging and electrophysiology, we have
uncovered a reduction in the size of the recycling synaptic vesicle pool and a defect in
endocytosis in mutant synapses. Dynamin-dependent endocytosis taking place during the
stimulus is particularly reduced in the absence of CSP-alpha. Such a defect could also explain
the reduced size of the recycling pool during prolonged stimuli. Our results reveal that CSP-
alpha function on the synaptic vesicle is broader than initially envisioned being CSP-alpha
required for the long term maintenance of synaptic exo- and endocytosis.
Supported by MICINN BFU2007-66008, JA P07-CVI-02854 and ISCIII




O-10.1
PKA anchoring via AKAP150 is required for sympathetic regulation of cardiac HCN4
pacemaker channels and heart rate in mice
Cathy Proenza
Department of Physiology and Biophysics, University of Colorado, Aurora, Colorado, USA
Hyperpolarization-activated cyclic nucleotide sensitive-4 (HCN4) channels are highly expressed
in pacemaker myocytes of the sinoatrial node of the heart, where they produce the cardiac
"funny current," If. The sympathetic nervous system stimulates HCN4 channels by increasing
intracellular cAMP levels, and the ensuring increase in If is thought to contribute to the sympathetic "fight-or-flight" increase in heart rate. It is generally believed that the mechanism for sympathetic regulation of HCN4 is via direct binding of cAMP to a conserved cyclic nucleotide binding domain in the carboxyl terminus of the channels. However, we have recently shown that protein kinase A (PKA) activity is required for b adrenergic regulation of If in sinoatrial myocytes, and that PKA can activate HCN4 by phosphorylation of the distal C-terminus (Liao et al., 2010, J Gen Physiol 136:247). Here we show further that PKA anchoring to A-kinase anchoring protein 150 (AKAP150) is required for sympathetic regulation of If, that HCN4 and AKAP150 associate in sinoatrial myocytes, and that PKA anchoring to AKAP150 is
required for sympathetic control of heart rate in mice.





O-10.2
Functional effects of a missense mutation in HERG associated with type 2 Long QT
syndrome
Tamargo J, Amorós I, Jiménez-Jáimez J, Tercedor L, Barana A, Gómez R, de la Fuente MG,
Dolz-Gaitón P, Alvarez M, Martínez-Espín E, Lorente JA, Melgares R, Caballero R, Delpón E
Department of Pharmacology. School of Medicine. Universidad Complutense. Madrid. Spain
BACKGROUND: Long QT syndrome (LQTS) is characterized by a prolonged QT interval that
can lead to severe ventricular arrhythmias (torsades de pointes) and sudden death. Congenital
LQTS type 2 (LQT2) is due to loss-of-function mutations in the KCNH2 gene encoding Kv11.1
channels responsible for the rapid component of the delayed rectifier current. This study was
performed to analyze determine the functional properties of the LQT2-associated mutation
p.E637G, located in the outer mouth of the pore at the beginning of S6, found in a Spanish
family.
METHODS: Wild-type (WT) and p.E637G Kv11.1 channels were transiently transfected in CHO
cells and currents were recorded using the patch-clamp technique.
RESULTS: p.E637G channels lost inward rectification and K+ selectivity generating small but
measurable slowly activating, non-inactivating currents. These alterations were not corrected
either by cotransfection with WT channels or by incubation at 27ºC or with pharmacological
chaperones (dofetilide, fexofenadine, terfenadine or 4-phenylbutyrate). As a consequence of its
effects on channel gating, the mutation significantly reduced the outward repolarizing current
during the cardiac action potential (AP), resulting in a marked lengthening of the duration of a
simulated human ventricular AP.
CONCLUSION: We have identified and characterized a LQT2-associated mutation that through
removal of C-type inactivation and reduction of K+ selectivity causes the QT prolongation
observed in the patients carrying the mutation. Moreover, the results obtained demonstrate the
importance of the glutamic acid at position 637 for the inactivation process and K+ selectivity of
Kv11.1 channels.



O-10.3
Channelopathies and sudden cardiac death
Ramón Brugada
Cardiovascular Genetics Center. IdIBGi-UdG, Girona.
Sudden Cardiac Death (SCD) is a major contributor to mortality in industrialized nations,
affecting 500,000 individuals annually in the Western World, and causing more deaths than
AIDS, lung and breast cancer and stroke together. Frequently the autopsy becomes the
principal diagnostic tool when macro and microscopic analyses provide a conclusive diagnosis
in cardiomyopathies. However, on average one third of autopsies do not identify a pathologically
defined cause of death and these deaths are classified as "natural" or arrhythmogenic. In the
athlete, the most common causes of sudden cardiac death are of inherited origin. This
represents a tremendous burden to families, community and health care.
There has been considerable progress in the understanding of these genetic components
thanks to research in familial forms of sudden death. Because these inherited diseases may
remain undetected by conventional clinical investigations, genetic testing has been recently
added to the clinical tools, improving significantly the detection of those individuals at risk. With
that new information the physician, the proband and his family members can make an informed
decision regarding their care and in the case of athletes, regarding continuation of competitive
training. While genetic technology has become essential to the diagnosis and subsequent
prevention strategies in carriers with borderline or abnormal phenotypes, the use of this
technology to guide clinical decisions in asymptomatic genetic carriers with normal phenotype is
not without controversy.





O-10.4
Role of Kv1.3 in vascular smooth muscle proliferation
J.R. López-López, P. Cidad, L. Jiménez-Pérez, E. Alonso, E. Miguel-Velado and M.T. Pérez-
García
Instituto de Biología y Genética Molecular, Universidad de Valladolid – CSIC, Valladolid, Spain
Phenotypic switch of vascular smooth muscle cells (VSMCs) involves changes in the
expression of membrane receptors and ion channels. Our previous work using in vivo and in
vitro animal models of VSMC proliferation demonstrated that functional expression of Kv1.3
channels associates with increased proliferation. In the present work we have explored if this
association is a conserved feature across vascular beds from different species and we have
investigate some of the possible mechanisms linking Kv1.3 expression and cell proliferation.
Our data comparing several contractile and proliferating VSMCs indicates the Kv1.5/Kv1.3 ratio
is a very good indicator of the phenotypic switch. Moreover, functional expression of Kv1.3
currents facilitates proliferation of VSMCs from several vascular beds while their blockade has
antiproliferative effects. These results can provide new therapeutical approaches for the
prevention and treatment of unwanted vascular remodelling.




O-10.5
Pre-mRNA splicing defects caused by exonic mutations in hereditary renal diseases
Félix Claverie-Martín
Unidad de Investigación, Hospital Universitario Ntra. Sra. de Candelaria, Tenerife, Spain
The accurate classification of mutations in terms of their actual mechanism of gene inactivation
is fundamental for understanding structure-function relationships in the corresponding protein,
for assessing risk in individuals with hereditary disease predisposition and for devising new
molecular therapies. A significant fraction of exonic mutations could be pathogenic by affecting
normal pre-mRNA splicing. To date there are very few data related to this type of mutation in
kidney disease.
We investigated the consequences of presumed missense and silent mutations in pre-mRNA
splicing of renal disease-associated genes including PKD1, PKD2, CLCN5, CLDN16 and
SLC12A1. Mutations with potential effect on splicing were selected using bioinformatics
analyses. The effect of these mutations was tested experimentally with a minigene system.
PCR-amplified DNA was inserted in the splicing reporter minigene, and the specific changes
were introduced by site-directed mutagenesis. Minigenes were transfected into kidney cell lines,
and the mRNAs were analysed by RT-PCR. The products with altered size were characterized
by DNA sequencing.
Our results show that several apparent missense and silent mutations can also alter pre-mRNA
splicing, resulting in aberrant mRNAs. The mutations can: (1) abolish a splice site leading to
skipping of an entire exon or incorporation of intronic sequences in the mRNA; (2) create
stronger splice sites within an exon resulting in mRNAs lacking part of that exon; or (3) create or
destroy splicing regulatory sequences leading to exon skipping. RNA analysis is therefore
essential for the assessment of the consequences of exonic mutations on splicing of renal
disease-associated genes.





O-10.6
Cancer therapies targeting KV10.1 channels
Luis. A. Pardo, Franziska Hartung, Araceli Sánchez, Fernanda Mello de Queiroz, Jasmin
Agarwal, Walter Stühmer
Max-Planck-Institute of Experimental Medicine, Göttingen, Germany
KV10.1 (Ether-á-go-go-1, Eag1) is a CNS-localized voltage-gated potassium channel that is
found ectopically expressed in a majority of extra-cranial solid tumors and a significant fraction of acute myeloid leukemias. Genetically modified mice overexpressing the channel show higher incidence of tumors, highlighting the relevance of the channel for tumor biology. Channel expression correlates with bad prognosis in several tumor types, and its inhibition is able to reduce tumor progression both in vitro and in animal models. We have generated specific monoclonal antibodies able to bind the channel and inhibit its function in intact cells. Although the antibody proves efficacious in xenograft models, it produces a modest inhibition of tumor progression, at least in immunocompromised mice. We therefore designed immunotargeted tools taking advantage of the facts that this protein is virtually not detected in normal tissue outside the central nervous system, but is expressed in over 70 % of tumors from different origins. As transmembrane proteins, ion channels are accessible from the external environment and can be targeted by antibodies. We designed a single-chain antibody (scFv) against the pore region of KV10.1 and fused it to the human soluble tumor necrosis factor-related apoptosis- inducing ligand (TRAIL). The KV10.1-specific scFv62-TRAIL antibody fusion was expressed in CHO-K1 cells. The purified construct was added to the medium of different prostate cancer cells, either positive or negative for KV10.1. scFv62-TRAIL induced apoptosis in KV10.1 positive cancer cells, but not in non-tumor or KV10.1-negative tumor cells. The construct also induced apoptosis in bystander, KV10.1-negative cancer cells, while normal prostate epithelia cells were not affected.



O-10.7
ORMDL3 expression levels affect lymphocyte physiology
Amado Carreras-Sureda*, Gerard Cantero-Recasens*, Fanny Rubio-Moscardo, Miguel A.
Valverde and Rubén Vicente
Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona,
Spain.
ORMDL3 belongs to a family of transmembrane proteins localized in the endoplasmic reticulum
the function of which is poorly understood. Recently, SNPs (Single Nucleotide Polymorfism) in
the regulatory region of ORMDL3 have been associated by Wide Genome Scan studies to
several pathologies like asthma, diabetes type I and Crohn disease whose common link is an
immune
In the present study we provide data indicating that ORMDL3 expression levels modify ICRAC and store-operated calcium entry (SOCE) signalling, a key pathway in the immune system development and functionality. NFAT translocation and IL-2 production levels were also evaluated in order to check the activation pathway in Jurkat cell line. These alterations might explain the recent association of ORMDL3 with proinflamatories and autoimmune diseases.

POSTER PRESENTATIONS



P.1
Role of the conformation of selectivity filter in KcsA stability and function
Estefanía Montoya, M. Lourdes Renart, Asia Fernández, M. Luisa Molina, José L. Ayala, José
A. Encinar, José A. Poveda, José M. González-Ros
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain
The potassium channel KcsA is an integral membrane protein from S.lividans. Since its atomic
structure was solved by X-ray diffraction, it has been used as a model system for studies on ion
channel and oligomeric membrane proteins. This channel is able to permeate K+ at high flux
rate and it is blocked by Na+. Fluorescence, circular dichroism and Fourier transform infrared
experiments carried out in our laboratory demonstrated that K+ and Na+ are able to bind to KcsA
in a competitive manner. This binding is indeed associated with conformational changes, which
seem
To further investigate the impact of the selectivity filter conformation on the structure, stability
and function of KcsA, we performed two different mutants representing functionally relevant
states. KcsA E71A showed a high open probability (Po), in contrast to the very low Po of the
wild-type channel. Thermal and chemical stability are diminished in the mutant channel and
some changes in tertiary and secondary structure were detected by fluorescence spectroscopy
and circular dichroism. On the other hand, KcsA M96V showed a non-conductive state in its
crystallographic structure. Interestingly, these channels did conduct K+ and even Na+, showing a
very low Po. Structural characterization showed that both secondary and tertiary structure are
very similar in the presence of K+ or Na+, in agreement with functional behavior.
Mutant channels with a structurally altered selectivity filter show that ion-induced global
conformational and stability changes are intimately associated to the conformation of this
selectivity filter (conductive and non-conductive states).




P.2
Salt-induced cooperative transport in a protein ion channel revealed by current noise,
conductance and selectivity experiments
María Queralt-Martín, Elena García-Giménez, Vicente M. Aguilella and Antonio Alcaraz
Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, Castellón,
Spain
The concept of positive cooperativity appeared in the study of oxygen uptake by hemoglobin to
explain that binding of a molecule of oxygen makes it easier for a second molecule to bind.
Quite the reverse, negative cooperativity refers to the situation where the presence of the first
molecule makes it more difficult for the second molecule to bind. We study here the effect of salt
on the pH titration of the OmpF channel, paying attention to the current noise, conductance and
ion selectivity that are analyzed in terms of the Hill formalism. In all cases, values of the Hill
coefficient lower than 1 are found, suggesting a negative cooperativity. Although OmpF porin is
a trimer, it was shown by a number of different methods that each monomer is identical and
functionally independent. Thus, the slowed-down channel titration is a property of each
monomer. Surprisingly, we find that increasing salt concentration promotes negative
cooperativity, which is seen as a salt-induced decrease of the Hill coefficient. This observation
seems to exclude direct electrostatic interactions between protonation sites as the source of the
phenomenon, suggesting another, more subtle mechanism(s). The binding of cations to certain
acidic residues has a crucial effect at low pH because it results in an inhibition of channel
conductance that additionally provides an anionic selectivity to the channel. This suggests that
the binding site could play a certain role in the protection of the bacteria against acidic media.





P.3
Molecular basis of the OmpF channel selectivity inversion induced by multivalent cations
Carmina Verdiá Báguena, María Queralt-Martín, Vicente M. Aguilella, Antonio Alcaraz
Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, Castellón,
Spain
The discrimination between charged solutes, known as ion selectivity, is a specialized
physiological function exerted by ion channels. We study here the bacterial porin OmpF, a
transmembrane protein that forms wide channels in the outer membrane of the E.coli. The
moderate cationic selectivity exhibited by OmpF channel in salts of monovalent cations (KCl,
NaCl) turns into anionic selectivity in the presence of salts of divalent and trivalent cations. Still,
previous studies indicate this process could be regulated by the cation/acidic residue particular
chemistry, although the molecular bases of the observed selectivity inversion are still unclear.
To gain new insights on the role of the channel charges and the counterion valence, we
investigate here the OmpF ion selectivity in salts of lanthanum chloride. The channel pH titration
in salts of multivalent cations is particularly challenging because of the cation hydrolysis and the
potential formation of insoluble species. However, we can take advantage here of the hydrolysis
phenomenon to investigate the cation/channel interaction both in solutions of trivalent cations
(La +
3 ) and divalent complexes (La(OH)2 ). The influence on selectivity inversion of the counterion charge and the lanthanum binding affinity for key acidic residues can then be
assessed. Preliminary measurements support our guess that channel charge inversion is a
residue-specific phenomenon.




P.4
Solubilization of inclusion bodies containing a peptidic fragment of KCNQ
Velázquez-Fernández Jesús B., Malo de la Fuente C., Villarroel A.
Autonomous University of Nayarit, Tepic, Mexico
The production of recombinant peptides by bacteria is a major tool in biophysics of proteins like
ion channels. Some peptides expressed by this mean could be confined in bacterial inclusion
bodies which are highly water insoluble. This makes the peptide hard to recover from bacterial
cultures. We have tested some conditions in order to solubilize proteins for further purification. It
seems that some but not all denaturing agents could improve solubilization.






P.5
A conserved positive charge in TM7: a key player in excitatory amino acid transporter
dual function
Delany Torres
Department of Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Excitatory Aminoacid Transporters (EAATs) are responsible for the clearance of glutamate after
synaptic release throughout the mammalian Central Nervous System. This energetically
demanding activity is crucial for maintaining precise neuronal communication and to maintain
the extracellular glutamate concentration below neurotoxic levels. Reductions in EAAT function
have been linked to a number of neurological disorders. In addition to their ability to re-capture
glutamate from the extracellular space, EAATs also shows a thermodynamically-uncoupled
glutamate gated chloride conductance. Major progress has been achieved over the last decade
in our understanding of the topology, structure and transport mechanism of these carriers.
However, the structural features required for the chloride conductance as well as its structural-
functional connection with substrate transport are yet poorly understood. Using site directed
mutagenesis and electrophysiological recordings in Xenopus oocytes we have identified a
positively charged residue in TM7 that plays a critical role in both glutamate translocation and
glutamate-mediated gating of the anion channel conductance. Mutating R388 to an acidic amino
acid small (Ala) or large non-polar (Phe) amino-acid reduces both the uptake activity and the
amplitude of the glutamate-gated anion current, suggesting that the nature of this residue is
relevant for both mechanisms. More importantly, when this residue is mutated in to a negative
charge, mutant EAATs are no longer able to either complete the full transport cycle or to open
the glutamate gated chloride conductance. Our results indicate that the positive charge in this
position is essential both for glutamate translocation and gating of the anion conductance, and
we propose that it may be an important element in the functional link connecting these two
processes.




P.6
Differential regulation of the dopamine transporter in response to dopaminergic lesion
Ignacio Cruz-Muros1, Javier Castro-Hernández1, Diego Álvarez de la Rosa2, Josmar Salas-
Hernández1, Teresa Giráldez3, Pedro Barroso-Chinea4 and Tomás González-Hernández1
1 Departamento of Anatomía, 2Departamento de Fisiología and Instituto de Tecnologías
Biomédicas, Universidad de La Laguna; 3 Unidad de Investigación, Hospital Universitario
Nuestra Sra. de Candelaria; 4 Centro de Investigación Médica Aplicada, Universidad de
Navarra, Pamplona
The dopamine transporter (DAT) is a membrane glycoprotein, responsible for the reuptake of
dopamine (DA) into presynaptic DA-terminals, which has been involved in DA-cell degeneration
in Parkinson's disease (PD). Previous studies suggest that DAT may be regulated in response
to dopaminergic injury, but relevant aspects concerning this phenomenon remain unknown. This
study has been focused on clarifying some of these by using molecular, morphological and
pharmacological techniques and animal (150-350 µg 6-OHDA i.c.v. injections in rats) and
cellular (MPP+, rotenone and 6-OHDA in rDAT expressing HEK293 cells) models of PD. The
results show that DAT is persistently down-regulated in surviving midbrain DA-neurons after
substantial (62%) loss of striatal DA-terminals, and transiently after slight (11%) loss of DA-
terminals in rats. Transient DAT down-regulation consisted of a decrease of glycosylated
(mature) DAT in the plasma membrane with accumulation of non-glycosylated (immature) DAT
in the endoplasmic reticulum-Golgi (ERG) compartment, and recovery of the normal expression
pattern five days after lesion. DAT redistribution to the ERG was also observed in rDAT-HEK
cells exposed to MPP. In contrast to other midbrain DA-cells, those in the ventrolateral region of
the substantia nigra do not regulate DAT and degenerate shortly after slight DA-lesion. These
data suggest that DAT down-regulation is a post-traslational event induced by DA-analogue
toxins, consisting of a stop in its glycosylation and trafficking to the plasma membrane. The fact
that neurons which do not regulate DAT die shortly after lesion suggests a relationship between
DAT down-regulation and neuroprotection.



P.7
Renal Na-K-Cl cotransporter (NKCC2) activity is modulated by phosphorylation at
residue Ser126
Ignacio Giménez, Carmen Ibañez
Aragon Health Sciences Institute, and Department of Pharmacology and Physiology, University
of Zaragoza, Spain
Kidney specific Na-K-2Cl cotransporter (NKCC2) is activated by phosphorylation of N-terminal
threonine residues (Thr99 and Thr104). An additional downstream conserved residue, Ser126,
was initially identified as being phosphorylated in vitro by AMP-activated kinase (AMPK).
Recent phosphoproteomic studies have shown Ser126 is phosphorylated in vivo in a regulated
fashion, likely mediated by PKA. To determine the relative importance of Ser126
phosphorylation in NKCC2 regulation, we have used site-directed mutagenesis and
heterologous expression in Xenopus oocytes. Our results suggest NKCC2 activity might depend
on Ser126 phosphorylation when Thr99 and Thr104 are not phosphorylated. In addition, Ser126
phosphorylation appears to modulate the phosphorylation of Thr99 and Thr104.
Supported by MICINN BFU2007/62119



P.8
Regulation of HpENA1 in the yeast Hansenula polymorpha
Celia Rodríguez, Rafaela González, Elisa Cabrera, José M. Siverio
Department of Biochemistry and Molecular Biology, University of La Laguna, Tenerife (Spain)
Maintenance of intracellular low sodium when sodium is present in excess in the external
medium relies heavily on the capacity to extrude this cation. One of the extrusion mechanisms
is based on a P-type ATPase pump encoded by ENA1. We show the regulation of ENA1 in the
yeast
Disruption of HpURE2 resulted in sensitivity to Li+ and Na+, no induction of ENA1, low levels of
the GATA-type transcription factor Gat1 and low intracellular Ca2+ levels. Gat1 levels were also
very low in a Δcnb1 mutant lacking the regulatory subunit of calcineurin. The strain Δure2 was
very sensitive to the calcineurin inhibitor FK506 and displayed several phenotypes reminiscent
of Δcnb1. The reporter 4xCDRElacZ, containing calcineurin-dependent response elements in its
promoter, revealed that calcineurin activation was reduced in HpΔure2. HpΔure2 showed an
increased expression of the gene PMR1 encoding the Golgi Ca2+-ATPase, whereas that of
PMC1 encoding the vacuolar Ca2+-ATPase remained unaltered. PMR1 up-regulation was
abolished by deletion of the GATA-type transcription factor GAT2 in a HpΔure2 genetic
background, and normal Ca2+ levels were recovered. Moreover, overexpression of GAT2 or
PMR1 yielded strains mimicking the phenotype of the HpΔure2. This suggests that the low Ca2+
levels in the HpΔure2 mutant are due to the high levels of Pmr1 that replenish the Golgi Ca2+
content, thus acting as a negative signal for Ca2+ entry into the cell. We conclude that HpUre2 is
involved in salt tolerance via Ca2+ homeostasis regulation and calcineurin activation, which
control the levels of Gat1.




P.9
Role of TRP domain in TRPV1 functionality
Lucia Gregorio-Teruel1, Pierluigi Valente1,2, and Antonio Ferrer-Montiel1.
1 Instituto de Biología Molecular y Celular. Universidad Miguel Hernández. 03202 Elche. Spain; 2 Department of Neuroscience and Brain Technologies, Italian Institute of Technology (IIT).
16163 Genova, Italy
TRPV1 (Transient receptor potential vanilloid 1) is a member of the TRP channel family
activated by physical and chemical stimuli. TRPV1 functions as a non selective tetrameric
cation channel with high permeability to calcium ions. Each subunit shows a topology of six a-
helical transmembrane segments with a pore region between the fifth and sixth segment. The
cytoplasmatic N- and C-termini contains several aminoacid residues involved in the modulation
of channel gating. Specifically, the C-terminal region contains the TRP domain (Glu684-
Arg721), a highly conserved sequence in the TRP channels family. This region is a molecular
determinant in the functional coupling of the channel. To further understand the role of this
region in the protein functionality we performed a site directed mutagenesis strategy on a non
functional TRPV1 chimera, TRPV1-AD2 that contains the TRP domain of TRPV2.
Complementary, we studied the role of residues I696, W697 and R701 in the functional coupling
of TRPV1. To perform these experiments we replaced each position by the all natural
aminoacids. We characterize the expression level and the electrophysiological properties of the
different mutants obtained for both strategies. Taking together, our data suggest that the TRP
domain region is critical for the functional coupling of the activating stimuli. Particularly, we
found that mutations of this region affected the energetic of channels opening. These results
demonstrate that the preservation of these positions is essential for the correct functionality of
TRPV1.
Funded by MICINN, CONSOLIDER-INGENIO 2010, ISCIII, Fundació La Marató de TV3
References
- García-Sanz et al. (2004) J. Neurosci., 24, 5307-5314
- García-Sanz et al. (2007) J. Neurosci., 27, 11641-11650
- Valente et al. (2008).FASEB J. 9, 3298-309


P.10
Activation of nociceptive TRPA1 channels by 1,4-dihydropyridines: role of permeating
calcium ions
Enoch Luis, Otto Fajardo, Víctor Meseguer, María José López, Carlos Belmonte & Félix Viana
Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones
Científicas. Sant Joan d'Alacant, 03550 Alicante.
Transient receptor potential type A1 (TRPA1) channels are cation permeable channels
activated by noxious cold temperatures, irritant chemicals and pungent compounds. Their
location in peptidergic sensory terminals innervating the skin and blood vessels makes them
important effectors of vasodilator responses of neural origin. 1,4-dihydropyridines are a class of
L-type calcium channel antagonists commonly used in the treatment of hypertension and
ischemic heart disease. Previously, we found that several 1,4-dihydropyridines (e.g. nifedipine,
nimodipine) and the structurally related L-type calcium channel agonist BayK8644, exert
powerful excitatory effects on TRPA1 channels (Fajardo et al. 2008) In a subclass of mouse
nociceptors expressing TRPA1 channels nifedipine also produces large elevations in [Ca2+]i.
These
Here, we investigated the effect of external calcium ions on nifedipine-evoked TRPA1 activity. The human wildtype TRPA1, or a Ca2+-impermeant pore mutant (D918A), was transiently transfected into HEK-293 and whole-cell ionic currents were monitored with ramp voltages from -100 to +100 mV. Recordings were started in zero Ca2+ conditions. Addition of 2.4 mM extracellular Ca2+ increased the nifedipine-evoked currents in wildtype TRPA1 but not in the mutant. The fold increase in current was 9.5 ± 2.3 at -80 mV (n = 8), while in the pore mutant the In conclusion, these findings identify TRPA1 channels as a new molecular target for the 1,4-dihydropyridine class of calcium channel modulators and demonstrate a role of permeating calcium Supported by MCINN (SAF2010-14990), CONSOLIDER_INGENIO 2010 (CS2007-00023) and
Generalitat Valenciana (Grisolía 2010-055).



P.11
Agonist-dependent internalization of TRPV1 targets the receptor to lysosomes for
degradation
Sanz-Salvador L1, Andrés-Borderia A1, Ferrer-Montiel A2 and Planells-Cases R1.
1 Centro de Investigación Príncipe Felipe, Valencia, Spain; 2 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández de Elche, Spain Regulation of Transient Receptor Potential Vanilloid type 1 (TRPV1) activity in nociceptor peripheral terminals appears to play an important role determining nociceptor sensitivity to noxious stimuli. Thus, whereas sensitization results from activation of different intracellular cascades triggering TRPV1 overactivity. In contrast, in the constant presence of noxious stimuli, nociceptor responses may become partial or totally diminished in a time-span that ranges between minutes and hours or up to days, a phenomenon known as desensitization or downregulation, phosphorylation/ dephosphorylation, and PKA-dependent processes have been described for acute TRPV1
desensitization, thus far little is known regarding long term desensitization of TRPV1. Given that
nociceptor function may be tightly regulated by equilibrating surface-expressed TRPV1, we
explored whether desensitization involved TRPV1-retrieval in an activity-dependent manner.
Here, we show that TRPV1 is internalized in an activity- and Ca2+-dependent manner upon
prolonged agonist exposure. Noteworthy, we found that TRPV1 is endocytosed through a
clathrin-independent pathway and directed to lysosomes for degradation. Our findings provide a
novel, previously unrecognized agonist-mediated regulatory mechanism of TRPV1 activity, that
most likely underlies the long-term effects of therapeutic vanilloid-induced receptor
desensitization.
References
- Ji, R. R., and Woolf, C. J. (2001) Neuronal plasticity and signal transduction in nociceptive
neurons: implications for the initiation and maintenance of pathological pain. Neurobiol Dis 8, 1-
10
- Vennekens, R., Owsianik, G., and Nilius, B. (2008) Vanilloid transient receptor potential cation
channels: an overview. Curr Pharm Des 14, 18-31
- Planells-Cases, R., Garcia-Sanz, N., Morenilla-Palao, C., and Ferrer-Montiel, A. (2005)
Functional aspects and mechanisms of TRPV1 involvement in neurogenic inflammation that
leads to thermal hyperalgesia. Pflugers Arch 451, 151-159
- Szallasi, A., and Blumberg, P. M. (1999) Vanilloid (Capsaicin) receptors and mechanisms.
Pharmacol Rev 51, 159-212
- Koplas, P. A., Rosenberg, R. L., and Oxford, G. S. (1997) The role of calcium in the
desensitization of capsaicin responses in rat dorsal root ganglion neurons. J Neurosci 17, 3525-
3537
- Bhave, G., Zhu, W., Wang, H., Brasier, D. J., Oxford, G. S., and Gereau, R. W. t. (2002)
cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by
direct phosphorylation. Neuron 35, 721-731
- Mohapatra, D. P., and Nau, C. (2005) Regulation of Ca2+-dependent desensitization in the
vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase. J Biol Chem 280,
13424-13432


P.12
Modulation of L-type calcium channels by urocortin in rat ventricular myocytes
Nieves Gómez-Hurtado1, Tarik Smani2, Eva Calderón-Sanchez2, María Fernández-Velasco1,
Victoria Cachofeiro1, Vicente Lahera1, Antonio Ordoñez2 and Carmen Delgado1,3.
1Departamentos de Farmacología y Fisiología, Facultad de Medicina, Universidad Complutense y 3Centro de Investigaciones Biológicas (CSIC), Madrid, Spain, 2Laboratorio de Investigación Cardiovascular, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Urocortins (Ucn) (I, II, III) are endogenous peptides, members of the corticotropin-releasing factor (CRF) family, that bind to G-protein-coupled receptors (CRF-R) 1 and -2. In the heart, Ucn binding to these receptors exerts beneficial action on the myocardium that might have a therapeutic potential in some pathological conditions. The aim of this study was to elucidate the signalling pathways implicated in the modulation of cardiac L-type Ca2+ channels by Ucn in ventricular Adult rat ventricular myocytes were stimulated in vitro with Ucn for 20-40 min. L-type calcium currents (ICaL) were measured with the patch-clamp technique. Ucn induced a significant increase in ICaL density, which was not prevented by the protein kinase A (PKA) inhibitor KT-5720 or the non-selective antagonist of guanine nucleotide exchange factor (GEF) blefeldin A (BFA), while Ucn effect was antagonized by astressin (CRF-R2 antagonist) and significantly reduced by protein kinase C (PKC) and ERK1/2 inhibitors. The cAMP analogue 8-pCPT-2'OMe-cAMP (8-CPT) that selectively activates the exchange protein activated by cyclic AMP (Epac) was ineffective to modify ICaL. Analysis of phospho-ERK1/2 showed that Ucn induced a significant activation of ERK1/2 pathway in ventricular myocytes and this effect was prevented by The present study provides evidence of new mechanisms involved in the modulation of L-type
Ca2+ channels by Ucn in adult ventricular myocytes. We propose that the marked increase in
ICaL density induced by Ucn is mediated through CRF-R2 and involves PKC dependent
activation of ERK1/2 pathway, while PKA and Epac-signalling are not implicated.




P.13
Cardiotrophin-1 enhances L-type Ca2+ current in adult rat ventricular myocytes
G. Ruiz-Hurtado1, N. Gómez-Hurtado2, J. Díez3, V. Cachofeiro4, A.M. Gómez1 and C. Delgado2.
1U-637, INSERM, Montpellier, France; 2Department of Pharmacology, School of Medicine, Universidad Complutense and CIB (CSIC), Madrid, Spain; 3Centre of Applied Medical Research, University of Navarra, Pamplona, Spain; 4Department of Physiology, School of Medicine, University of Complutense, Madrid, Spain Cardiotrophin-1 (CT-1) is a cytokine member of the interleukin-6 superfamily that was originally discovered as a factor that can induce hypertrophy in cardiac myocytes. Plasma levels of CT-1 are elevated in patients with hypertension and heart failure suggesting that CT-1 plays an important role in structural LV remodelling. Little information exists about the possible influence of this cytokine on cardiomyocyte electrical activity. The present study was designed to determine the role of CT-1 on whole-cell L type Ca2+ current (ICaL) in isolated adult rat ventricular myocytes using the patch-clamp technique. We found that incubation of cells with 1 nM CT-1 for 15-40 min increases the density of ICaL significantly (At -10 mV, CT-1: -16.2 ± 0.7 pA/pF, n=32 vs., Control: -11.8 ± 0.6 pA/pF, n=37; p<0.01). This effect was not prevented by the specific inhibitor of ERK1/2, PD98059 (At -10 mV, PD98059: -13.5 ± 0.7 pA/pF, n=21 vs. PD98059 + CT-1: -17.6 ± 1.8 pA/pF, n=12; p<0.05) indicating that this phenomena is independent of MEK/ERK pathway. However, pre-incubation with the protein kinase A (PKA) inhibitor KT-5720 (At -10 mV, KT-5720: -12.1 ±1.5 pA/pF, n=16 vs. KT-5720 + CT-1: -12.7±0.9 pA/pF, n=13) or the calmodulin kinase II (CaMKII) inhibitor (AIP) (At -10 mV, AIP: -11.0±1.9 pA/pF, n=10 vs. AIP + CT-1: -8.8 ± 1.0 pA/pF, n=11) completely inhibited the increase in ICaL induce We conclude that CT-1 is able to increase ICaL density through the activation of PKA and
CaMKII in cardiomyocytes.





P.14
The Parkinson´s disease-associated GPR37 receptor oligomerize with the adenosine A2A
receptor
Francisco Ciruela, Víctor Fernández-Dueñas, Victoria Wong, Igor Stagljar, Jorge Gandía.
Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de
Medicina-Bellvitge, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona,
Spain.
The substrate of parkin, the orphan G protein-coupled receptor 37 (GPR37), form insoluble
aggregates that accumulate in brain tissue of Parkinson's disease patients (e.g. Lewy bodies
and neurites). By means of membrane yeast two-hybrid (MYTH) screen, GPR37, was identified
as a binding partner for the adenosine A2A receptor (A2AR). Co-expression and co-
immunoprecipitation experiments showed a close and specific interaction between GPR37 and
A2AR in transfected HEK-293. Furthermore, bioluminescence resonance energy transfer
(BRET) experiments showed that GPR37 and A2AR are in close proximity, thus allowing us the
detection the GPR37-A2AR oligomers in living cells. Interestingly, the interaction of GPR37 with
A2AR modulated the cell surface expression of A2AR. Overall, these results show that A2AR
interacts with GPR37 in living cells and that the interaction is relevant for A2AR cell surface
expression.





P.15
Effects of β-adrenoceptor stimulation on human atrial outward K+ currents
Ricardo Gómez, Ricardo Caballero, Marta G. de la Fuente, Irene Amorós, Adriana Barana,
Pablo Dolz, Lourdes Osuna, Juan Tamargo, Eva Delpón
Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid
It has been proposed that β-adrenergic stimulation has profound influence in the genesis and
maintenance of atrial fibrillation (AF). We studied the effects produced by isoproterenol (Iso, 1
nM), a β-adrenoceptor agonist, on the transient outward (Ito), the ultrarapid (IKur) and the slow
(IKs) delayed rectifier K+ currents recorded in enzymatically dissociated myocytes obtained from
right (RAA) and left (LAA) atrial appendages from patients in sinus rhythm (SR) and AF, using
the patch-clamp technique.
In myocytes from SR patients, Iso slightly inhibited the Ito (10.1±6.6% in RAA and 15.6±3.3% in
LAA at +30 mV), whereas in AF patients, the Ito inhibition reached a 24.9±6.2% in RAA (P<0.05
vs SR) and was even greater in LAA (36.5±4.9%, P<0.05 vs RAA). In RAA and LAA moycytes
from SR and AF patients, Isus was not modified. Finally, Iso augmented the IKs in SR
(4.5±2.6% in RAA and 6.6±1.4% in LAA at +30 mV), whereas in AF the increase reached a
51.8±6.2% in RAA and was even greater in LAA (78.0±12.4%, P<0.05 vs RAA). Both in SR and
AF, the β1-adrenoceptor antagonist atenolol abolished Iso effects on Ito and IKs. A real-time q-
PCR analysis demonstrated that the β1-adrenoceptor mRNA expression was significantly
higher in AF than in SR samples.
We concluded that AF potentiates the β-adrenergic effects on Ito and IKs. Moreover, this
potentiation was greater in LAA than in RAA myocytes, increasing the atrial electrical
heterogeneity and revealing a possible mechanism predisposing to AF.





P.16
Receptor-mediated effects of endocannabinoids on human cardiac Kv1.5 and Kv4.3
channels
Adriana Barana, Irene Amorós, Ricardo Gómez, Marta Gónzalez de la Fuente, Pablo Dolz,
Lourdes Osuna, Ricardo Caballero, Juan Tamargo, Eva Delpón
Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid
Endocannabinoids are synthesized in human myocardium where they exert cardioprotective
and antiarrhythmic actions. In pathologies such as atrial fibrillation (AF), myocardial synthesis of
endocannabinoids is increased.
We studied the effects of the activation of the cannabinoid CB1 (r-CB1) and GPR55 (r-GPR55)
receptors on human cardiac Kv4.3 and Kv1.5 channels, which are the main responsibles for
atrial repolarization. Kv4.3 (IKv4.3) and Kv1.5 (IKv1.5) currents were recorded using the patch-
clamp technique in r-CB1 (CHO-CB1) and r-GPR55 (CHO-GPR55) stably-expressing CHO cells
that were transiently transfected with the cDNA encoding Kv4.3 and Kv1.5 channels. The
presence of r-CB1 and r-GPR55 in human atrial samples was assessed by a PCR analysis.
The results demonstrated that r-CB1 and r-GPR55 expression was significantly increased in
samples from AF patients.
In CHO-CB1 cells, the r-CB1 activation with WIN55,212-2 inhibited the IKv4.3 in a
concentration-dependent manner (IC50=1.6±0.3 nM), whereas no r-CB1-mediated IKv1.5
effects were observed. In IKv4.3, r-CB1 activation produced a leftward shift in the midpoint of
the inactivation curve ( 20 mV) and significantly accelerated the inactivation process.
In CHO-GPR55 cells, the r-GPR55 activation with LPI inhibited the IKv4.3 and IKv1.5 in a
concentration-dependent manner (IC50=0.1±0.02 µM and 3.2±0.3 µM, respectively). The r-
GPR55 activation shifted the activation curve of Kv1.5 channels to more negative potentials,
without modifying the voltage dependence of Kv4.3 channels inactivation.
Our results demonstrated that, in AF patients, endocannabinoids may exert an important role by
modulating the duration of refractoriness of atrial tissue.




P.17
A non-genomic induction of LC20 phosphorylation and calcium sensitization by
androgens in the colonic smooth muscle
María C. González-Montelongo, Raquel Marín, Tomás Gómez and Mario Díaz
Department of Animal Biology, University of La Laguna, La Laguna, Tenerife, Spain
Androgens (testosterone and 5alpha-dihydrotestosterone) acutely provoke the potentiation of
spontaneous and agonist-induced contractile activity in colonic smooth muscle, independently
of transcriptional events. In permeabilized smooth muscle preparations, we could demonstrate
that these effects are entirely due to calcium sensitization. The effect of androgens was
mimicked by the polyamine putrescine, and was accompanied by increased ornithine
decarboxilase (ODC) activity and RhoA translocation. In addition, DHT (10 nM) increased
phosphorylation of both LC20 (regulatory subunit of myosin light chain, MLC) and CPI-17 (an
endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of PKC, ROK
(Rho-associated Rho kinase) and ODC with chelerythrine, Y27632 and berenyl, respectively,
prevented LC20 phosphorylation and abolished calcium sensitization. The results demonstrate
that androgens induce sensitization of colonic smooth muscle to calcium through activation of
polyamine synthesis, followed by activation of RhoA which, in turn, stimulates PKC and ROK.
Activation of these two kinases induce a potent steady stimulation of LC20 by displacing the
equilibrium towards its phosphorylated state.





P.18
Diversity-oriented synthesis from amino acid-derived β–ketoesters: Finding new TRPV1
antagonists
Paula Pérez-Faginas1, M. Isabel García-Aranda1, M. Teresa Aranda1, M. Jesús Pérez de
Vega1, M. Teresa García-López1, Susana Quirce2, Roberto de la Torre Martínez2, Asia
Fernández-Carvajal2, A. Ferrer-Montiel2, R. González-Muñiz1
1Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain; 2Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain Structural diversity is essential for hit generation in chemical genetics and drug discovery. Within the different strategies to get this end, Diversity Oriented Synthesis (DOS), aimed at synthesizing collections of compounds that differ substantially in their molecular structure, is one of the most promising approaches.(1) The highest level of structural diversity will consist of compounds with different molecular scaffolds, stereochemistries, and functional groups. DOS approaches to diverse small molecules are still a challenge to synthetic chemists, requiring new stereocontrolled, diversity-oriented synthetic methods, using simple starting material. In our lab, natural and non-proteinogenic amino acid derivatives have been used to the generation of different kinds of heterocyclic compounds.(2) Among them, β-ketoesters derived from amino acids and dipeptides were the starting point to pyrido[1,2-c]pyrimidine and oxopipearzine scaffolds.(3) In this communication we exploit the versatility of simple highly functionalized amino acid β-ketoesters to the preparation of small libraries of β,γ-diamino esters, indole and pyrrole derivatives. Though the synthetic procedures are quite simple, the stereochemical issues still need to be solved within these set of compounds. The components of these libraries are being tested for the modulation of different ion channels and other ion channel-related targets. Preliminary results indicated that some of these compounds displayed potent and selective TRPV1 antagonist activity, constituting new hits towards References
(1) Stuart L. Schreiber. Molecular diversity by design. Nature, 2009, 457, 153-154.
(2) a) David Andreu, et al., J. Am. Chem. Soc. 1997, 119, 10579-10586. b) Guillermo Gerona-
Navarro, et al., J. Org. Chem. 2001, 66, 3538-3537. c) Paula Pérez-Faginas, et al., Adv. Synth.
Catal. 2008, 350, 2279-2285.
(3) a) Mercedes Martín-Martínez, et al., J. Med. Chem. 1997, 40 (21), 3402–3407. b) Rosario
Patino-Molina, et al., Tetrahedron 1999, 55, 15001-15010.




P.19
New pharmacological tools for drug characterization
Beltrán, B.; González-Morales, Y.; Pereda, D.; Machado, J.D. and Borges, R.
Laboratorio de Farmacología, Instituto de Bioorgánica Antonio González y Unidad de
Farmacología, Facultad de Medicina, Universidad de La Laguna. Tenerife. Spain
Drug screening always requires the evaluation of the effects of a given substance on biological
systems. It usually sequentially involves on-silicon single molecule-, cell based-, isolated
organs- and whole animal-screenings. However, the enormous amount of new drugs produced
by pharmaceutical chemists has become unviable the development of a whole sequence
described above. Along the last decades the use of whole organs and animals has been
reduced to a minimum, the result is a drastically reduction of new drugs arriving to clinical trials.
As one of the problems working with classical pharmacological preparation all efforts should be
put in increasing the speed of drug evaluation accompanied with the reduction in their cost of
equipment, bench space and personnel.
We present several improvements carried out in our laboratories that include bath and perfusion
organs as well as automatic analysis of animal behaviour.
Acknowledgments: JDM and BB have CONSOLIDER contracts (CSD2008-00005). DP is
recipient of a FPI fellowship from the Spanish Ministry of Science and Innovation (MICINN).
Supported by MICINN Grant BFU2007-64963 (RB) and Canary Agency for Research,
Innovation and Society of Information (ACIISI) and FEDER PI 2007/017 (JDM).





P.20
Design and evaluation of new TRPV1 Open Channel Blockers
Asia Fernández-Carvajal1, Miquel Vidal2, Ana Gomis3, Pierluigi Valente1, Felix Viana3, Rosa
Planells-Cases4, José M. González-Ros1, Carlos Belmonte3, Jordi Bujons2, Angel Messeguer2
and Antonio Ferrer-Montiel1.
1IBMC-UMH, Alicante, Spain; 2IQAC-IC, Barcelona, Spain; 3IN-CSIC, Alicante, Spain; 4Centro
de Investigación Príncipe Felipe, Valencia, Spain.
Vanilloid receptor subtype 1 (VR1) appears to play a fundamental role in the transduction of
peripheral tissue injury and/or inflammation responses. This receptor is implicated in thermal
and chemical nociception and has been suggested to contribute to thermal hyperalgesia. Thus,
this channel has been validated as a therapeutic target for analgesic drug development.
Paradoxically, a large number of high affinity competitive antagonists have failed during clinical
assays for their undesirable side effects. Therefore, there is yet a need to identify new
pharmacophoric scaffolds that lead to the development of antagonists with better therapeutic
index. Uncompetitive antagonists acting as open channel blockers may provide activity-
dependent blockers that preferentially modulate the activity of overactive channels, thus
displaying fewer side effects.
We report here the identification of a family of novel open channel blockers of this receptor that
exhibit in vivo anti-nociceptive and analgesic activity. These compounds blocked capsaicin-
operated ionic currents with nanomolar efficacy and strong voltage dependency. Analysis of the
mechanism of action showed that the drug binding site is located relatively deep in the aqueous
pore of the channel. These compounds attenuated capsaicin-induced Ca2+ influx in trigeminal
nociceptors in culture, and eliminated the vanilloid stimulated nerve activity in afferent fibers
without affecting mechanically evoked responses. Intraperitoneal administration resulted in a
significant analgesic activity against inflammatory pain in model animal of chronic liver failure
that produces inflammation and TRPV1 overactivity. Taken together, these compounds
represent a new class of activity-dependent TRPV1 antagonists with in vitro and in vivo
properties that could be used to expand the family of TRPV1 open channel blockers with the
aim of evolving a lead for analgesic drug development.
Funded by: MICINN, Consolider-Ingenio 2010, Fundació La Marató de TV3.




P.21
MaxiK channel-dependent effects of tungstate in heterologous expression system and in
the vasculature
Ana Isabel Fernández-Mariño, Cristina Porras, Juan Ureña, Antonio Castellano, Jana Selent,
Manuel Pastor, Miguel A. Valverde and José M. Fernández-Fernández
Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona,
Spain.
Tungstate (WO 2-
4 ) is able to reduce blood pressure in spontaneously hypertensive rats although its mechanism is not well understood. We have evaluated the impact of tungstate on BK
channels, which are key regulators of vascular tone regulation. Electrophysiological studies of
heterologously expressed BK channels and vascular contractility in mouse models were run to
determine the impact of tungstate in the vasculature.





P.22
Lidocaine effects on GABAA and Kainate receptors microtransplanted from rat brain to
Xenopus oocyte membrane
Rocío Pérez, Armando Alberola-Die, Juan Martínez-Pinna, Isabel Ivorra and Andrés Morales
Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain
We have recently shown that lidocaine has multiple inhibitory actions on muscle-type nicotinic
acetylcholine receptors (nAChRs), including open- and closed-channel blockade and changes
in desensitisation (Alberola-Die et al. 2010; J. Mol. Neurosci. 40: 248-250). Since several
allosteric modulators of nAChRs have also effects on other ligand-gated ion-channels (LGIC),
we have now studied the effect of lidocaine on GABAA and Kainate (Ka) receptors (GABAARs;
KaRs) microtransplanted from brain sinaptosomal membranes to Xenopus oocytes. Membrane
currents elicited by 1 mM GABA (IGABA) or 100 microM Ka (IKa) either alone or co-applied with
lidocaine (up to10 mM) were recorded at a membrane potential of -60 mV. Microtransplanted
GABAAR and KaR were fully functional, retaining IGABA and IKa their characteristic properties.
Direct co-application of GABA and lidocaine (10 mM) slightly decreased IGABA peak (less than
10%), but had no effect on the desensitisation pattern. However, when lidocaine was pre-
applied before its co-application with GABA, IGABA blockade was more pronounced and the
blocking effect was dependent on the time of pre-application, reaching a maximum of 30% of
inhibition at 30 seconds. In contrast, lidocaine showed no significant effects on IKa amplitude or
desensitisation, even at high concentrations. These results indicate that lidocaine has inhibitory
effects restricted to certain neuroreceptors and its mechanisms of action are specific for each
subtype of LGIC.





P.23
Role of store-operated calcium channels and mitochondria in the inhibition of human
coronary smooth muscle cells migration and proliferation by NSAIDs
Eva Muñoz, Lucía Núñez, Carlos Villalobos
Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid y Consejo
Superior de Investigaciones Científicas (CSIC).Valladolid, Spain
Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and
proliferative disorders of vessel wall. Salicylate and other non-steroideal anti-inflammatory drugs
(NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory
activity. In search for this mechanism, we have studied the effects of salicylate and other
NSAIDs on subcellular Ca2+ homeostasis and Ca2+-dependent cell proliferation in human
coronary,vascular smooth muscle cells (hcVSMCs). We found that hcVSMCs displayed both
store-operated Ca2+entry (SOCE) and voltage-operated Ca2+entry (VOCE). Interestingly, the
ratio of SOCE to VOCE and cell proliferation decreased with increased pass number, possibly
due to the differentiation towards a contractile phenotype. Inhibition of SOCE by specific Ca2+
release-activated Ca2+ (CRAC/Orai) channels antagonists prevented hcVSMC proliferation. We
also found that NSAIDs inhibited SOCE and thereby Ca2+-dependent proliferation and
migration. In search for the action mechanismwe found that NSAIDs prevented mitochondrial
Ca2+ uptake in hcVSMCs, thus facilitating the Ca2+-dependent inactivation of store-operated
Ca2+ entry. We conclude that NSAIDs inhibit human coronary vascular smooth cell migration
and proliferation by enabling the Ca2+-dependent inactivation of store-operated Ca2+ entry
regulated
This work has been funded by grants CSI12A08 from Junta de Castilla y León and BFU2009-
08967 from Ministerio de Ciencia e Innovación





P.24
Estrogen receptor beta (ERb) mediates rapid insulinotropic effects in pancreatic beta
cells: possible implications for diabetes treatment
Paloma Alonso-Magdalena, Sergi Soriano, Marta García-Arévalo, Ivan Quesada, Esther
Fuentes and Angel Nadal
CIBER of Diabetes and Associated Metabolic Diseases, CIBERDEM, lnstitute of
Bioengineering, University Miguel Hernandez of Elche, Elche, Spain.
Type 2 diabetes mellitus constitutes one of the most challenging health problems on a global
scale. It is characterized by the emergence of fasting hyperglycemia, which is associated with
an inadequate insulin secretion from pancreatic b-cells to compensate for insulin-resistance in
peripheral tissues. The ATP-potassium channel (KATP channel) is the key molecule involved in
glucose-stimulated insulin secretion (GSIS). We have previously demonstrated that
physiological concentrations of 17b-estradiol (E2) decrease KATP channel activity thus
enhancing insulin secretion and that estrogen receptor beta (ERb) is involved in this
phenomenon.
Here we evaluate the potential clinical use of ERb agonists as rapid insulinotropic agents. In
vitro studies demonstrate that low doses of WAY 200070, an ERb specific agonist, decrease
KATP channel activity and enhance GSIS in the presence of stimulatory (7 and 16 mM glucose)
but not low glucose concentrations. In a set of in vivo experiments we show that a single-
administration of WAY 200070 leads to an improvement in the plasma glucose response to a
glucose challenge with a concomitant increase in insulin level. Thus, we next study the potential
clinical use of ERb agonists by using streptozotocin-nicotinamide-induced mildly diabetic mice.
These animals exhibit moderate hyperglycaemia and impaired glucose tolerance because of the
loss of early-phase insulin secretion. One-week treatment with the ERb agonist caused a
significant improvement in glucose tolerance, with a significant increase in the plasma insulin
and
We conclude that ERb agonists should be considered as new antidiabetic drugs.




P.25
Insights into ASIC1 gating: Open and Closed conformations of the ion pore
Tianbo Li, Youshan Yang and Cecilia M. Canessa
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut,
USA
ASIC1 is a proton-activated sodium channel found in the plasma membrane of most neurons in
the mammalian nervous system. ASIC1 modulates synaptic transmission, induction of fear and
memory, and nociception in peripheral neurons. Structurally, ASIC1 is a trimer of identical or
homologous subunits. Each subunit has two transmembrane segments, short intracellular
amino- and carboxytermini, and a large extracellular domain. This architecture is the hallmark of
all members of the ENaC/Degenerin family regardless of their particular function and mode of
activation. We screened TM1 and TM2 by SCAM and MTS reagents of lamprey ASIC1, a
channel with little intrinsic desensitization and with higher tolerance to cysteine mutagenesis
than the mammalian counterpart. The effect of MTS reagents applied to the external or internal
side of the channel (outside-out or inside-out patches) in either the open or closed
conformations revealed a pattern of reactivity consistent with an open architecture having the
narrowest segment at the level of residues G443A444S445 in TM2. In the closed conformation
MTSEA modifies outer residues of TM2 (429-to-432) when applied from the external side but when applied from the internal side it reaches position 440 indicating that the constriction made by G443A444S445 widens in the open conformation allowing the entrance of the reagent deeper into the ion pathway. In addition, substitutions of residues G443A444S445 either abolish current or change ion conductance consistent with forming the selectivity filter. We conclude that the open configuration has a constriction at the level of the selectivity filter (G443A444S445) whereas the closed configuration has a constriction at the level of G432-to-Q437. These findings imply: first that the selectivity filter is the narrowest segment of the pore in the open conformation but it widens and disassembles in the closed state. Second, that the constriction formed by G432-to-Q437 functions as the opening, closing and desensitization gates, and third, that the transition from
closed to open requires straightening of the 50o angle of TM2.




P.26
Differential modulation of Nav1.5 channels by the β2 subunit in two different cell lines.
Helena Riuró, Pedro Beltran, Oscar Campuzano, Guillermo Pérez, Fabiana Scornik and
Ramon Brugada
Cardiovascular Genetics Center. IdIBGi-UdG, Girona.
Cardiac sodium channels are composed mainly of a pore-forming α-subunit, and auxiliary β-
subunits. Mutations in Nav1.5 and β-subunits have been identified in different channelopathies
such as Long QT syndrome and Brugada Syndrome. In addition, different studies have shown
that modulation of Nav1.5 channels by β1 and β2 is affected by post-transcriptional changes
which might differ among cell lines. The aim of our study was to investigate the differences in
modulation of Nav1.5 currents by the β2 subunit heterologously expressed in either tsa201 or
CHO cells. We performed patch clamp studies of sodium currents in tsa201 or CHO cells
transiently expressing Nav1.5 a subunit alone, or together with the b2 subunit. We analyzed the
changes in current density, voltage dependence of activation and inactivation, recovery from
inactivation and inactivation time course. Peak sodium current amplitude recorded at -20 mV
from tsa201 cells increased significantly when the a subunit was expressed together with the β2
subunit (-56.65±11.83 and -105.13±14.31 pA/pF, a and a+β2, respectively, n=10, p>0.00408).
Recovery from inactivation was shifted to negative values in the presence of the β2 subunit
(τ1/2=19.98±1.89ms, and 11.58±1.40ms, a and a+β2, respectively, n=10, p>0.0011). Similar
studies performed in CHO cells (n=3 of each type) did not show any difference in currents
recorded from cells expressing a or a+β2 subunits. These preliminary results reinforce the
concept that β2 subunit modulation of Nav1.5 currents is dependent on cell background,
suggesting that the cell model might be critical for the analysis of mutations related to sodium
channelopathies.





P.27
Regulation of the cardiac sodium channel by protein-protein interactions and post-
translational modifications
Pedro Beltran, Guillermo Pérez, Fabiana Scornik and Ramon Brugada
Cardiovascular Genetics Center. IdIBGi-UdG, Girona.
The cardiac sodium channel (Nav1.5) is composed of a pore-forming α-subunit, auxiliary β-
subunits and a range of regulatory proteins. Mutations in Nav1.5 and interacting proteins have
been identified in different channelopathies such as Long QT syndrome type 3, and Brugada
Syndrome. In addition, early studies have identified post-translational modifications in Nav1.5
that might affect its function. To date, knowledge about post-translational modification sites has
been limited to phosphorylation and glycosylation of key residues in the intracellular or
extracellular domains of Nav1.5. The aim of our study was doublefold. First, to investigate the
protein-protein interactions of Nav1.5, second, to analyze the post-translational modifications of
the sodium channel. To address the first objective, we performed pull-down studies using the
intracellular domains of Nav1.5 as baits in affinity co-purification experiments after incubation
with soluble and membrane heart fractions. To investigate the post-translational modification
pattern of Nav1.5, we created a stable HEK293 tsa201 cell line expressing Nav1.5 and a GFP
gene reporter in a bicistronic construct. We report here a battery of protein-protein interactions
strategies as well as preliminary results from our post-translational modifications studies that
introduce a new point of view towards the understanding of the regulation of Nav1.5.





P.28
Selective formation of oligomeric Kv7.5 (KCNQ5)/KCNE1 and Kv7.5 (KCNQ5)/KCNE3
channels. Differential targeting to membrane surface microdomains
Laura Solé1, Meritxell Roura-Ferrer1,2, Anna Oliveras1, Albert Vallejo1, Núria Comes1, Álvaro
Villarroel2 and Antonio Felipe1
1Molecular Physiology Laboratory, Departament de Bioquímica i Biologia Molecular, Universitat
de Barcelona, Barcelona, Spain; 2Unidad de Biofísica, CSIC-UPV/EHU, Universidad del País
Vasco, Leioa, Spain
Kv7 (KCNQ) proteins form a family of voltage-gated potassium channels that is comprised of
five members, Kv7.1–Kv7.5. While Kv7.1 is crucial in the heart, the Kv7.2-Kv7.5 channels
contribute to the M-current in the nervous system. In addition, Kv7.5 is expressed in muscles,
where its physiological role is currently under evaluation. Kv7 associations with KCNE
accessory subunits (KCNE1–5) enhance channel diversity. KCNE peptides control the surface
expression, voltage-dependence, kinetics of gating, unitary conductance, ion selectivity and
pharmacology of several channels. KCNE subunits have been primarily studied in the heart;
however, their activity in the brain and in many other tissues is being increasingly recognized.
Here, we found that Kv7.5 and KCNE subunits are present in myoblasts. Therefore, oligomeric
associations may underlie some Kv7.5 functional diversity in skeletal muscle. Expression in
Xenopus oocytes and HEK-293 cells demonstrates that KCNE1 and KCNE3, but none of the
other KCNE subunits, associate to Kv7.5. While KCNE1 slows activation and suppresses
inward rectification, KCNE3 inhibits Kv7.5 currents. Furthermore, KCNE1 increases Kv7.5
currents in HEK cells. The membrane targeting is also affected. Biochemical isolation of lipid
rafts demonstrates that Kv7.5 barely locates in rafts. KCNE1 and KCNE3 show differential
targeting. While KCNE3 targets to rafts, KCNE1 does not. Association of Kv7.5 with KCNE3
impairs KCNE3 targeting to rafts and this is further supported by FRAP analysis. Our results
have physiological relevance since Kv7.5 is abundant in skeletal and smooth muscle and its
association with KCNE peptides may fine-tune cellular responses.




P.29
A splice variant-insert drives basolateral targeting of MaxiK channel in polarized
epithelial cells.
1,*Araceli Morales, 1Ning Zhu, 1,2Enrico Stefani and 1,3Ligia Toro. UCLA, 1Departments of Anesthesiology, Division of Molecular Medicine, 2Physiology, and 3Molecular & Medical Pharmacology, Los Angeles, CA, USA (* present address: Unidad de
Investigación, Hospital Universitario de Canarias – Universidad de La Laguna, Tenerife, Spain)
Splice variation can modify MaxiK channel traffic by retention in intracellular stores. In Madin
Darby canine kidney (MDCK) cells, expressed MaxiK is targeted to the apical surface, whereas
in human bronchial epithelial (16HBE14o-) cells channel activity was reported at the basolateral
membrane. Thus, we tested the hypothesis that splice variants can explain this difference.
Using high-resolution confocal microscopy and specific antibodies, we confirmed that in
polarized MDCK cells endogenous Slo is mainly expressed at the apical surface; on the other
hand, 16HBE14o- cells showed expression at both apical and basolateral surfaces. The
presence of endogenous Slo was confirmed by Western blot and RT-PCR demonstrated that
16HBE14o- cells have robust expression of a 29 amino acid spliced exon (SV29) but not MDCK
cells where its expression was minimal. Quantitative real-time PCR results showed that the
amount of this insert is >95% more abundant than insertless human Slo (hSlo) in 16HBE14o-
cells. The role of this spliced exon was tested using viral constructs of Slo attached to EGFP
with and without SV29 (Slo-EGFP and Slo-SV29-EGFP). After viral infection of MDCK cells
under non-saturating conditions, Slo-EGFP showed apical expression, meanwhile, Slo-SV29-
EGFP was also expressed in the basolateral surface suggesting an intrinsic basolateral signal
in SV29.
Thus, our data indicate that the splice variant insert SV29 can drive basolateral expression of
Slo channel in MDCK cells, suggesting a new role for Slo splice variants in regulating polarized
targeting.
Supported by NIH (ES, LT).



P.30
Constitutive calmodulin tethering is not required for Kv7 channel function
Juan Camilo Gomez-Posada, Paloma Aivar, Alessandro Alaimo, Araitz Alberdi, Juncal
Fernández-Orth, Ainhoa Etxeberria, Pilar Areso and Alvaro Villarroel
Unidad de Biofísica CSIC-UPV/EHU, Leioa (Bizkaia), Spain
M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve
as important regulators of neuronal excitability. Ensuring that the correct number of these
channels are present at the plasma membrane is crucial to control cell excitability. Calmodulin
binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin
binding reduce surface expression and cause Benign Familial Neonatal Convulsions (BFNC), a
dominantly inherited human epilepsy. However, the mechanisms by which calmodulin regulates
surface expression remain largely unknown. The Kv7.2 calmodulin binding site is made up of
two discontinuous alpha helices (helix A and helix B) and in helix B we have found a trafficking
domain using Tac chimeras. Furthermore, we identified a presumably phosphomimetic mutation
in the first PKC site within this helix B that permits calmodulin-independent surface expression.
Thus, our data reveal a critical role for helix B of Kv7.2 in calmodulin-dependent trafficking, and
they demonstrate that constitutive tethering of calmodulin is not required for M-channel function.




P.31
Phosphorylation changes in ERG channels regulation by TRH
Luis Carretero, Pedro Casado, Pedro R. Cutillas, Jorge Fernández-Trillo, Angeles Machín,
Rosa Alvarez, Pilar de la Peña, Francisco Barros, Pedro Domínguez
Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Oviedo, Spain
The ERG K+ channels are key proteins in cardiac physiopathology and are also essential for
the electrophysiological characteristics of many cells. In anterior pituitary GH3 cells and HEK-
H36/T1 cells expressing HERG channels and TRH receptors, ERG currents are inhibited by
activation of the G protein-coupled TRH receptor, this regulation being mediated by a signaling
cascade in which a phosphorylation/dephosphorylation cycle is involved, triggered by still
unknown protein kinase(s) and or phosphatase(s), acting on the channel itself and/or other
proteins. For this reason we have studied the phosphorylation state of the ERG channel using a
phosphoproteomic approach, in which we used TiO2 affinity after protease treatment of cell
extracts in order to enrich samples in phosphopeptides, which were later identified by LC-
MS/MS and quantified using TIQUAS technology. Using these techniques we have identified
multiple phosphorylation sites in the HERG channel, most of which are located in the proximal
domain of the amino terminus, previously described by our group as a determinant of the
electrophysiological properties of the channel and also recognized as necessary for the
regulation of HERG by TRH. We have also found that the phosphorylation levels of two sites
from that domain increase upon TRH treatment. We have also studied the changes in the
phosphorylation state of other cell proteins after TRH treatment. Both in GH3 and HEK-H36/T1
previous findings of our lab involving the MAPK cascade in TRH signal transduction.




P.32
Regulation of hERG by a1-adrenoceptor is mediated by depletion of PIP2 and PKC-
mediated phosphorylation.
Janire Urrutia, Aintzane Alday, Mónica Gallego, Oscar Casis
Departamento de Fisiología, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, Bilbao,
Spain
Human ether-á-go-go related gene (hERG) K+ channels have a critical role in cardiac
repolarisation. It is known that this current is regulated by a1-adrenoceptors (a1-AR), but the
mechanism is controversial. The aim of this work is to clarify the intracellular pathway
connecting the a1-AR and hERG current regulation. Methods: Stable HEK cell line expressing
hERG was transiently transfected with a1-AR and GFP. The whole cell version of the patch-
clamp technique was used in all experiments. The specific agonist Phenylephrine (20 mM) was
used to stimulate the receptor. Results: a1-AR activation with Phenilephrine shifted the HERG
activation half voltage (Vh) to more positive potentials. Blockade of the components of the
typical a1-AR pathway, Gaq with GPant2A, PLC with Neomycin and PIP2 with an anti-PIP2
antibody prevented the effect of Phenylephrine on HERG activation Vh. Prevention of PIP2
depletion with PIP2-DIC8 also eliminated Phenylephrine effect. Genystein did not prevent the
effect of Phenylephrine, excluding Tyrosine kinases from the intracellular pathway. PKC
blockade with Bisyndolylmaleimyde-1 only partially prevented the Phenylephrine-induced shift in
HERG activation Vh. Conclusion: Our results suggest that the regulation of hERG current by a1-
AR depends mainly on the consumption of PIP2 and in a lower extent on PKC-mediated
phosphorylation.




P.33
Expression of large conductance calcium-activated (BK) channels in porcine intracardiac
ganglia.
Elisabet Selga, Alexandra Pérez-Serra, Ramon Brugada, Guillermo J. Pérez and Fabiana S.
Scornik.
Cardiovascular Genetics Center. IdIBGi-UdG, Girona.
Intracardiac ganglia provide the inhibitory drive to the heart. Our work is directed towards
understanding the signaling of principal neurons in mammalian intracardiac ganglia. In the
present study, we investigated the expression of BK channels from porcine intracardiac ganglia.
First, we studied outward currents from porcine isolated intracardiac neurons in whole cell
configuration. Total outward current showed a fast transient component and a slow, sustained
component similar to what we previously observed in canine intracardiac neurons. To determine
the contribution of the BK current to the total outward current, we performed recordings in the
absence and after the addition of 1mM TEA to the bath. In the presence of TEA, mean
maximum amplitude decreased to almost 55% of the control value (78.33 ± 6.91 and 42.98 ±
6.41 pA/pF, control and TEA, respectively; n=5). Second, we performed RT-PCR and
sequencing analysis to confirm the expression of the BK channel in this tissue. Interestingly, we
found a BK channel transcript sequence that includes a 81bp intronic insertion between exons
24 and 25. Splice variants described in neighboring regions have been shown to play a role in
determining cell surface expression and function of BK channels. Further studies are needed to
determine how this insertion may affect BK channel activity in porcine intracardiac ganglia.




P.34
Essential Role of Intracellular Calcium Handling in Heart Protection Induced by Urocortin
Eva Calderón-Sánchez1, Gema Ruiz-Hurtado2, Tarik Smani1, Jean Pierre Benitah2, Ana María
Gómez2, Antonio Ordóñez1
1Instituto de Biomedicina de Sevilla, Spain; 2INSERM, U637, Université Montpellier, France Ischemia/reperfusion (I/R) damage in the heart occurs mainly during the first minutes of reperfusion, which involve intracellular Ca2+ increase. Urocortin (Ucn) is a member of the corticotrophin-releasing factor that has been identified as a potent endogenous cardioprotector peptide when used in pre- and postconditioning protocols. However, the underlying mechanisms are not completely elucidated. Here, we focused on intracellular calcium ([Ca2+]i) handling We used Langendorff-perfused rat hearts to determine hemodynamics parameters, and isolated cardiomyocytes loaded with fluorescence Ca2+ dye fluo-3AM and viewed with confocal microscope We found that heart contractility and relaxation was fully recovered in isolated hearts submitted to I/R and treated with Ucn at the onset of reperfusion. In isolated myocytes, following ischemia we observed that the diastolic [Ca2+]i was increased, the systolic [Ca2+]i transients amplitude were depressed and sarcoplasmic reticulum (SR) Ca2+ load was reduced. This was correlated to a decrease in the Na+/Ca2+ exchanger (NCX) activity. Importantly, Ucn applied at reperfusion produced a full recovery in diastolic [Ca2+]i and [Ca2+]i transient amplitude, which were In Conclusion, we demonstrated that [Ca2+]i handling plays an essential role in Ucn cardioprotective postconditioning. Acknowledgements: This study was supported by grants Red RECAVA [RD06-0014-0020];
from the Government of Andalucía (Consejería de Innovación Ciencia y Empresa [P09-CTS-
4715]; and FIS (grant no. PS09-02287) E.C. is researcher from RECAVA and T.S. from "La
Fundación Pública Andaluza Progreso y Salud".



P.35
Regulation of vascular tone by urotensin-II is mediated by STIM1 and ORAI1
Díaz I, Domínguez-Rodríguez A, Antonio Ordoñez, Tarik Smani
Instituto de Biomedicina de Sevilla, Sevilla, Spain
Circulating neuro-hormone Urotensin-II which acts through the de-orphanized G-protein coupled
urotensin receptor (UTS2R) has been characterized as the most potent mammalian
vasoconstrictor identified so far. Although the mechanism of it action is still under debate. Here
we examined the role of Store Operated Calcium Channels (SOCC) in the Urotensin-II-induced
vasoconstriction
Coronary artery and isolated smooth muscle cells (SMC) from Wistar rats were used in this study. Vessel myograph, Ca2+ imaging, pharmacology and molecular approaches were utilized to determine the role STIM1, the sensor of store depletion, and Orai1, the pore forming subunit, in vasoconstriction. We found that Urotensin-II induced dose dependent vasoconstriction that was inhibited by UTS2R antagonist and was sensitive to store operated Ca2+ channels (SOCC) inhibitors. In isolated SMC, Urotensin-II elicited Ca2+ and Mn2+ influx that have similar characteristics as SOC influx induced by the passive store depletion with thapsigargin (TG). Ca2+ and Mn2+ influx evoked by Urotensin-II have similar sensitivity to GD3+, 2APB and DES. Moreover, transfection of SMC with small interfering RNA (siRNA) to Orai1 and Stim1 impaired significantly Urotensin-II induced Ca2+ and Mn2+ entry; and coronary arterial ring transfection with Orai1 siRNA decreased In summary, we demonstrated that SOCE is required for UII-induced vasoconstriction of coronary Acknowledgements: This study was supported by MICINN (BFU-2010-21043-C02-02), ISCIII
(PS09-02287 and Recava RD06-0014-0020) and from la Junta de Andalucía (P08-CVI-3913).




P.36
The calcium homeostasis imbalance caused by ORMDL3 is not shared by the rest of the
ORMDL family members
Gerard Cantero-Recasens, Amado Carreras-Sureda, Fanny Rubio-Moscardo, César Fandos,
Miguel A. Valverde and Rubén Vicente.
Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain
The ER depends upon a specialized environment of high Ca2+ concentration to fold and
assemble newly synthesized proteins together with the synthesis of lipids like sphingolipids and
phospholipids destined for insertion into one of several cellular membranes. The ORMDL family,
composed by three members, seems to play a pivotal role in the ER homeostasis since their
yeast homologous, ORM1 and ORM2, regulate the lipid synthesis. In addition they participate in
the unfolded protein response (UPR) and in humans one of its members, ORMDL3, is
implicated in the calcium cellular homeostasis. This family has focus recently the attention
because ORMDL3 has been associated by genetic studies with several pathologies like
asthma, diabetes type I, Crohn's disease and colitis ulcerosa. In the present work we do an
exhaustive study of the entire human ORMDL family looking at the three aspects: the calcium
homeostasis, the lipid synthesis and the participation in the UPR. Our results show that,
contrary to their yeast homologous, ORMDL1, ORMDL2 and ORMDL3 present different
behaviour regarding the calcium ER handling and the UPR response. Therefore we suggest
that the unique association of ORMDL3 with several pathologies might be due to the specific
roles of this protein which do not share with the other members of the ORMDL family.




P.37
TREKing through the autonomic nervous system
Diego Fernández-Fernández, Alba Cadaveira-Mosquera, Antonio Reboreda, Paula Rivas-
Ramírez, Vanesa Domínguez and J. Antonio Lamas
Laboratory of Neuroscience, Department of Functional Biology, Faculty of Biology, University of
Vigo, Spain
Potassium channels of the K2P family are widely expressed in the central nervous system and
peripheral somatosensory ganglia. Recently we proved, for the first time, that K2P channels of
the TREK subfamily are also functionally expressed in the autonomic nervous system (mouse
sympathetic superior cervical ganglion). These channels generate a riluzole-activated outward
current insensitive to most classic potassium channel blockers but strongly inhibited by
fluoxetine
Neurosci., In this work we used patch-clamp, qRT-PCR and immuno-cytochemical techniques to investigate the presence and significance of TREK channels in cultured vagal sensory afferents of the Nodose Ganglion (NG). qRT-PCR experiments showed a high expression of TREK-1 channels but also, at a lesser extent, of TREK-2 and TRAAK (TREK-1>TREK-2>TRAAK). Consistently, application of riluzole to neurons of the NG in culture induced a potassium outward current in a high percentage of them. The effect of riluzole was dose-dependent and seems not to be related with the presence of TTX-resistant sodium currents, characteristic often used Our results suggest that the TREK subfamily of the K2P channels may be important in the performance Our group has been supported by grants from MICINN (BFU2008-02952/BFI and CSD2008-
00005 SICI CONSOLIDER), XUNTA DE GALICIA (INBIOMED 2009/063 and IN845B-2010/148)
and UNIVERSITY OF VIGO (00VI 131H 641.02).




P.38
Inhibition of TREK currents by fluoxetine modulates the excitability and resting
membrane potential of sympathetic neurons
Alba Cadaveira-Mosquera, Antonio Reboreda, Paula Rivas-Ramírez, Diego Fernández-
Fernandez, Vanesa Domínguez and J.Antonio Lamas.
Laboratory of Neuroscience, Department of Functional Biology, Faculty of Biology, University of
Vigo, Spain
The 6 subfamilies of two-pore-domain K+ channels (K2P) are widely expressed in the nervous
system and they are modulated by several biophysical and pharmacological agents. The
modulation by different agents allows K2P channels to participate in distinctive functional roles.
One of the most physiologically relevant is the modulation of the resting membrane potential
and
We have recently reported (J. Neurosci., in press) the presence of the three members of the
TREK subfamily (TREK-1, TREK-2 and TRAAK) in neurons cultured from the mouse superior
cervical ganglia (SCG). In voltage-clamp experiments, TREK currents were activated by
riluzole, linoleic acid, mechanical stimulation and intracellular acidification. All these stimuli
induced an outward potassium current very sensitive to the blockade by fluoxetine. The riluzole-
activated current was also inhibited by muscarinic agonists through M1 receptors. Moreover,
single channel experiments showed that inclusion of fluoxetine in the recording electrode
dramatically decreased the open probability of TREK-2 channels. In current-clamp experiments,
low concentrations of fluoxetine (10 µM) provoked a reduction in the latency of the first action
potential while higher concentrations (100 µM) caused a depolarization of the membrane resting
potential.
Our data showed that the resting membrane potential and the excitability of SCG neurons are
modulated by TREK currents sensitive to the antidepressant agent fluoxetine.
Our group has been supported by grants from MICINN (BFU2008-02952/BFI and CSD2008-
00005 SICI CONSOLIDER), XUNTA DE GALICIA (INBIOMED 2009/063 and IN845B-2010/148)
and UNIVERSITY OF VIGO (00VI 131H 641.02).



P.39
TRESK channel contribution to DRG sensory neuron hyperexcitability following nerve
injury
Astrid Tulleuda1, Barbara Cokic2, Gerard Callejo1, Barbara Saiani1, Jordi Serra2 and Xavier
Gasull1
1Neurophysiology Lab, Medical School, University of Barcelona-IDIBAPS, Barcelona, Spain; 2Neuroscience Technologies, Barcelona Science Park, Spain
Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In
invertebrate nociceptors, the S-type leak K+ channels (analogous to TREK-1) plays a critical role
in determining neuronal excitability following nerve injury (J Neurophysiol 2002;87:2408-20).
Few data are available on the role of leak K2P channels after peripheral axotomy in mammals.
Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory
neurons and decreases TRESK (K2P18.1) expression, a channel that has a major contribution
to total leak current in DRGs. While the expression of other channels from the same family did
not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly
acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and
similar total background currents compared to neurons injured in vivo. In addition, the sanshool
derivative IBA, which blocked TRESK currents in HEK-transfected cells and DRG neurons,
increased intracellular calcium in 49% of DRG neurons. Most IBA-responding neurons (71%)
also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors.
Additional proof of a biological role of TRESK was provided by behavioral evidence of pain
(flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following
IBA injection in the rat hindpaw and increased sensitivity to painful pressure after TRESK
knockdown in vivo or IBA injection. In summary, our results clearly support an important role of
TRESK channels in determining neuronal excitability in specific DRG sensory neuron
subpopulations, and show that axonal injury down-regulates TRESK channels, therefore
contributing to neuronal hyperexcitability.
Supported by FIS PI080014; Retic RD07/0062 and 2009SGR-869, Spain.




P.40
Subcellular localization of small conductance calcium-activated potassium (SK) channels
in the hippocampus
R. Luján1; C. Ballesteros-Merino1, C. Ferrándiz-Huertas1; M. Watanabe2; J.P. Adelman3
1Departamento de Ciencias Médicas, IDINE-Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spain; 2Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan; 3Vollum Institute, OHSU, Portland, OR, USA Small-conductance, Ca2+-activated K+ (SK) channels containing the SK2 (KCNN2) subunit modulate synaptic transmission and plasticity in CA1 pyramidal neurons, and influences hippocampus-dependent memory encoding. Here, we investigated the temporal and spatial expression of SK2 in the developing mouse hippocampus using molecular and biochemical techniques, quantitative immunogold electron microscopy and electrophysiology. The mRNA encoding SK2 was expressed in the developing and adult hippocampus. Western blotting and immunohistochemistry showed that SK2 protein increased with age. This was accompanied by a shift in subcellular localization. Early in development (P5), SK2 was predominantly localized to the endoplasmic reticulum in the pyramidal cell layer. But by P30 SK2 was almost exclusively expressed in the dendrites and spines. The level of SK2 at the postsynaptic density (PSD) also increased during development. In the adult, SK2 expression on the spine plasma membrane showed a proximal-to-distal gradient. Within individual dendritic spines the expression level of SK2 positively correlated with spine size. Consistent with this developmental redistribution and expression gradient of SK2, the selective SK channel blocker apamin increased evoked excitatory postsynaptic potentials (EPSPs) only in CA1 pyramidal neurons from mice older than P15. In adults, apamin increased the magnitude of long-term potentiation only at distal but not at proximal CA3-CA1 synapses in the stratum radiatum. These results show a developmental increase and gradient in SK2-containing channel surface expression that correlate with their influence on neurotransmission and synaptic plasticity, and that may contribute to increased memory Support Contributed By: PAI08-0174-6967 and CSD2008-00005



P.41
Disrupted NMDA receptors trafficking underlies synapse elimination in Huntington´s
disease models
S. Marco, J. Torres-Peraza, A. Giralt, R.K. Graham, M.R. Hayden, J. Alberch and I. Pérez-
Otaño
Departamento de Neurociencias, CIMA, Spain
Alterations in NMDA-type glutamate receptors (NMDARs),are implicated in the cell-specific
neurodegeneration, cognitive decline and psychiatric impairment characteristic of Huntington's
disease (HD). Yet, it is unknown how the mutations in htt are linked to alterations in NMDAR
function. We previously discovered that the huntingin-interacting protein PACSIN1/syndapin1
behaves as an endocytic adaptor for NMDARs by targeting the NMDAR subunit NR3A.
Interestingly, the interaction of PACSIN1 is enhanced for mutant htt, and its overexpression
rescues the HD phenotype. We hypothesized that mutant htt might disrupt NMDAR trafficking
by altering PACSIN1 function, which could lead to enhanced surface expression of NMDAR
subtypes
Here we show that N-terminal fragments of mutant htt transfected into striatal neurons sequester PACSIN1 into intracellular aggregates, limiting its dendritic availability and causing an accumulation of NR3A-containing NMDARs at the plasma membrane. Consistent with in vitro data, young R6/1 mice expressing exon1 of mutant htt and YAC128 mice expressing full-length mutant htt (with 115 and 128 CAG repeats, respectively) displayed reduced PACSIN1 and increased NR3A levels in striatal synaptic plasma membranes. Cross-linking studies of surface receptor pools suggest an underlying defect in NR3A internalization. Enhanced NR3A is observed in medium-sized spiny neurons, the vulnerable neuronal population in HD, and correlates with decreases in spine density that can be fully reversed in YAC128 mice lacking NR3A. Thus, aberrant expression of juvenile NMDR subtypes could explain several of the synaptic and behavioral deficits observed in HD human patients and transgenic models, and might Funded by HDF, Comunidad de Trabajo de los Pirineos, Gobierno de Navarra and
CONSOLIDER—Ingenio CSD2008-00005.




P.42
Dynamin-dependent endocytosis of freshly exocytosed synaptopHluorin at the
neuromuscular junction during nerve stimulation
Pedro Linares-Clemente1, José L. Rozas1, Pablo García-Junco-Clemente1, José A. Martínez-
López1, M. Eugenio Vázquez2, C. Oscar Pintado3 and Rafael Fernández-Chacón1
1Instituto de Biomedicina de Sevilla, IBiS, Hosp.Univ. Virgen del Rocío/CSIC/Universidad de
Sevilla and Dept. Fisiología Médica y Biofísica, and CIBERNED, Seville, Spain; 2Dpto. Química
Orgánica y Centro Singular de Investigación en Química Biológica y Materiales Moleculares,
Universidad de Santiago de Compostela, Santiago de Compostela, Spain; 3Centro Producción y
Experimentación Animal, Universidad de Sevilla, Seville, Spain
Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by
exocytosis. Here, at the neuromuscular junction (NMJ) of synaptopHluorin (spH) transgenic
mice, we have used imaging to study exo- and endocytosis simultaneously occurring during
nerve stimulation. We have found two endocytosis components that occurs sequentially. The
early component of endocytosis internalizes spH-molecules freshly exocytosed, it is sensitive to
the blocking of GTPase activity of dynamins with dynasore and resistant to myristyl trimethyl
ammonium bromide (MiTMAB), a competitive agent that inhibits dynamin by blocking dynamin
binding to phospholipid membranes. The late component of endocytosis, internalizes spH
molecules that pre-exist at the plasma membrane before stimulation starts and it is blocked by
impairing dynamin binding to phospholipid membranes with MiTMAB. The mechanism
responsible for early endocytosis is tightly coupled to exocytosis activation and late endocytosis
likely emerges upon saturation of the early component. Our study demonstrate the co-
existence of two sequential synaptic endocytosis mechanisms: one that internalizes vesicular
components immediately after they are released and another that internalizes vesicular
components pre-existing at the plasma membrane surface.





P.43
A New Kinetic Framework for Vesicle Trafficking
Thomas Gabriel1, Kashif Mahfooz1, Elizabeth Garcia-Pérez1, Rebeca Martínez-Turrillas1, Isabel
Pérez-Otaño1, Joaquín Goñi1,2, Donald C. Lo3, John F. Wesseling1
1Departamento de Neurociencias, CIMA, Spain; 2Departamento de Física y Matemática
Aplicada, Universidad de Navarra, Pamplona, Spain; 3Center for Drug Discovery and
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina, USA
The current concept that the kinetics of presynaptic vesicle trafficking depend on the number of
reserve vesicles by mass-action rules is not compatible with recent measurements of the timing
of induction and recovery of short-term depression, which instead suggest a simpler
mechanism. Here we report a previously unknown additional kinetic constraint that seems to
simplify the problem even further. We then develop a re-conceptualized kinetic model which is
formalized as a countable set of Markov chains and contains only four parameters, the values of
which are fully constrained by previous empirical results. Finally, we analyze the deficit
exhibited by synapsin 1 and 2 knockout synapses against the model and find that the deficit can
be fully characterized by changing the value of a single one of the four parameters in the model
from 4 to 2.6, whereas key activity/residual Ca2+ dependent mechanisms remain intact in the
knockout. To guide intuitions, a physical interpretation of the new model is proposed that
ascribes the slowest to recover component of short-term depression to depletion of a new kind
of reserve pool consisting of four vesicles localized to individual release sites in the active zone
whereas alternative interpretations in which inhibitory mechanisms unrelated to reserve-pool
depletion cause the deepest form of short-term depression are ruled out.





P.44
Phenotypes of chromogranins knocking out mice
Pereda, D.; Pardo, M.R.; Beltrán, B.; Machado, J.D. and Borges, R.
Laboratorio de Farmacología, Instituto de Bioorgánica Antonio González y Unidad de
Farmacología, Facultad de Medicina, Universidad de La Laguna. Tenerife. Spain.
rojas
Chromogranins (Cgs) are the major soluble proteins present inside large secretory vesicles.
Beside their intravesicular functions Cgs are released pro-hormones with many documented
biological functions. Besides that, Cgs are tumour markers and have been involves in diseases
like hypertension, schizophrenia, epilepsy, Parkinson's, and Alzheimer's disease, as well as
amyotrophic lateral sclerosis. Recently, three new mouse strains lacking Cgs (A & B) have
become available from these, we created the double CgA&B-KO mouse. Having these animals
in our laboratory we have conducted several strategies for their phenotypical characterization,
which include: single cell, isolated organ contraction, biochemical and behavioural studies.
We will present the results obtained to date with these animals.
Acknowledgments: We thank the personnel of the animal house of the ULL for the maintenance
of the mouse strains. JDM and BB have CONSOLIDER contracts (CSD2008-00005). MRP is
contracted by the Canary Agency for Research, Innovation and Society of Information (ACIISI).
DP and YGM are recipients of FPI fellowship from the Spanish Ministry of Science and
Innovation (MICINN). Supported by MICINN Grant BFU2007-64963 (RB) and (ACIISI/FEDER)
PI 2007/017 (JDM).





P.45
On the calcium entry pathway induced by Aβ1-42 oligomers in Alzheimer´s disease
Erica Caballero, Sara Sanz-Blasco, Carlos Villalobos, Lucía Núñez
Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid y Consejo
Superior de Investigaciones Científicas (CSIC).Valladolid, Spain
Alzheimer's disease (AD) is characterized by a dramatic cognitive decline due to neuronal
dysfunction and death associated to amyloid plaques and neurofibrillary tangles. Previous
evidence from our lab (Sanz-Blasco et al. 2008) and others suggest that Aβ may promote Ca2+
influx and cell death but the responsible Ca2+ entry pathway remains controversial. We found
that Aβ oligomers, but not fibrils, induce a massive entry of Ca2+ into primary neurons. This
entry promotes mitochondrial Ca2+, apoptosis and cell death. We studied the effects of different
Ca2+ channel antagonist on calcium entry induced by Aβ. We found that Ca2+ entry induced by
Aβ was prevented partially by NMDA receptor antagonist MK-801, the amyloid channel inhibitor
NA7 and ω-agatoxin, a specific blocker of voltage-gated Ca2+ channel (VOCC) type P but not
nifedipine, a blocker of type L VOCCs, suggesting the Ca2+ influx takes place through different
pathways including those recruited by neuronal networking activity. The effects of Aβ were also
prevented by tetrodotoxin. Accordingly, we asked whether synaptic activity is required for Aβ
induced entry of Ca2+. We tested the effects of NMDA and Aβ in neuronal cultures before (1
DIV) and after (7 DIV) neuronal networking activity. We found that responses to oligomers, but
not those to NMDA, were largely reduced at 1 DIV relative to those at 7 DIV. In addition, the
effects of Aβ were prevented yet partially by MK-801 and NA7 but not by P/Q type channel
antagonist or tetrodotoxin. We tested also the effects of Aβ and NMDA on a series of cell lines
expressing or lacking NMDA receptor. Our results suggest that Ca2+ influx induced by Aβ is
started likely by formation of amyloid channels, a step that seemingly requires the functional
expression of NMDA receptor. This Ca2+ entry promoting activity is furtherly enlarged by
recruiting
This work has been funded by ISCIII (PI07/0766), MCIN (BFU2009-08967) and JCyL (VA-
088/A06).




P.46
A Missense Mutation in CALHM1 found in an Early Onset Alzheimer's Disease Patient
Alters Calcium Homeostasis and APP Processing
N. Setó-Salvia1,2, F. Rubio-Moscardo3, M. Pera1, C. Plata3, G. González-Gené3, O. Dols-Icardo1,
A. Frank4,5, M.J. Bullido5,6, M. Boada7, P. Pastor5,8, L. Samaranch5,8, M. Baquero9, L. Tàrraga7,
P. Martínez-Lage7, J. Masdeu5,8, J.L. Molinuevo10, J. Pérez-Tur5,11, R. Sánchez-Valle10, F.
Valdivieso5,6, T. Gómez-Isla1,12, R. Blesa1,5, F.J. Muñoz3, A. Lleó1,5, M.A. Valverde3, J.
Clarimón1,5
1Neurology Department, Hospital de Sant Pau; 2Departament d'Autoecologia Humana, Universitat Rovira i Virgili – IPHES; 3Department of Experimental and Health Sciences, University Pompeu Fabra; 4Neurology Department, Hospital Universitario La Paz, Barcelona; 5Center for Networked Biomedical Research into Neurodegenerative Diseases (CIBERNED); 6Centro de Biología Molecular Severo Ochoa, Madrid; 7Alzheimer Research Center, Memory Clinic. Fundació ACE, Barcelona; 8Neurogenetics Laboratory, Division of Neurociences, Center for Applied Medical Research (CIMA) University of Navarra Medical School, Pamplona; 9Neurology Department, Hospital Universitario "La Fe", Valencia; 10Neurology Department, Hospital Clinic Barcelona; 11Institut de Biomedicina de Valencia-CSIC, Barcelona, Spain; 12Massachusetts General Hospital, Harvard Medical School, MassGeneral Institute for
Neurodegenerative Disease, Boston, MA, USA
Over the last 15 years, many different studies have supported the hypothesis that Ca2+
homeostasis dysregulation may play an important role in the pathogenesis of Alzheimer's
disease (AD). Recently, a nonsynonimous genetic polymorphism (p.P86L) of the Ca2+
homeostasis modulator gene, CALHM1, has been shown to alter plasma membrane Ca2+
permeability, increase amyloid-β peptide (Aβ) levels and the risk of late-onset AD in several
population studies, but not in others. In the present report, we sequenced all CALHM1 coding
regions in a well-characterized cohort of 98 early onset AD (EOAD) patients without mutations
in the amyloid precursor protein (APP) and presenilin genes. We found a novel mutation
(p.G330D) in an EOAD patient, that was absent in 637 healthy controls. We also found that this
mutation had profound effects on calcium homeostasis and APP processing. The mutation
significantly reduced Ca2+ permeability, both at the plasma membrane and endoplasmic
reticulum, and increased extracellular Aβ levels by enhancing the b-secretase cleavage of APP.
The p.P86L polymorphism had intermediate effects between the wild-type and the p.G330D-
mutated protein. Our data indicate that rare genetic variants in CALHM1 may result in Ca2+
dysregulation and enhanced amyloidogenic processing of APP. We propose that theses
disturbances may be relevant pathophysiological processes in EOAD.



P.47
Gain of Function in Ito Secondary to Genetic Variations in SCN1B, SCN3B and KCNE1
Underlie Brugada Syndrome Phenotype
Janire Urrutia, Aitor Martínez, Dan Hu, Hiart Alonso, Aintzane Alday, Mónica Gallego, Hector
Barajas-Martinez, Charles Antzelevitch, Oscar Casis
Universidad del País Vasco, Department of Physiology, Leioa, Spain; Molecular Genetics
Laboratory, Masonic Medical Research Laboratory, Utica, New York, USA.
Brugada Syndrome (BrS) is an inherited channelopathy associated with life-threatening
ventricular arrhythmias. Recent studies have identified variations in accessory subunits of
sodium and potassium channels associated with BrS. These variants in SCN1B, SCN3B and
KCNE1 however were found to produce changes in sodium and potassium channel currents
insufficient to explain the development of a BrS phenotype. Hypothesis: This study examines
the hypothesis that mutations or rare polymorphisms in these sodium and potassium accessory
subunit genes lead to a gain of function in Ito contributing to BrS manifestation. Methods: Wild-
type and rare variants in these Brugada-susceptibility genes were co-expressed with human
KCND3 in HEK293 cells and studied using patch-clamp and co-immunoprecipitation (Co-IP)
techniques. Results: Functional studies expressing these subunits with KCND3 showed robust
macroscopic Ito. Co-expression of SCN1Bb-WT or SCN3B-WT with KCND3 caused a 1.5-fold
increase in Ito. The SCN1B-R214Q and SCN3B-L10P mutations increased Ito 3-fold compared
to KCND3 alone. Co-expression of KCNE1-WT with KCND3-WT increased Ito by 1.3-fold. The
polymorphism KCNE1-D85N produced a 4.5-fold gain-of-function compared to KCND3 alone.
Co-IP results showed a structural interaction of KCND3-WT with SCN1B-WT, SCN1B-R214Q,
SCN3B-WT, SCN3B-L10P, KCNE1-WT and KCNE1-D85N in HEK293 cells. Conclusions: Our
results, for the first time, support the hypothesis that accessory subunits presumed to associate
exclusively with sodium and slowly activating potassium channels can associate with Ito
channels and that mutations and rare SNPs in these accessory subunits augment Ito and thus
can be responsible for the manifestation of a BrS phenotype.



P.49
A channelopathy responsible for sudden death in the young in a large family from the
Canary Islands
Cristina Bosch Calero, Guillermo J. Pérez, Fabiana S. Scornik, Yijun Tang, S. R. Wayne Chen,
Paola Berne, Josep Brugada, and Ramon Brugada
Cardiovascular Genetics Center. IdIBGi-UdG, Girona.
We have identified a family originating from the south of Gran Canaria island with over 1000
members with a history of a rare fatal disease. We have partially reconstructed the pedigree of
this family and its origins date back to the eighteen hundreds. Individuals of this large family
suffer from sudden cardiac death at early ages of life (32 patients with sudden death at young
age 17±8 years). This family presents an arrhythmogenic syndrome characterized by the
development of adrenergically mediated ventricular tachycardia in young individuals with an
apparently normal heart. This type of syndrome is known as Catecholaminergic Polymorphic
Ventricular Tachycardia (CPVT), and can be the manifestation of a cardiac ion channel genetic
defect. We analyzed biological samples from some of the deceased members and living
relatives with a history of syncope. We identified a mutation in the gene that codifies for the
cardiac type ryanodine receptor (RyR2), with autosomal dominant transmission, as a likely
cause of sudden death in the family. Our sequencing analysis revealed a nucleotide change in
the position 1069 G>A of the RyR2 gene corresponding to the amino acid change G357S in the
protein sequence. We have developed an experimental cell line model to establish the
mechanistic molecular basis of the disease. The study of this extended family will help to
determine possible correlations between genotype and phenotype including identification of
possible modulatory factors. This will lead us to identify markers of risk and / or response to
treatment of this lethal channelopathy.
LAST NAME
FIRST NAME EMAIL
UNIVERSITY/
INSTITUTION
[email protected] Weill Cornell Medical College Universitat Jaume I [email protected] University of British Columbia [email protected] Universitat de Girona Universitat Jaume I [email protected] Universidad de la Laguna [email protected] Institute of Animal Physiology, Justus-Liebig University of Giessen Alvarez de la Rosa [email protected] Universidad de la Laguna University of Pittsburgh [email protected] Universidad Complutense de Madrid [email protected] Universidad del País Vasco Bahamonde Santos Maria Isabel [email protected] Institute for Bioengineering of Catalonia (IBEC) Universidad Complutense de Madrid [email protected] Hospital Ramón y Cajal IRYCIS [email protected] Universidad de Oviedo Universidad de La Laguna [email protected] Universitat de Girona Universidad de La Laguna [email protected] Universitat de Girona Universidad Austral de Chile Brugada Terradellas Ramon [email protected] Universidad de Girona [email protected] Nanion Technologies GmbH Caballero Collado [email protected] Universidad Complutense de Madrid Caballero Ortega [email protected] Universidad de La Laguna [email protected] Universidad de Vigo Calderón-Sánchez IBiS-Hospital Virgen del Rocío [email protected] Universitat de Barcelona Univ. Extremadura [email protected] [email protected] [email protected] Universidad del País Vasco IBIS- Universidad de Sevilla University of Wisconsin-Madison Universitat de Barcelona Justus-Liebig-University Giessen [email protected] Hospital Universitario Ntra [email protected] Universitat de Girona [email protected] Universitat de Barcelona Neuroscience Institute [email protected] University of Medicine and Dentistry of New Jersey [email protected] Universidad Miguel Hernández-CSIC [email protected] Universidad de La Laguna, Tenerife, Spain De la Peña Cortines Mª Pilar [email protected] Universidad de OVIEDO Hydra Biosciences [email protected] Universidad Complutense/CSIC [email protected] Universidad Complutense de Madrid Universidad de La Laguna [email protected] [email protected] Universidad Complutense de Madrid. [email protected] Universidad de Oviedo Domínguez Reyes [email protected] Universidad de La Laguna [email protected] Instituto de Biomedicina de Sevilla Hospital Universitario Virgen del Rocio University of barcelona Estévez Herrera Universitaat de Barcelona Centro de Investigacion Principe Felipe [email protected] IES SAN VICENTE/ UA [email protected] University of Vigo Fernández Fernandez_Ballester Gregorio Miguel Hernandez Fernández-Carvajal Asia [email protected] Universidad Miguel Hernández Fernandez-Chacon IBISInstituto de Biomedicina de Sevilla (IBIS) Universitat Pompeu Fabra Fernández Fernández-Mariño Universitat Pompeu Fabra Universidad Miguel Hernández, Elche, Spain Miguel Hernandez University [email protected] [email protected] Universidad de Valladolid [email protected] Hospital Vall d'Hebron Universitat Pompeu Fabra Universitat de Barcelona [email protected] Universidad de Zaragoza [email protected] Hospital Universitario Ntra Sra Candelaria Universidad de La Laguna Universidad Complutense [email protected] Universidad Complutense Universidad Miguel Hernández-CSIC [email protected] Universidad Complutense Fuente Gonzalez Gené [email protected] Universidad de La Laguna. Departamente de Bioquímica y Biología Molecular. Instituto Universitario de Tecnologías Biomédicas. González Muñiz [email protected]. Instituto de Química Médica es UNIVERSIDAD POLITECNICA DE MADRID [email protected] La Laguna University González-Rodríguez Patricia [email protected] Universidad de Sevilla-IBiS [email protected] IBMC, Universidad Miguel Hernández [email protected] Unidad de Biofisica (CSIC-UPV/EHU) Hernández-Díaz [email protected] Universidad de La Laguna Justus-Liebig University of [email protected] Jorquera Martínez- [email protected] Universidad Miguel [email protected] Institute of Animal Physiology, Justus-Liebig University of Giessen [email protected] Nanion Technologies GmbH José Antonio [email protected] University of Vigo [email protected] UMH-CSIC (Instituto de Neurociencias López Hernández [email protected] Universidad de Barcelona [email protected] [email protected] U. of Valladolid [email protected] Instituto de Neurociencias de Alicante-CSIC [email protected] Universidad de Castilla-La Mancha [email protected] Oviedo/Departamento de Bioquimica y Biología Molecular Macias Martínez [email protected] Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM) Jorge Nicolás [email protected] Universidad de La Laguna Martín Martínez [email protected] Instituto de Química Médica (CSIC) [email protected] University of Extremadura Universidad de La Laguna Martínez de la Cruz Luis Alfonso [email protected] Juan Enrique [email protected] Universidad de Alicante Messeguer Peypoch Angel [email protected] [email protected] [email protected] Universidad de La Laguna IBMC, Universidad Miguel Hernández [email protected] IBMC- Inst.Biologia Molecular e Celular, Porto, Portugal Universidad de La Laguna, Tenerife, Spain [email protected] [email protected] [email protected] [email protected] Miguel Hernandez University Centro Interd. De Neurociencia de Valparaiso [email protected] Weill Cornell Medical College [email protected] Universidad de Valladolid [email protected] Universitat de Girona Max-Planck-Institute of Experimental Medicine [email protected] Universitat de Girona Albert Einstein College of Medicine [email protected] Universidad de La Laguna [email protected] Universitat de Girona [email protected] Instituto de Química Médica (CSIC) [email protected] F.C.V. Centro de Investigación Príncipe Felipe [email protected] Universitat de Girona [email protected] U. of Valladolid [email protected] Centro de Investigación Príncipe Felipe Plata Fernández [email protected] [email protected] University of Extremadura University of Colorado Instituto de Biología Molecular y Celular, Universidad Miguel Hernández Rendón Ramírez Adela Leonor [email protected] Unidad de Biofísica UPV-EHU [email protected] Universitat de Girona Rodríguez Artalejo Universidad Complutense de Madrid Rodríguez Moyano [email protected] IBIS Hospital Virgen del Rocío Rodríguez Pardo [email protected] Universidad de La Laguna, Tenerife, Spain Universidad de La Laguna Departamento de Bioquímica y Biología Molecular / Instituto Universitario de Tecnologías Biomédicas Universidad de Santiago de Chile [email protected] Temple University School of Medicine [email protected] University of Extremadura [email protected] Universidad Complutense Facultad de Farmacia [email protected] Universitat de Girona [email protected] Universitat de Girona [email protected] University of Texas Health Science Center, San Antonio Universidad de La Laguna, Tenerife, Spain Instituto de Biomedicina de Sevilla, IBiS/ Hospital Universitario Virgen del Rocio [email protected] Universitat de Barcelona U. Barcelona - IDIBELL [email protected] Medical College of Wisconsin Max-Planck-Institute of Experimental Medicine [email protected] [email protected] [email protected] University of Pittsburgh [email protected] Universidad Complutense de Madrid Universidad del Pais Vasco/Euskal Herriko Unibertsitatea FONDAZIONE ISTITUTO ITALIANO DI TECNOLOGIA [email protected] [email protected] Autonomous University of [email protected] Universitat Jaume I [email protected] Instituto de Neurociencias de Alicante [email protected] Villalobos Jorge [email protected] Universidad del País Vasco Vinals Izquierdo Maria Teresa [email protected] [email protected] [email protected] CIMA, Universidad de Navarra University of Waschington

Source: http://sici.umh.es/Cursos/RECI3/Abstract_RECI3.pdf