a TR P to Spain

International Workshop on Transient Receptor Potential Channels

12th – 14th September 2012 Valencia, Spain

www.trp2012.com

Schedule and Abstracts book

September 2012

Dear participants,

Travelling to faraway places in search of spiritual or cultural enlightenment is a millennium old human activity. In their travels, pilgrims brought with them news, foods, music and traditions from distant lands. This friendly exchange led to the cultural enrichment of visitors and the economic flourishing of places, now iconic, such as Rome, Santiago, Jerusalem, Mecca, Varanasi or Angkor Thom. The dissemination of science and technology also benefited greatly from these travels to remote locations.

The new pilgrims of the Transient Receptor Potential (TRP) community are also very fond of travelling. In the past years they have gathered at various locations around the globe: Breckenridge (USA), Eilat (Israel), Stockholm (Sweden) and Leuven (Belgium) come to mind. These meetings, each different and exciting, have been very important for the dissemination of TRP research.

We are happy to welcome you in Valencia (Spain) for TRP2012. The response to our call has been extraordinary, surpassing all our expectations. The speakers, the modern bards, readily attended our request to communicate their new results. At last count we were already more than 170 participants, many of them students, and most presenting their recent work in the form of posters or short oral presentations. At least 25 countries are sending TRP ambassadors to Valencia, making this a truly international meeting.

We like to thank the staff of the Cátedra Santiago Grisolía, Fundación Ciudad de las Artes y las Ciencias for their dedication and excellence in handling the administrative details of the workshop. We also like to thank all the institutional and private sponsors for their generous support. In the rather depressed economic climate that we happened to be immersed in, we know that their effort is specially valuable and significant. Thanks to them we were able to balance our budget and offer 30 travel fellowships.

Finally, we thank you all for coming and wish that you will find your stay in Valencia fruitful and enriching, not only in the academic but also at the personal level. We are firmly convinced that the field of TRP channels will continue to expand, with new and exciting discoveries, and we hope to learn about them from all of you in future meetings.

Welcome to Valencia and we wish you have a very pleasant stay,

The Scientific Committee TRP2012

International Workshop on Transient Receptor Potential (TRP) Channels i

ii International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation

International Workshop on Transient Receptor Potential (TRP) Channels

September 12 th - 14 th , 2012 Valencia, Spain

Scientific Committee

Antonio Ferrer-Montiel Universidad Miguel Hernández, Elche, Spain.

Sven-Eric Jordt Yale University, New Haven, USA.

Rosa Planells-Cases Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany.

Félix Viana Instituto de Neurociencias de Alicante, UMH-CSIC, Alicante, Spain.

Thomas Voets KULeuven, Leuven, Belgium.

TRP Workshop Secretariat

Cátedra Santiago Grisolía Fundación Ciudad de las Artes y las Ciencias – Comunitat Valenciana Telephone: 0034 96 197 4670 Fax: 0034 96 197 4598 E-mail: [email protected] Web: www.trp2012.com

Venue

Santiago Grisolía Auditorium Science Museum Príncipe Felipe City of Arts and Sciences Avinguda del Professor López Piñero (Historiador de la Medicina) nº 7 46013 - Valencia (Spain)

International Workshop on Transient Receptor Potential (TRP) Channels iii

iv International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation

Index

Schedule ...... 2

Lectures ...... 7

Opening ...... 8

Session 1 ...... 9

Session 2 ...... 15

Session 3 ...... 24

Session 4 ...... 31

Posters ...... 39

Alphabetical list of authors ...... 139

Alphabetical list of participants ...... 143

International Workshop on Transient Receptor Potential (TRP) Channels Schedule

Wednesday 12 th , September 2012

17.30 - 19.00 Registration

19.00 - 19.15 Opening

19.15 - 20.15 . Dept. of Physiology, University of California, San Francisco, CA, USA. Probing the structural basis of TRP channel thermo- and chemo- sensitivity

20.15 Cocktail

Thursday 13 th , September 2012

Session 1

Chair: Aziz Moqrich. Dept. of Neurobiology, Institut of Developmental Biology Marseille, CNRS, Marseille, France.

09.00 - 09.40 Rachelle Gaudet. Dept. of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA. Structural determinants of TRPV channel activation and desensitization

09.40 - 10.20 Daniel Minor. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA. Structural insights into ion channel structure and modulation

10.20 - 11.00 Jorg Grandl. Ion Channel Research Unit & Dept. of Neurobiology, Duke University Medical Center, Durham, NC, USA. The temperature-activation mechanism of TRP channels

11.00 - 11.30 Coffee break and posters view

11.30 - 12.10 Stuart Bevan. School of Biomedical Sciences, King's College London, London, UK. TRP channel-mediated sensory transduction in normal and pathophysiological conditions

12.10 - 12.50 Michael J. Caterina. Dept. of Biological Chemistry and Dept. of Neuroscience, Center for Sensory Biology. The Johns Hopkins School of Medicine, Baltimore, MD, USA. TRP channels in the skin

12.50 - 13.30 Diana Bautista. Dept. of Molecular & Cell Biology, University of California, Berkeley, CA, USA. Roles of TRP channels in itch transduction

13.30 - 15.00 Lunch

2 International Symposium on Foundation Schedule

Session 2

Chair: Viktorie Vlachova. Dept. of Cellular Neurophysiology, Institute of Physiology AS CR, Prague, Czech Republic.

15.00 - 15.20 Indu S. Ambudkar. Molecular Physiology and Therapeutics Branch, NIDCR, NIH, Bethesda, MD, USA. Resolving the contributions of TRPC1 and Orai1 to regulation of salivary gland fluid secretion

15.20 - 15.40 Albert Gonzales. Vascular Physiology Research Group, Dept. of Biomedical Science, Colorado State University, Fort Collins, CO, USA. STIM1 is essential for the coupling of sarcoplasmic reticulum calcium stores to TRPM4 and BK Ca channel activity

15.40 - 16.40 Peter Zygmunt. Dept. of Laboratory Medicine, Clinical Chemistry & Pharmacology, Lund University, Lund, Sweden. hits the TRPs V1 and A1

16.40 - 17.20 Coffee break and posters view

17.20 - 17.40 Vera Y. Moisenkova-Bell 1 / Gregorio Fernández Ballester 2 1Dept. of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, USA. / 2Instituto de Biologia Molecular y Celular, Universidad Miguel Hernández, Elche, Alicante Spain. Structural insights into the dynamics of the TRPA1 activation mechanism

17.40 - 18.00 Víctor Meseguer. Instituto de Neurociencias de Alicante, Universidad Miguel Hernández y CSIC, Alicante, Spain. TRPA1 channels are neuronal sensors for bacterial endotoxins

18.00 - 18.20 Susan D. Brain. Vascular Biology Group and Centre for Integrative Biomedicine; Cardiovascular Division, King’s College London, London, UK. TRPA1: A link between pain exacerbation in the arthritic joint in cold environments

18.20 - 18.40 Debapriya Ghosh. Laboratory of Ion Channel Research, Dept. of Molecular Cell Biology, Katholieke Universiteit Leuven, Leuven, Belgium. Characterization of the trafficking of human transient receptor potential melastatin 8 (hTRPM8) channel by total internal reflection fluorescence (TIRF) microscopy

18.40 - 19.00 Xuming Zhang. Dept. of Pharmacology, University of Cambridge, Cambridge, UK. Modulation of the cold-activated TRPM8 channel: The role of Gq protein and PIP2

19.00 - 19.20 Rosa Señarís. Dept. of Physiology (CIMUS), Universidad de Santiago de Compostela, Spain. TRPM8-deficient mice develop obesity and metabolic syndrome. Role of TRPM8 in the regulation of energy homeostasis

19.20 - 19.25 Announcements

International Workshop on Transient Receptor Potential (TRP) Channels 3 Schedule

Friday 14 th , September 2012

Session 3

Chair: Scott Earley. Vascular Physiology Research Group, Dept. of Biomedical Science, Colorado State University, Fort Collins, CO, USA.

09.00 - 09.40 Miguel A. Valverde. Laboratory of Molecular Physiology and Channelopathies, Universitat Pompeu Fabra, Barcelona, Spain. TRPV4 regulation and population genetics

09.40 - 10.20 Maria G. Belvisi. Imperial College London, London, UK. TRP channels and sensory reflexes in the lung

10.20 - 11.00 Antti Pertovaara. Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland. TRPA1 in peripheral diabetic neuropathy

11.00 - 11.30 Coffee break and posters view

11.30 - 12.10 René J.M. Bindels. Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. New insights in the regulation of mineral TRP's

12.10 - 12.50 Thomas Gudermann. Walther-Straub-Institut of Pharmacology and Toxicology, University of Munich, Munich, Germany. A TR(i)P to magnesium homeostasis

12.50 - 13.30 Haoxing Xu. Dept. of Molecular, Cellular, and Developmental Biology, University of Michigan , Ann Arbor, MI, USA. Mucolipin TRP channels in the lysosome: opening the gate to the cell's recycling center

13.30 - 15.00 Lunch

Session 4

Chair: Heather Bradshaw . Dept. of Psychological and Brain Sciences at Indiana University, Bloomington, IN, USA.

15.00 - 15.40 X. Z. Shawn Xu. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA. TRP channels in C. elegans sensory transduction

15.40 - 16.40 Craig Montell. Dept. of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA. TRP channels: From animal behavior to neurodegenerative disease

16.40 - 17.20 Coffee break and posters view

17.20 - 18.00 Roger Hardie. Dept. of Physiology Development and Neuroscience, Cambridge University, UK. TRP channels in Drosophila phototransduction.

4 International Symposium on Foundation Schedule

18.00 - 18.20 Baruch Minke. Dept. of Medical Neurobiology, Faculty of Medicine, Hebrew University, Jerusalem, Israel. Signal dependent hydrolysis of PI(4,5)P2 without activation of phospholipase C: implications on the gating of the Drosophila TRPL channel

18.20 - 18.40 Bimal N. Desai. Dept. of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA, USA. TRPM7 channel - turned on by cleavage

18.40 - 19.00 Johannes Oberwinkler. Institut fur Physiologie und Pathophysiologie, Philipps-Universitat Marburg, Hessen, Germany. Regulation of TRPM3 activity in pancreatic β cells by intracellular signaling cascades

19.00 - 19.20 Lisa Alenmyr . Clinical Chemistry & Pharmacology, Dept. of Laboratory Medicine, Lund University, Lund, Sweden. TRPV4 regulates calcium influx and ciliary beat frequency activity in human airway ciliated epithelial cells

19.20 - 19.25 Announcements

21.00 Farewell dinner

International Workshop on Transient Receptor Potential (TRP) Channels 5 Schedule

6 International Symposium on Foundation

Lectures

International Workshop on Transient Receptor Potential (TRP) Channels Opening

Probing the structural basis of TRP channel thermo- and chemo-sensitivity

Julio Cordero-Morales, Erhu Cao, Elena Gracheva and David Julius Department of Physiology, University of California, San Francisco, San Francisco, CA 94158-2610 USA

TRP channels of the somatosensory system, including TRPV1, TRPA1, and TRPM8, are among the best-characterized members of the vertebrate TRP channel family. Their widely validated roles in pain physiology, together with the availability of potent and selective pharmacological agents (natural and synthetic) make each a ‘poster child’ for elucidating basic principles underlying TRP channel pharmacology, structure, and regulation.

In addition to these experimental advantages, the analysis of channel orthologues has helped to identify protein domains that track with species-specific functional properties, illustrating the power of evolutionary genetics in the study of TRP channel structure and function. Among somatosensory TRP channels, this strategy has been applied most intensively to the study of the receptor (TRPV1) and the receptor (TRPA1). These channels show robust sensitivity to thermal stimuli and/or chemical irritants in a species-specific manner, facilitating the mapping of domains that account for these functional attributes.

Findings will be presented in which structure-function, biophysical, and biochemical experiments are used to delineate TRPV1 and TRPA1 domains that are required for the detection of chemical or thermal stimuli, or which tune channel sensitivity in a physiologically relevant manner. Emphasis will be placed on regions of the presumptive cytoplasmic N- and C-termini, which our data implicate as being particularly important in these mechanisms.

8 International Foundation Session 1

Structural determinants of TRPV channel activation and desensitization

Rachelle Gaudet Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA

My lab is broadly interested in the mechanisms of signaling and transport across cellular membranes. Much of our research centers on TRP channels and their role in sensory perception. We focus on temperature-sensitive ion channels, particularly TRPV1 and TRPA1. TRP channels are challenging structural biology targets because they are large multidomain eukaryotic membrane proteins and are not naturally abundant. We take complementary approaches to obtain structural and functional information on TRP channels. One strategy is to divide and conquer: determine crystal structures of isolated domains of TRP channels. The results can then combined with genetic, biochemical and physiological data to advance our understanding of TRP channel function. It also provides valuable insights as we tackle the structure determination of whole TRP channels.

TRPV channels play key roles in pain, thermo- and mechanosensation, and calcium homeostasis. The N-terminus of TRPV channels contains six ankyrin repeats, short sequence motifs often involved in protein-ligand interactions. We determined structures of several TRPV ankyrin repeat domains (ARDs). The isolated ARDs do not oligomerize, suggesting that they interact with regulatory factors instead. The accumulated data from our lab provide information about how regulatory molecules like ATP and calmodulin interact with the ARD and alter the sensitivity of TRPV1 and other TRPV channels. More recently, we have performed biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases, suggesting molecular mechanisms for the diseases. We are now expanding these studies to the ankyrin repeats of other TRP channels.

International Workshop on Transient Receptor Potential (TRP) Channels 9 Session 1

Structural insights into ion channel structure and modulation

Daniel Minor Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA

Ion channels are fundamental components of biological electrical signaling networks. These proteins control the passage of ions across the cell membrane and generate the bioelectricity that is essential for life. Much of the future of neuroscience, cardiovascular research, and design of biomimetic membrane systems lies in understanding the molecular details of how these protein machines function, how they are regulated, and how they are integrated into large, macromolecular complexes within the cell. My laboratory seeks to uncover the basic mechanisms by which ion channels act. We use a multidisciplinary approach that includes genetic selections, X- ray crystallography, solution biophysical methods, small molecule screening, and electrophysiology. We are interested in understanding the high-resolution structures of channel proteins, their regulatory factors, and the conformational changes that accompany channel action as well as in the development novel means to manipulate channel action in vivo. Recent progress from the laboratory's efforts in these areas, particularly with respect to temperature gating of ion channels, will be presented.

10 Session 1

The temperature-activation mechanism of TRP channels

Jorg Grandl , Ph.D. Ion Channel Research Unit & Department of Neurobiology, Duke University Medical Center, Durham, USA

ThermoTRPs are multimodal receptors that are activated by temperatures with distinct thresholds and a variety of physically distinct stimuli such as chemicals, voltage and pH. Whereas the mechanisms of ion channel gating by chemicals and voltage are understood in principal, the logic of temperature-activation remains unknown. An important first step towards our understanding of how temperature activates ion channels mechanistically would be the identification of structures (residues/domains) that are specifically involved in temperature-activation. Using unbiased random mutagenesis screens we identified single point-mutations in TRPV3 and TRPV1 that specifically alter temperature-sensitivity, without affecting activation by chemicals. The identified mutations are not randomly distributed throughout the protein, but cluster in the pore domain, suggesting a mechanistic involvement of the pore domain in temperature-activation. However, these results are not strict proof that during temperature-activation conformational changes occur within the pore domain: for example, point mutations might act allosterically on structures outside the pore and thus affect the temperature-gating transition. Therefore, we used targeted mutagenesis of single amino-acids or larger domains within the pore domain to investigate structural changes during the temperature-gating. Our data collectively suggest that the outer pore domains of TRPV3 and TRPV1 undergo conformational changes upon temperature-activation.

International Workshop on Transient Receptor Potential (TRP) Channels 11 Session 1

TRP channel-mediated sensory transduction in normal and pathophysiological conditions

Stuart Bevan Wolfson Centre for Age Related Diseases, King’s College London, London, UK

Studies over the last 15 years have revealed the important roles that TRP channels play in transduction mechanisms in peripheral sensory neurons. Data from many laboratories have shown that TRP channels act as sensors and transducers for stimulation by noxious heat, warm and cold termperatures. Furthermore, modifications in TRP channel properties are thought to be, at least partly, responsible for hypersensitivities to thermal stimuli in condition such as inflammation. The key roles of TRP channels in sensory transduction have prompted activities to develop selective channel antagonists as analgesic drugs. To date these activities have not yielded any clinically used drugs. However, our recent collaborative studies have revealed that the analgesic activity of a commonly used analgesic/anti-pyretic drug, acetaminophen, exerts its action by an interaction of key metabolites with TRPA1 and TRPV1. We have also discovered that the hypothermic effects of acetaminophen is absent in mice lacking functional TRPA1 channels; however hypothermia evoked by a range of other agents such as ethanol and receptor agonists is similar in wild-type and TRPA1-deficient mice. Acetaminophen evoked hypothermia is also present in mice lacking either TRPV1 or TRPM8, ruling out a significant role of these temperature sensitive channels in the hypothermic actions of this anti-pyretic drug. Our studies have also revealed a role for TRPA1 in animal models of painful diabetic neuropathy. The diabetogenic agent, streptozotocin, evokes a rapidly appearing acute phase of painful mechanical hypersensitivity which is inhibited by prior administration of a TRPA1 antagonist, AP18, and is abolished in TRPA-deficient mice. Furthermore TRPA1 acts as a sensor for several endogenous chemicals produced during diabetes. These include reactive oxygen species and (MG). MG acts as a TRPA1 agonist and intraplantar injection of MG elicits an early phase of pain behaviours that are absent in TRPA1-deficient mice. In addition, we studied the effects of long term administration (up to 2 weeks) of an inhibitor of glyoxylase 1, which is responsible for degrading/detoxifying endogenously produced MG. This treatment produced time dependent heat, cold and mechanical sensitivities in wild-type mice that were not found in TRPA1 knockout mice. These data suggest that TRPA1 has an important role in the development of painful hypersensitivities in diabetes.

12 Session 1

TRP channels in the skin

Michael J. Caterina Department of Biological Chemistry and Department of Neuroscience, Center for Sensory Biology. The Johns Hopkins School of Medicine, Baltimore, MD, USA

Keratinocytes are epithelial cells that constitute the stratified epidermis of the skin. These cells are best recognized for their contributions to the formation of the cutaneous barrier. However, there is increasing evidence that these cells interact dynamically with the external environment and with numerous other cell types in the skin. Among the latter are sensory neurons of the peripheral nervous system. Although many of the properties of these neurons can be recapitulated by culturing dissociated sensory ganglia, the intimate apposition of these neurons to skin keratinocytes and other skin structures raises the possibility that such nonneuronal elements actively communicate with neurons to shape our sensory experience. Further support for this notion has come from the observation that keratinocytes express many ion channel proteins, including members of the transient receptor potential (TRP) and voltage-gated sodium channel families, which have the capacity to recognize and respond to physical and chemical stimulation. Yet, direct evidence for acute keratinocyte to neuron communication in vivo has been lacking. We have utilized knockout and transgenic mouse approaches to interrogate keratinocyte communication with cutaneous sensory neurons.

International Workshop on Transient Receptor Potential (TRP) Channels 13 Session 1

Roles of TRP channels in itch transduction

Diana Bautista Department of Molecular & Cell Biology, University of California, Berkeley, CA, USA

Pruritus, or itch, is associated with many inflammatory conditions including insect bites, atopic dermatitis and psoriasis. Acute itch serves an important protective function by warning against harmful agents in the environment such as insects, toxic plants or other irritants. In contrast, pruritus can also be a debilitating condition that accompanies numerous skin, systemic, and nervous system disorders. Approximately 5–20% of primary afferent C-fibers are activated by endogenous itch-producing compounds released by non-neuronal cells in the skin (Ikoma et. al., 2006; Davidson & Giesler, 2010). While many itch pathways involve histamine signaling, there are clearly other key neural pathways as most pathophysiological itch conditions are insensitive to antihistamine treatment and novel therapeutic targets have yet to be identified (Ikoma et. al., 2006; Davidson & Giesler, 2010). Members of the Mas-Related G Protein coupled Receptor (Mrgpr) family have emerged as key mediators of histamine- independent itch. Mast cells secrete a variety of peptides that directly activate MrgprC11 to evoke itch. Likewise, MrgprA3 is activated by the antimalaria drug, chloroquine, which causes antihistamine-insensitive intolerable itch. We now show that MrgprA3 and MrgprC11 couple to TRPA1 in heterologous systems and sensory neurons, providing a molecular mechanism of Mrgpr-evoked excitation. TRPA1- deficient neurons display clear signaling deficits in response to chloroquine and the MrgprC11 pruritogen, BAM 8-22. Indeed, our data definitively show that TRPA1 is the primary contributor to MrgprA3 and MrgprC11- evoked itch, as animals lacking this channel show a huge reduction in chloroquine and BAM 8-22 sensitive neurons. This is also matched by a profound alteration in behavior, such that TRPA1-deficient mice display no scratching following chloroquine or BAM 8- 22 injection. Likewise, we also show that TRPA1 is essential for itch-evoked scratching behaviors in an experimental dry-skin mouse model of chronic itch. Thus TRPA1 may define a new signaling pathway that mediates histamine-independent, chronic itch.

14 Session 2

Resolving the contributions of TRPC1 and Orai1 to regulation of salivary gland fluid secretion

Indu S. Ambudkar Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, NIH, Bethesda MD 20892

Agonist stimulation of cells leads to the generation of intracellular Ca 2+ signals that are decoded for the regulation of various cellular functions, including salivary gland fluid secretion and Ca 2+ -dependent gene expression. Two STIM1-regulated Ca 2+ entry channels, Orai1 and TRPC1, are activated in response to agonist-induced depletion of 2+ 2+ salivary gland cells and contribute to sustained [Ca ]i elevation by mediating Ca influx into cells. We have previously reported that TRPC1 is a critical component of SOCE in these cells and that salivary acinar cells from TRPC1-/- mice display reduced SOCE which accounts for loss of sustained K Ca activation and, consequently, salivary fluid secretion in the animals. While Orai1 controls the activation of TRPC1 in response to store depletion, the individual Ca 2+ signals generated by the two channels and their impact on cell function is not yet known. We have studied the individual contributions of TRPC1 and Orai1 to agonist-stimulated cytosolic calcium signals and salivary gland function by using mice that lack TRPC1 or those in which Orai1 expression was suppressed by in vivo knockdown of the channel (by delivery Adeno-CRE vector to the salivary glands from Orai1 fl/fl ) mice. Together, our findings suggest that Orai1 and TRPC1 mediate distinct local and global Ca 2+ signals that determine the channel specificity in the regulation of cell function. These recent studies will be discussed.

International Workshop on Transient Receptor Potential (TRP) Channels 15 Session 2

STIM1 is essential for the coupling of sarcoplasmic reticulum calcium stores to TRPM4 and BK Ca channel activity

Albert L. Gonzales and Scott Earley Vascular Physiology Research Group, Department of Biomedical Science, Colorado State University, Fort Collins, CO 80523 USA

In cerebral arterial myocytes, pharmacologically distinct Ca 2+ release events from the 2+ + sarcoplasmic reticulum (SR) activate large-conductance Ca -activated K (BK Ca ) channels and melastatin transient receptor potential 4 (TRPM4) channels located on the plasma membrane. The stromal-interacting molecule, STIM1, is a single transmembrane protein consisting of a luminal Ca 2+ -sensing EF-hand and cytosolic interacting domains, and is localized in the SR membrane juxtaposed to the plasma membrane. To investigate the role of STIM1 in regulating Ca 2+ -activated ion channels, we used RNAi-mediated protein suppression and STIM1- interaction inhibitory peptides in rat cerebral artery smooth muscle cells. STIM1 siRNA treatment decreased STIM1 but not TRPM4 or BK Ca protein expression. Using patch clamp electrophysiology, we 2+ found that Ca -dependent TRPM4 and BK Ca channel activity was diminished following STIM1 siRNA treatment. Additionally, STIM1 inhibitory peptides targeting the STIM1- STIM1 or STIM1-ORAI1 interacting domains decreased TRPM4 channel activity. To investigate SR Ca 2+ store load, we examined cyclopiazonic acid-induced Ca 2+ release, and saw no difference between treatments. Using membrane specific fluorescent staining of the SR and plasma membranes, we found that SR membrane architecture and coupling with the plasma membrane was disrupted following our STIM1 siRNA or inhibitory peptide treatments. Thus, following down-regulation or inhibition of STIM1; SR Ca 2+ stores were maintained; SR and plasma membrane coupling was disrupted; and Ca 2+ -dependent activation of plasma membrane ion channels were lost. This is the 2+ first evidence of a novel role for STIM1 in physically coupling SR Ca stores with BK Ca and TRPM4 channel activity in smooth muscle cells. RO1HL091905 (SE); F31HL094145-01 (AG).

16 Session 2

Paracetamol hits the TRPs V1 and A1

Peter M. Zygmunt & Edward D. Högestätt Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, Lund, Sweden.

Although paracetamol (acetaminophen) is one of the most consumed over-the-counter analgesic and antipyretic agents, its mechanism of action is unclear. The metabolism and action of paracetamol is complex and possibly dependent on the dose and route of administration.

We have found that the paracetamol metabolite p-aminophenol is metabolised by fatty acid amide hydrolase (FAAH) to the TRPV1 activator N-arachidonoylphenolamine (AM404) in the central nervous system. The antinociceptive effect of paracetamol is absent in FAAH and TRPV1 knockout mice and in wild type mice given the TRPV1 antagonist intracerebroventricularly, suggesting that activation of TRPV1 on descending inhibitory neurons in the brain are involved in the antinociceptive effect. In another study we have shown that paracetamol via some of its electrophilic metabolites is antinociceptive via activation of TRPA1 in the mouse spinal cord. However, electrophilic compounds are tissue damaging and not suitable as drugs. It is therefore of interest that intrathecal administration of 9-, a cannabinoid derivative without CB1/CB2 receptor agonistic properties, also produced spinal TRPA1-mediated antinociception.

Our studies show that paracetamol is antinociceptive via activation of TRPV1 and TRPA1. We provide a conceptual framework for the use of 1) drug molecules that are converted to bioactive N-acylamines acting on TRPV1 and other molecular targets involved in noxious signalling in the central nervous system, and 2) non-electrophilic TRPA1 activators to achieve analgesia at the level of the spinal cord.

International Workshop on Transient Receptor Potential (TRP) Channels 17 Session 2

Structural insights into the dynamics of the TRPA1 activation mechanism

Teresa L. Cvetkov 1, Kevin W. Huynh 1, Liwen Wang 2, Gregorio Fernandez-Ballester 3, Antonio Ferrer-Montiel 3, Mark R. Chance 2, Vera Y. Moiseenkova-Bell 1,2 1Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, USA 2Center for Proteomics, School of Medicine, Case Western Reserve University, Cleveland, USA 3Department of Biochemistry and Molecular Biology, University Miguel Hernandez , Elche, Spain

TRPA1 is a Ca 2+ -permeable, non-selective cation channel and one of the key pain sensors in mammals. Pain sensation mediated by TRPA1 involves modification of N- terminal cysteine residues on the channel by thiol-reactivecompounds and inflammatory mediators.Binding of thiol-reactive compounds to the channel in the resting state leads to channel activation, a state of high cation conductance, followed rapidly by desensitization, a low-conductance state averse to opening in response to additional agonists. Recent mutagenesis and chimeric studies have suggested that N- terminal cysteine residues (C622, C642, C666) located on the flexible linker region of the channel are involved in channel activation and desensitization, but how conformational changes in the flexible linker region lead to activation and desensitization is unresolved. Capitalizing on our ability to isolate pure and functional TRPA1 channel protein, we recently provided the first insight into the channel architecture by reconstructing a TRPA1 structure to16 Å resolution in resting state using electron microscopy (EM). Fitting TRPA1 homology model into the EM density lead us to hypothesizethat the critical cysteines on the flexible linker region (C622, C642, C666) are in close proximity to one another and that covalent modification of cysteineswithin this pocket could promote conformational changes leading to channel gating and desensitization.Furthermore, we performed a mass spectrometry analysis of the in vivo TRPA1 thiol status and discovered that, when treated with a thiol-reactive TRPA1 agonist,C622 and C666 could form a disulfide bond with each other or with two other cysteine residues. This disulfide rearrangementcould lead to channel activation and the rapid desensitization observed in electrophysiological studies. Our current structural and biophysical analyses using EM, mass spectrometry and homology modeling areelucidatingligand-induced conformational changes in the channel’s flexible linker region that occur during activation and desensitization.These insights bring us a greater understanding of the structural rearrangements involved in the gating and desensitization mechanisms of the TRPA1 ion channel.

18 Session 2

TRPA1 channels are neuronal sensors for bacterial endotoxins

Victor Meseguer 1, Yeranddy A. Alpizar 2, Otto Fajardo 1, Sendoa Tajada 3, Bristol Denlinger 1, Enoch Luis 1, Jan-Albert Manenschjin 1, Carlos Fernández Acuña 1, Arturo Talavera 5,2 , Tatiana Kichco 4, Peter Reeh 4, María Teresa Pérez-García 3, José Ramón López López 3, Thomas Voets 2, Carlos Belmonte 1, Karel Talavera 1,2 , Félix Viana 1 1Instituto de Neurociencias de Alicante, Universidad Miguel Hernández y CSIC, Alicante, Spain. 2Laboratory of Ion Channel Research, Department of Cell and Molecular Biology, KULeuven, Leuven, Belgium 3Departamento de Bioquímica y Biología Molecular y Fisiología e Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid y CSIC, Valladolid, Spain 4Department of Physiology and Pathophysiology, University of Erlangen-Nuremberg, Erlangen, Germany 5Vicepresidencia de Investigaciones. Instituto Finlay, La Habana

Many Gram-negative bacterial infections are accompanied by somatic or visceral pain and irritation. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells through activation of the Toll-like- receptor 4 (TLR4) signaling pathway by lipopolysaccharide (LPS), a toxic byproduct of bacterial lyses. Here we show that LPS exerts direct, powerful excitatory actions on TRPA1, a transient receptor potential cation channel that is critical for translating many environmental irritant stimuli into nociceptor activity. Using intracellular , we found that LPS extracted from E. coli increased intracellular calcium in wild type mouse cultured visceral and somatic sensory neurons. These responses were nearly abolished by pre-application of the TRPA1 inhibitor HC- 030031, and sensory neurons from Trpa1 KO mice responded to LPS with significantly lower incidence. LPS induced a dose-dependent and reversible increase of intracellular calcium in CHO cells expressing recombinant mouse TRPA1 channels, and stimulation of TRPA1 by LPS was confirmed in whole-cell and excised patch-clamp experiments in which extracellular application of LPS increased TRPA1 activity. Furthermore, we unveil a tight correlation between the ability of a given LPS to produce mechanical strain in the plasma membrane with its potency on TRPA1 activation. Finally, we investigated the possible role of TRPA1 in well-known effects of LPS, including pain and vasodilation. We found that LPS-induced nocifensive responses were almost abolished in Trpa1 KO mice. Moreover, LPS induced a strong dilation in WT mouse mesenteric arteries pre-contracted with the alpha1 adrenergic agonist phenylephrine. Notably, this effect was reduced significantly by HC-030031 and by genetic ablation of TRPA1. These findings may have important relevance for the treatment of symptoms caused by Gram-negative bacterial infections and offers new insights into the pathogenesis of inflammatory pain, and endotoxic shock.

International Workshop on Transient Receptor Potential (TRP) Channels 19 Session 2

TRPA1: A link between pain exacerbation in the arthritic joint in cold environments

Elizabeth S. Fernandes 1, Jennifer V. Bodkin 1, Claire Sand 1, Robin Salamon 1 and Susan D. Brain 1,2 1Vascular Biology Group and 2Centre for Integrative Biomedicine; Cardiovascular Division, King’s College London, London, England

We have previously shown that TRPV1 deletion reduces the severity of arthritis in mice, preventing inflammatory hyperalgesia caused by CFA (Keeble et al., 2005). In a more recent research, TRPA1 was suggested to play a role in the progression of arthritic pain in animals intra-articularly (i.art.) injected with CFA (Fernandes et al., 2011). In addition, it was described that TRPV1 and TRPA1 play a distinct role in TNF α-induced arthritic pain, with both centrally located TRPV1 and peripherally located TRPA1 contributing for pain sensation (Fernandes et al., 2011). We are further investigating the role of TRPA1 in arthritis. TRPA1 is a receptor known to be activated by cold (Karashima et al., 2009) and has been implicated on the perception of pain induced by cold stimuli under inflammatory conditions (Chen et al., 2011). We are interested in understanding the link between the exposure to cold environments and arthritic pain; especially the role of TRPA1 activation in this scenario. For this, 8-10 week old male CD1 and TRPA1KO and WT mice were used. Mice were i.art. injected with CFA (ipsilateral joint; 10 µg/10µl) and saline (contralateral joint; 10 µl/joint). Primary mechanical hyperalgesia was measured by pressure assessment meter over 4 weeks. Animals were exposed to cold (10 oC) for 1 h prior to hyperalgesia measurements. Arthritic animals exposed to ambient temperature (22-23 oC) were used as control. We found that cold exposure increases (P<0.05) primary mechanical hyperalgesia 2 weeks after arthritis induction when compared to control animals. This deleterious effect of cold on primary hyperalgesia was observed in both contralateral and ipsilateral joints. Our results suggest that there is a link between increased primary mechanical hyperalgesia and TRPA1 in arthritic animals exposed to a cold environment. The specific mechanisms involved in the TRPA1 participation remain to be investigated.

This work is supported by the Arthritis Research UK, a capacity building award led by the BBSRC and the BHF.

Keeble et al., 2005. Arthritis Rheum. 52: 3248-3256. Karashima et al., 2009. Proc Natl Acad Sci U S A. 106: 1273-1278. Chen et al., 2011. Pain. 152: 1165-1172. Fernandes et al., 2011. Arthritis Rheum. 63: 819-829.

20 Session 2

Characterization of the trafficking of human Transient Receptor Potential melastatin 8 (hTRPM8) channel by Total Internal Reflection Fluorescence (TIRF) Microscopy

Debapriya Ghosh 1, Grzegorz Owsianik 1, Pieter Vanden Berghe 2, Joris Vriens 1, Andrei Segal 1 and Thomas Voets 1 1 Laboratory of Ion Channel Research, Department of Molecular Cell Biology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium. 2 Laboratory of Translational Research in Gastrointestinal Disorders, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.

Transient Receptor Potential (TRP) channels form a superfamily of channels that can be considered as multiple signal integrators. TRP channels play vital roles in perception of all major classes of external stimuli, like thermosensation, chemosensation and mechanosensation. These channels also help individual cells with the ability to sense changes in the local environment, such as alterations in osmolarity. There is very little knowledge of the kinetics and molecular mechanism regarding the intracellular trafficking of TRP channels. Till date, only a handful of proteins are known to interact with TRP channels and to influence their trafficking. However, it is becoming increasingly clear that the dynamic modulation of the number of active TRP channels in the plasma membrane and intracellular organelles represents an important mechanism to regulate channel activity. A better knowledge of the fundamentals of TRP channel trafficking are therefore essential to our understanding of the role of these channels in various physiological/pathophysiological processes. We focused our study on TRPM8 - a cation channel activated by cold and the cooling compounds and . With the help of TIRF Microscopy- a state of art high resolution microscopic system, which allows the detection of individual fluorophores within 100 nm of the cell surface, we present for the first time a detailed depiction of TRPM8-vesicles in the near-membrane field. TRPM8 is present in intracellular structures with diverse morphological characteristics. We observed that a large fraction of TRPM8 resides in highly mobile late endosomal and lysosomal vesicles whereas less than 1% of TRPM8 resides in clathrin and caveolin coated vesicles or early endosomal vesicles. We found that TRPM8 vesicle movement is dependent on microtubular tracks while actin filaments could be involved in near membrane movements. Calcium entry as a result of TRPM8 activation seems to alter the dynamics of TRPM8 protein movement. Our results fetched significant insights regarding the movement pattern and habitation of TRPM8 within the intracellular compartment and further study need to be persisted to unveil its physiological importance.

International Workshop on Transient Receptor Potential (TRP) Channels 21 Session 2

Modulation of the cold-activated TRPM8 channel: The role of Gq protein and PIP 2

Dr. Xuming Zhang Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK

Peripheral endings of somatosensory neurons transduce cold mainly through activation of TRPM8 ion channels. Activation of TRPM8 is involved in multiple and even antagonistic cold signallings such as cold hypersensitivity and cold analgesia. It remains unknown how TRPM8 is regulated to fulfil its multiple functions. Membrane lipids PIP2 is essential for TRPM8 activity. Depletion of PIP 2 followed by activation of Gq-coupled GPCRs is assumed to cause the inhibition of TRPM8 under inflammatory condition. Here we show, however, that inflammatory mediators such as and histamine inhibit TRPM8 without the participation of conventional signalling pathways downstream of G-protein coupled receptors. Activated G-protein subunit Gq instead directly inhibits TRPM8 activation by binding to the channel. Deletion of Gq/11 largely abolished inhibition of TRPM8, and inhibition was rescued by a Gq chimera whose ability to activate PIP2 and downstream signalling pathways is completely ablated. Activated Gq protein potently inhibits TRPM8 in excised patches. We conclude that Gq pre-forms a complex with TRPM8, and inhibits TRPM8 by a direct action following activation of G-protein coupled receptors.

22 Session 2

TRPM8-deficient mice develop obesity and metabolic syndrome. Role of TRPM8 in the regulation of energy homeostasis

A. Reimúndez 1, B. Navia 1, Ch. Davletova 1, R. Gallego 2, JL Relova 1, C. Fernández- Peña 3, F. Viana 3, R. Señarís 1 1Department of Physiology (CIMUS) and 2Department of Morphological Sciences. University of Santiago de Compostela (Spain). 3Institute of Neuroscience. CSIC-UMH. Alicante (Spain).

Environmental temperature influences energy homeostasis through the regulation of energy expenditure, heat production and food intake. As TRPM8 and TRPA1 are considered the main peripheral cold thermosensors, and recent data suggest a role of TRPM8 in brown adipose tissue (BAT) thermogenesis, the aim of this study was to evaluate the possible role of these two ionic channels in the regulation of energy balance and metabolism “in vivo”. For this objective, TRPM8 and TRPA1-deficient mice were used. Animals of both sexes were grown at room temperature (22ºC) with a standard diet and followed over a period of 12 months starting at weaning time. We evaluated body weight gain and adiposity, food intake, energy expenditure, locomotion, and BAT physiology. We also determined serum hormonal, and biochemical metabolic parameters. We demonstrate that TRPM8–deficient mice develop the classical hallmarks of obesity and metabolic syndrome, reduced energy expenditure and locomotion, and an altered BAT physiology. In contrast to these results, TRPA1-deficient mice do not exhibit any change in the various parameters evaluated in this study. In summary, we demonstrate that the lack of functional TRPM8 channels is associated with a phenotype of obesity and metabolic syndrome, indicating that TRPM8 but not TRPA1 channels are essential to maintain a normal energy homeostasis.

International Workshop on Transient Receptor Potential (TRP) Channels 23 Session 3

TRPV4 regulation and population genetics

Miguel A. Valverde Laboratory of Molecular Physiology and Channelopathies, Universitat Pompeu Fabra, C/ Dr. Aiguader 88, Barcelona 08003, Spain

Transient receptor potential cation channels (TRP) participate in the generation of Ca 2+ signals at different locations of the respiratory system thereby controlling its correct functioning. I will comment on the association of non-synonymous and intronic single nucleotide polymorphisms (SNP) of TRPV1, TRPV4 and TRPA1 with asthma and how the genetic studies have led us to focus on the N-terminal tail as an important regulatory region of the TRPV4 channel. The TRPV4 channel was initially described as an osmosensor, activated by changes in environmental osmolarity. Although it is now known that the channel activation occurs down-stream of the activation of phospholipase A2, it is still not known what domains of the channel are necessary for its gating.

Funded by the Spanish Ministry of Science and Innovation, Red HERACLES (ISCiii), FEDER Funds and Generalitat de Catalunya.

24 Session 3

TRP channels and sensory reflexes in the lung

Maria G. Belvisi Imperial College London, UK

Cough is a protective reflex and defence mechanism in healthy individuals, which helps clear excessive secretions and foreign material from the lungs [1]. However, cough is also the most common respiratory complaint for which medical attention is sought [2] and often presents as the first and most persistent symptom of many respiratory diseases (e.g. common cold, lung infections, asthma, COPD, pulmonary fibrosis, bronchiectasis, lung cancer) and some non-respiratory disorders (gastro-oesophageal reflux, post-nasal drip). Patients with chronic cough probably account for 10–38% of respiratory outpatient practice in the USA [3]. Chronic cough of various aetiologies is a common presentation to specialist respiratory clinics, and is reported as a troublesome symptom by 7% of the population [4]. Treatment options are limited. A recent meta- analysis concluded that over the counter (OTC) cough remedies are ineffective [5] and there is increasing concern about the use of OTC therapies in children. Despite its importance our understanding of the mechanisms which provoke cough is poor. The respiratory tract is innervated by sensory afferent nerves which are activated by mechanical and chemical stimuli [6]. Activation of capsaicin-sensitive C-fibres and acid- sensitive, capsaicin-insensitive mechanoreceptors innervating the larynx, trachea, and large bronchi regulate the cough reflex [6,7]. Endogenous inflammatory mediators are often elevated in respiratory disease states. For example, higher concentrations of PGE 2 [8] and BK [9] have been found in the airways of patients with asthma and chronic obstructive pulmonary disease (COPD). Both PGE 2 and BK are also known to cause cough by stimulating airway sensory nerves [10, 11]. Furthermore, increased PGE 2 levels have been found in idiopathic cough and cough associated with post nasal drip, gastroesophageal reflux disease, cough variant asthma and eosinophilic bronchitis [12]. Although we do have some information regarding which G protein- coupled receptors (GPCRs) are activated by these endogenous tussive agents it is still unclear what post receptor signalling pathways are involved. Recently, ion channels of the Transient Receptor Potential (TRP) class such as TRPV1 have been implicated in the afferent sensory loop of the cough reflex [13, 14] and in the heightened cough sensitivity seen in disease [15]. TRPA1 is a Ca 2+ -permeant non- selective channel with 14 ankyrin repeats in its amino terminus which also belongs to the larger TRP family. TRPA1 channels are activated by a range of natural products found in oil, and [16-18] and by environmental irritants (eg. ) [19-21], and is primarily expressed in small diameter, nociceptive neurons [22]. It has been demonstrated that stimulating TRPA1 channels activates vagal broncho-pulmonary C-fibers in rodent lung [21-23] causing cough both in guinea-pig models and in normal human volunteers [24]. The TRPV4 channel is also widely expressed in mammalian tissues including lung, heart, kidney, sensory neurons, sympathetic nerves, brain, skin, intestine, salivary gland, sweat glands, inner ear, endothelium and fat tissue. In the lung, TRPV4 is detected by RT-PCR in a human bronchial epithelial cell line and in cultured human airway smooth muscle cells [25-27]. In addition, we have preliminary data to suggest that TRPV4 may be present on vagal sensory nerve endings and may be involved in the activation of lung specific afferents in response to endogenous stimuli such as hypotonicity. Although many exogenous stimuli are known to activate particularly TRPA1 and TRPV1, it is still unknown how cough and other reflexes are elicited in health and disease by endogenous agents, and whether these ion channels are involved. We have provided evidence that TRP ion channels may have a role as common effectors for tussive agents. Furthermore, models of exaggerated cough have now been developed in our laboratory which may help to identify novel disease relevant targets.

International Workshop on Transient Receptor Potential (TRP) Channels 25 Session 3

References: 1. Widdicombe JG. (1995) Neurophysiology of the cough reflex. Eur. Resp. J. 8 , 1193-1202. 2. Cherry DK, Burt CW, and Woodwell DA. (2003) National ambulatory medical care survey: 2001 summary. Adv. Data . 337 , 1-44. 3. Irwin RS, Corrao WM, Pratter MR. Chronic persistent cough in the adult: the spectrum and frequency of causes and successful outcome of specific therapy. Am Rev Respir Dis 1981; 123 : 413–417. 4. Ford AC, Forman D, Moayyedi P et al. Cough in the community: a cross sectional survey and the relationship to gastrointestinal symptoms. Thorax 2006; 61 : 975-979. 5. Schroeder K, Fahey T. Systematic review of randomised controlled trials of over the counter cough medicines for acute cough in adults. B.M.J. 2002; 324 : 329-331. 6. Canning BJ, Chou Y-L. Cough sensors. I. Physiological and pharmacological properties of the afferent nerves regulating cough. Handb Exp Pharmacol . 2009; 187 : 23-47. 7. Nasra J, Belvisi MG. Modulation of sensory nerve function and the cough reflex: understanding disease pathogenesis. Pharmacol Ther . 2009; 124 : 354-375. 8. Profita M, Sala A, Bonanno A, et al. Increased prostaglandin E 2 concentrations and cyclooxygenase-2 expression in asthmatic subjects with sputum eosinophilia. Journal of Allergy and Clinical Immunology 2003; 112 : 709-716. 9.Baumgarten CR, Lehmkuhl B, Henning R, et al. Bradykinin and other inflammatory mediators in BAL-fluid from patients with active pulmonary inflammation. Agents Actions Suppl 1992; 38 : 475- 481. 10. Fox AJ, Lalloo UG, Belvisi MG, e al. Bradykinin-evoked sensitization of airway sensory nerves: a mechanism for ACE-inhibitor cough. Nat Med 1996; 2: 814-817. 11. Maher, S.A., Birrell, M.A., & Belvisi, M.G. Prostaglandin E 2 mediates cough via the EP 3 receptor: Implications for future disease therapy. Am J Respir Crit Care Med 2009; 180 : 293-298. 12. Birring SS, Parker D, Brightling CE, et al. Induced sputum inflammatory mediator concentrations in chronic cough. Am J Respir Crit Care Med. 2004; 169 : 15-19. 13. Laude EA, Higgins, KS, Morice AH. A comparative study of the effects of citric acid, capsaicin and on the cough challenge in guinea-pig and man. Pulm Pharmacol 1993; 6: 171- 175. 14.Lalloo UG, Fox AJ, Belvisi MG, et al.. Capsazepine inhibits cough induced by capsaicin and citric acid but not by hypertonic saline in guinea pigs. J Appl Physiol 1995; 79 : 1082-1087. 15. Groneberg DA, Niimi A, Dinh QT, et al.. Increased expression of transient receptor potential vanilloid-1 in airway nerves of chronic cough. Am J Respir Crit Care Med 2004; 170 : 1276-1280. 16. Story GM, Peier AM, Reeve AJ, et al. Patapoutian A. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003; 112 : 819-829. 17. Jordt SE, Bautista DM, Chuang HH, et al. Mustard oils and excite sensory nerve fibres through the TRP channel ANKTM1. Nature 2004; 427 : 260-265. 18. Bandell M, Story GM, Hwang SW, et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004; 41 : 849-857. 19.Bautista DM, Jordt SE, Nikai T, et al., TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 2006; 124 : 1269-1282. 21.Andrè E, Campi B, Materazzi S, et al. Cigarette smoke-induced neurogenic inflammation is mediated by alpha, beta-unsaturated aldehydes and the TRPA1 receptor in rodents. J Clin Invest 2008; 118 : 2574-2582. 22. Nassenstein C, Kwong K, Taylor-Clark T, et al. 2008. Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs. J Physiol 2008; 586 : 1595-1604. 23.Taylor-Clark TE, McAlexander MA, Nassenstein C et al. Relative contributions of TRPA1 and TRPV1 channels in the activation of vagal bronchopulmonary C-fibres by the endogenous autocoid 4-oxononenal. J Physiol 2008; 586 : 3447-3459. 24.Birrell MA, Belvisi MG, Grace M, et al. TRPA1 agonists evoke coughing in guinea pig and human volunteers. Am J Respir Crit Care Med 2009; 180 : 1042-1047. 25. Wolfgang Liedtke and S.A. SimonAm J Physiol Lung Cell Mol Physiol. 2004, A possible role for TRPV4 receptors in asthma. Am J Physiol Lung Cell Mol Physiol 287:L269- L271. 26. Liedtke W, Simon SA. D. Ni, Q. Gu, H.Z. Hu, N. Gao, M.X. Zhu, L.Y. Lee. Thermal sensitivity of isolated vagal pulmonary sensory neurons: role of transient receptor potential vanilloid receptors. Am. J. Physiol., Regul. Integr. Comp. Physiol., 291 (2006), pp. R541–R550 27. Jia, YL, Lee L-Y. Role of TRPV receptors in respiratory disease. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1772, Issue 8, August 2007, Pages 915–927

26 Session 3

TRPA1 in peripheral diabetic neuropathy

Antti Pertovaara 1 & Ari Koivisto 2 1Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland & 2OrionCorporation, OrionPharma, Turku, Finland

Peripheral diabetic neuropathy (PDN) is a devastating complication of diabetes mellitus (DM). Here we test the hypothesis that the transient receptor potential ankyrin 1 (TRPA1) ion channel on primary afferent nerve fibers is involved in the pathogenesis of PDN, due to sustained activation by reactive compounds generated in DM. DM was induced by streptozotocin in rats that were treated daily for 28 days with a TRPA1 channel antagonist (Chembridge-5861528) or vehicle. Pain hypersensitivity was assessed behaviorally with a paw pressure test. Laser Doppler flow method was used for assessing axon reflex induced by intraplantar injection of a TRPA1 channel agonist () and immunohistochemistry to assess -like innervation of the skin. In vitro calcium imaging and patch clamp were used to assess whether endogenous TRPA1 agonists (4-hydroxynonenal and methylglyoxal) generated in DM induce sustained activation of the TRPA1 channel. During the first two weeks of DM, the animals developed an early pain hypersensitivity that was reversed by acute and prevented by prolonged treatment with a TRPA1 channel antagonist. Axon reflex induced by a TRPA1 channel agonist in the plantar skin was suppressed and the number of substance P-like immunoreactive nerve fibers was decreased four weeks after induction of DM. Prolonged treatment with Chembridge-5861528 reduced the DM- induced attenuation of the cutaneous axon reflex and loss of substance P-like immunoreactive nerve fibers. Moreover, in vitro calcium imaging and patch clamp results indicated that reactive compounds generated in DM (4-hydroxynonenal and methylglyoxal) produced sustained activations of the TRPA1 channel, a prerequisite for adverse long-term effects. In line with this, intraplantar methylglyoxal in healthy control animals produced sustained pain behavior that was accompanied by mechanical pain hypersensitivity. Together the results indicate that the TRPA1 channel exerts an important role in the pathogenesis of PDN. Blocking the TRPA1 channel provides a selective disease-modifying treatment of PDN.

International Workshop on Transient Receptor Potential (TRP) Channels 27 Session 3

New insights in the regulation of mineral TRP's

René J.M. Bindels Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Ca 2+ and Mg 2+ are of great physiological importance in their function in neural excitability, muscle contraction, blood coagulation, bone formation, hormone secretion and cell adhesion. The human body is equipped with an efficient negative feedback system counteracting variations of the Ca2+ and Mg 2+ balance. These divalents are maintained within a narrow range by the small intestine and kidney which both increase their fractional (re)absorption under conditions of deprivation. Rapid progress has recently been made in identification and characterization of the Ca 2+ and Mg 2+ transport proteins contributing to the delicate balance of divalent cations. Expression cloning approaches in combination with knockout mice models and genetic studies in families with a disturbed Mg 2+ balance revealed novel gatekeeper proteins that belong to the super family of the transient receptor potential (TRP) channels. These epithelial Ca 2+ (TRPV5) and Mg 2+ channels (TRPM6) form prime targets for hormonal control of the active Ca 2+ and Mg 2+ flux from the urine space or intestinal lumen to the blood compartment. The characteristics of these TRP’s will be discussed and in particular the distinctive molecular regulation of these new epithelial Ca 2+ and Mg 2+ channels in (patho)physiological situations will be highlighted.

28 Session 3

A TR(i)P to magnesium homeostasis

Vladimir Chubanov 1, Annika Wisnowsky 1, Silvia Ferioli 1, Susanna Zierler 1, Renate Heilmaier 1, Ludmila Sytik 1, David Simmons 2, Thomas Hofmann 3, and Thomas Gudermann 1 1Walther-Straub-Institut of Pharmacology and Toxicology, University of Munich, Germany 2School of Biomedical Sciences, The University of Queensland, Brisbane, Australia 3Institute of Pharmacology, Medical Faculty, University of Marburg, Germany

Divalent cation-selective outwardly rectifying currents induced upon removal of intracellular Mg 2+ have been described in all mammalian cells examined so far. Accordingly, these currents were referred to as magnesium-inhibited currents (MIC) or magnesium nucleotide-regulated metal ion currents (MagNuM). TRPM6 and TRPM7 (melastatin-related members of the transient receptor potential gene family) were identified as molecular candidates mediating MIC/MagNuM. Recent studies provided evidence that TRPM7 is essential for the cell viability, embryonic development and Mg 2+ homeostasis. Loss-of-function mutations in the human TRPM6 gene result in a human disease, hypomagnesemia with secondary hypocalcemia (HSH). HSH is an autosomal recessive disorder characterized by low serum Mg 2+ and Ca 2+ levels. TRPM6 is specifically expressed in the intestinal epithelium and the distal convoluted tubule in the kidney. Collectively, these findings strongly suggest that TRPM6 can directly participate in Mg 2+ uptake by renal and intestinal epithelial cells. This concept, however, is surrounded by considerable controversy. In order to investigate the role of TRPM6 in the pathomechanism of HSH, we initiated a phenotypic analysis of TRPM6 gene deficient mice carrying a LacZ reporter sequence and of additional mouse strains with TRPM6 genes conditionally inactivated in different organs. In stark contrast to what we know about the clinical picture of HSH, homozygous TRPM6-deficient mice die at a mid-gestational stage. Tracking LacZ expression and using TRPM6-specific antibodies, we observed an expression profile of TRPM6 much broader than that a previously assumed, supporting the notion that the phenotype of HSH patients only partially depicts the physiological relevance of TRPM6.

International Workshop on Transient Receptor Potential (TRP) Channels 29 Session 3

PIP 2 isoforms determine compartment-specific TRP channel activity

Haoxing Xu The Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 3089 Natural Science Building (Kraus), 830 North University, Ann Arbor, MI 48109, USA

Phosphoinositides serve as address labels for recruiting peripheral cytoplasmic proteins to specific subcellular compartments and as endogenous factors for modulating the activity of integral membrane proteins. Phosphatidylinositol 4,5- bisphosphate (PI(4,5)P 2) is a plasma-membrane (PM)-specific phosphoinositide and a positive cofactor required for the activity of most PM channels and transporters. This requirement for phosphoinositide cofactors has been proposed to prevent PM channel/transporter activity during passage through the biosynthetic/secretory and endocytic pathways. To determine whether intracellularly-localized channels are similarly “inactivated” at the PM, we studied the PIP 2 modulation of intracellular TRPML1 channels. TRPML1 channels are primarily localized in lysosomes, but can also be detected temporarily in the PM upon lysosomal exocytosis. By directly patch- clamping isolated lysosomes, we previously found that lysosomal, but not PM- localized, TRPML1 is active with PI(3,5)P 2, a lysosome-specific PIP 2, as the underlying positive cofactor. Here we found that “silent” PM-localized TRPML1 could be activated by depleting PI(4,5)P 2 levels and/or by adding PI(3,5)P 2 to inside-out membrane patches. Unlike PM channels, surface-expressed TRPML1 underwent a unique and characteristic run-up upon patch excision, and was potently inhibited by a low micromolar concentration of PI(4,5)P 2. Conversely, depletion of PI(4,5)P 2 by either depolarization-induced activation or chemically-induced translocation of 5’- phosphatase potentiated whole-cell TRPML1 currents . PI(3,5)P 2 activation and PI(4,5)P 2 inhibition of TRPML1 were mediated by distinct basic amino acid residues in a common PIP 2-interacting domain. Thus, PI(4,5)P 2 may serve as a negative cofactor for intracellular channels such as TRPML1. Based on these results, we propose that phosphoinositide regulation sets compartment-specific activity codes for membrane channels and transporters.

30 Session 4

TRP channels in C. elegans sensory transduction

X.Z. Shawn Xu , Ph.D. Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA

The nematode C. elegans has emerged as a popular model for the study of various phenomena in neurobiology because of its simple and well characterized nervous system and amenability to genetic manipulation. The C. elegans genome encodes 17 TRP channel genes that fall within all of the seven TRP subfamilies and are highly homologous to their vertebrate counterparts. Genetic analyses in C. elegans have implicated TRP channels in a wide spectrum of behavioral and physiological processes, ranging from sensory transduction to drug dependence, fertilization, organelle biogenesis, apoptosis, gene expression, and neurotransmitter/hormone release. Many C. elegans TRP channels share similar activation and regulatory mechanisms with their vertebrate counterparts. Studies in C. elegans have also revealed some previously unrecognized functions and regulatory mechanisms of TRP channels. C. elegans represents an excellent genetic model organism for the study of function and regulation of TRP channels in vivo . Here we will discuss our recent work on the role of TRP channels in sensory physiology. Using a combination of genetic, behavioral and electrophysiological assays, we have characterized the in vivo function and regulation of a number of TRP channels, particularly TRPV, TRPA, and TRPN channels. Our data show that these TRP channels are involved in regulating sensory physiology, including mechanosensation, thermosensation, and chemosensation.

International Workshop on Transient Receptor Potential (TRP) Channels 31 Session 4

TRP channels: From animal behavior to neurodegenerative disease

Craig Montell Johns Hopkins University School of Medicine, Baltimore, MD, USA

Transient Receptor Potential (TRP) cation channels are notable in contributing to virtually every sensory modality, and in controlling a daunting array of behaviors. Flies encode 13 TRPs, most of which are expressed and function in sensory neurons, and impact behaviors ranging from phototaxis to thermotaxis, the avoidance of noxious chemicals and proprioception. In this presentation, I will describe recent work from my laboratory demonstrating how TRP channels impact on a wide variety of animal behaviors, using flies as a model organism. In many cases, TRP channels function through G-protein coupled signaling cascades that are initiated by rhodopsin. Thus, while rhodopsin was formerly thought to function exclusively in light detection, we found that different rhodopsins also initiate signaling cascades that participate in thermotaxis, chemotaxis and other behaviors. Many diseases result from defects in TRP channels, including the childhood neurodegenerative disease, MLIV, which results from mutations in TRPML1. Based on insights from a fly model for this disease, I will describe a concept for a therapy for this disease.

32 Session 4

TRP channels in Drosophila phototransduction

Roger Hardie Cambridge University, Department of Physiology Development and Neuroscience. Downing Street, CB2 3EG Cambridge, U.K.

Drosophila TRP is the founding member of the TRP superfamily and together with its homologue, TRPL, mediates the light-sensitive transducer current in the photoreceptors. Like other members of the TRPC subfamily, the light-sensitive TRP and TRPL channels are activated via a canonical phospholipase C (PLC β) signaling pathway, but the mechanism of channel activation downstream of PLC remains unresolved. Along with rhodopsin and other components of the Gq-protein coupled phototransduction cascade, the channels are localized in ~30000 tightly bundled microvilli (each ~50 nm in diameter), together forming a light-guiding rod-like stack – the “rhabdomere”. PLC’s obvious action is to hydrolyze PIP 2, generating DAG and InsP 3; but in addition PLC activity results in a simultaneous reduction in PIP 2 and release of a proton. Recently we found that light-induced PLC activity led to a rapid acidification of the microvillar rhabdomere. Furthermore, both TRP and TRPL could be 1 activated by the combination of PIP 2 depletion and protonophores ; but how PIP 2 depletion might contribute to channel gating was unclear. New results suggest that the effects of PIP 2 depletion may be mediated mechanically. The data indicate that by cleaving PIP 2’s bulky head group from the tightly curved inner leaflet of the lipid bilayer in the microvilli, PLC induces a small, but rapid reduction in microvillar diameter, resulting in measurable contractions of the entire cell. We suggest that the resultant physical changes in the lipid bilayer may contribute to gating the light-sensitive TRP and TRPL channels in a mechanical sense, in combination with the PLC-mediated acidification of the microvillar compartment.

International Workshop on Transient Receptor Potential (TRP) Channels 33 Session 4

Signal dependent hydrolysis of PI(4,5)P 2 without activation of phospholipase C: Implications on the gating of the Drosophila TRPL channel

Shaya Lev, Ben Katz, and Baruch Minke Department of Medical Neurobiology and Institute of Medical Research Israel-Canada (IMRIC) and the Edmond & Lily Safra Center for brain sciences (ELSC), Faculty of Medicine of the Hebrew University, Jerusalem 91120, Israel

In Drosophila , a PLC-mediated signaling cascade links photo-excitation of rhodopsin to the opening of the Transient Receptor Potential (TRP) and TRP-Like (TRPL) channels. A lipid product of phospholipase C (PLC), diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, can one rule out that increase of the many signaling molecules downstream of PLC activity or other changes in phosphoinositides or pH are essential for gating the TRP/TRPL channels instead of or together with the loss of PI(4,5)P 2? To answer this question we co-expressed the TRPL channels, together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method to reduce plasma membrane–associated PI(4,5)P 2 in HEK cells within seconds, without activating PLC. Upon addition of a dimerizing drug, PI(4,5)P 2 was selectively hydrolyzed in the cell membrane without producing DAG, IP 3, or calcium signals. We show that PI(4,5)P 2 is not an inhibitor of TRPL channel activation. While PI(4,5)P 2 hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, PUFA did activate them. Moreover, a reduction in PI(4,5)P 2 level or inhibition of DAG lipase during PLC activity suppressed the PLC activated TRPL current, suggesting that PI(4,5)P 2 is a crucial substrate for PLC mediated activation of the channels, while PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.

34 Session 4

TRPM7 channel – turned on by cleavage

Bimal N. Desai PhD Assistant Professor, Department of Pharmacology, University of Virginia School of Medicine 1340 Jefferson Park Avenue, Jordan Hall - Room 5026, PO Box 800735, Charlottesville, VA 22908-0735, USA

Transient Receptor Potential Melastatin-like 7 (TRPM7) is a channel protein that also contains a regulatory serine-threonine kinase domain. Here, we find that Trpm7 -/- T- cells are deficient in Fas-receptor induced apoptosis; TRPM7 channel activity participates in the apoptotic process and is regulated by caspase-dependent cleavage. This function of TRPM7 is dependent on its function as a channel, but not as a kinase. TRPM7 is cleaved by caspases at D1510, disassociating the carboxy-terminal kinase domain from the pore without disrupting the phosphotransferase activity of the released kinase, but substantially increasing TRPM7 ion channel activity. Furthermore, we show that TRPM7 regulates endocytic compartmentalization of the Fas receptor following receptor stimulation, an important process for apoptotic signaling through Fas receptors. These findings raise the possibility that other members of the TRP channel superfamily are also regulated by caspase-mediated cleavage with wide-ranging implications for cell death and differentiation.

International Workshop on Transient Receptor Potential (TRP) Channels 35 Session 4

Regulation of TRPM3 activity in pancreatic β cells by intracellular signaling cascades

1,2 1 2 1 Florian Mohr , Kerstin Hartwig , Anita Leist , Johannes Oberwinkler 1 Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, Germany 2 Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Germany

Insulin secretion from pancreatic β cells is a very complex and highly regulated process involving the concerted action of many different ion channels. Previously we showed that TRPM3 proteins, forming calcium permeable, non-selective cation channels are expressed in pancreatic β cells. Their activation by the endogenous steroid 2+ leads to an increased Ca influx in – and subsequently to an enhanced -stimulated insulin release from – pancreatic β cells. However, the precise function of TRPM3 channels in pancreatic β cells and the physiological regulation of their activity are not yet known. To better understand the physiological role of TRPM3 in pancreatic β cells we examined whether TRPM3 is subject to regulation by intracellular signaling cascades in β cells. In rat insulinoma cells (Ins-1) and mouse primary pancreatic β cells, application of noradrenaline or adrenaline strongly inhibited TRPM3 activity by activating α2- adrenoreceptors. Experiments with pertussis toxin (PTX), an inhibitor of G i/o -proteins, revealed that the mechanism is Gprotein coupled. Furthermore, using IBMX, a nonselective phosphodiesterase inhibitor, and forskolin, an activator of the adenylyl cyclase, showed that the inhibitory effect of the catecholamines on TRPM3 channels is still detectable when the intracellular cAMP concentration is increase. This proves that catecholamines cause their effect independently of the intracellular cAMP concentration. Thus questioning the classical G αi/o pathway, we investigated whether active G αi or G αo subunits alone are sufficient to inhibit TRPM3 channels. The results showed that neither constitutive active G αi nor constitutive active G αo subunits reduced TRPM3 channel activity. In contrast, overexpression of β1 and γ2 subunits led to a strong reduction of the TRPM3 channel activity, indicating that β/γ subunits are at least partially involved in the inhibition mechanism. This hypothesis was further strengthened by the observation that overexpression of the β/γ-scavenging peptides myr-β-ARKct or myrphosducin (that bind β/γ-subunits of G-proteins particularly at the plasma membrane, due to their myristoylation tag) strongly reduced the inhibitory effect of catecholamines on TRPM3 channels. Finally, the effect of β/γ-subunits was specific, in the sense that only β1 proteins could inhibit TRPM3 channels, but not β3, β4 or β5 proteins. Changing γ2 for other γ subunits did not affect the capacity of the complex to inhibit TRPM3 channels. Our results therefore favor a model in which certain β/γ subunits, released after G i/o activation, cause the inhibition of TRPM3 channels. Experiments are underway to test whether or not the action of β/γ subunits on TRPM3 channels is direct.

36 Session 4

TRPV4 regulates calcium influx and ciliary beat frequency activity in human airway ciliated epithelial cells

Lisa Alenmyr 1, Lena Uller 2, Lennart Greiff 3, Edward D. Högestätt 1 and Peter M. Zygmunt 1 1Clinical Chemistry & Pharmacology, Department of Laboratory Medicine, Lund University, Lund, Sweden 2Cell and Tissue Biology, Department of Experimental Medical Science, Lund University, Lund, Sweden 3Department of ORL, Head & Neck Surgery, Skåne University Hospital, Lund, Sweden

Aim: The transient receptor potential vanilloid 4 (TRPV4) is a calcium permeable ion channel expressed in airway epithelial cells. Based on studies in cell lines or animals, TRPV4 has been suggested to play a role in the regulation of cell volume and ciliary beat frequency (CBF). Whether the same is true in human ciliated epithelial cells is not known. Therefore, we examined the expression and function of TRPV4 in native human nasal ciliated epithelial cells.

Methods: Expression of TRPV4 in nasal epithelial cells as well as in the cell lines BEAS2B and 16HBE was estimated using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). In addition, TRPV4 expression in nasal epithelial cells was studied using immunohistochemistry. Responses to pharmacological modulation of TRPV4 were assessed with calcium imaging in hTRPV4-expressing HEK293 cells, BEAS2B cells, 16HBE cells and freshly isolated nasal ciliated epithelial cells. Furthermore, CBF measurements were performed on epithelial cells exposed to the TRPV4 agonist GSK1016790A in the absence or presence of either the TRPV4 antagonist HC067047 or the TRP channel blocker .

Results: TRPV4 mRNA was found in native nasal epithelial, BEAS2B and 16HBE cells, and a marked apical TRPV4 immunoreactivity was detected in nasal epithelial cells. GSK1016790A produced concentration-dependent calcium responses in hTRPV4- expressing HEK293, BEAS2B and 16HBE cells, and HC067047 caused a rightward shift of the concentrations-response curves for GSK1016790A. Native ciliated epithelial cells responded to GSK1016790A (1 and 10 nM) with increases in intracellular calcium concentration and CBF, effects that were inhibited by HC067047 (1 or 10 M) or ruthenium red (10 M). After approximately 10-15 min, the cilia stopped beating and cell death was confirmed by trypan blue staining.

Conclusion: TRPV4 is expressed in human primary nasal epithelial cells and modulates epithelial CBF. Thus, TRPV4 may participate in mucociliary clearance and airway protection. However, exaggerated activation of TRPV4 may result in epithelial cell death and disrupted barrier. Further studies are needed to elucidate the role of TRPV4 in airway physiology and its potential role in airway diseases.

International Workshop on Transient Receptor Potential (TRP) Channels 37

Posters

International Workshop on Transient Receptor Potential (TRP) Channels

List of posters

Nº 1 Peripheral AMPA receptors differentially modulate TRPV1 function in the context of heat and algogen-mediated nociceptor sensitization Vijayan Gangadharan, Rui Wang, Nitin Agarwal , Bettina Ulzhofer, Gary Lewin and Rohini Kuner

Nº 2 Effects of cannabinoids on transient receptor potencial channels (TRP) in Alzheimer disease Aguirre-Rueda D. , Paredes-Brunet P., Gil-Bisquert A. and Vallés S.L.

Nº 3 Characterization of voltage-activated calcium currents in TRPM8-expressing cold thermoreceptors L. Almaraz , C. Morenilla-Palao, J.A. Manenschijn, F. Viana

Nº 4 Anesthetic effect of the TRPA1 specific agonist cinnamaldehyde Yeranddy A. Alpizar , Laura van Gerven, Wouter Everaerts, Brett Boonen, François Vermeulen, Dirk De Ridder, Peter Hellings, Thomas Voets, Karel Talavera

Nº 5 Cross-talk between ααα1d -adrenergic receptor ( ααα1d -AR) and Transient Receptor Potential Vanilloid 1 (TRPV1) triggers the proliferation of PC-3 prostate cancer cells Amantini C. , Farfariello V., Morelli M.B., Nabissi M., Liberati S., Santoni M., Ranzuglia V., Cardinali C., Filosa A., Pieramici T., Ranaldi R., Piergentili L., Quaglia W. and Santoni G.

Nº 6 The diabetic marker methylglyoxal produces painful neuropathy by stimulating TRPA1 David A. Andersson , Clive Gentry, Angelika Bierhaus, Thomas Fleming, Peter P. Nawroth and Stuart Bevan

Nº 7 Role of TRPA1 in the peripheral vasculature in vivo : Increasing evidence for a CGRP and nitric oxide-sensitive mechanism Aisah A. Aubdool , Bodkin J.V., Xenia Kodji, Ross King, Clive Gentry, Elizabeth S. Fernandes, Stuart Bevan and Susan Brain

Nº 8 D-series resolvins potently suppressed sensory TRP activities leading to multiple anti-nociception Sangsu Bang , Sungjae Yoo, Tae-Jin Yang, Ji Yeon Lim, Sun Wook Hwang

Nº 9 TRPV1 receptor potentiation contributes to pruritogenesis and thermal hypersensitivity in a rat model of liver disease Majedeline Belghiti , Judith Estévez-Herrera,Carla Giménez-Garzó, Alba González- Usano,Carmina Montoliu, Antonio Ferrer-Montiel, Vicente Felipo, Rosa Planells- Cases

Nº 10 Modulation of voltage-dependent sodium currents by the TRPA1 agonist cinnamaldehyde Brett Boonen , Bristol Denlinger, Yeranddy A. Alpizar, Thomas Voets, Victor M. Meseguer, Carlos Belmonte, Karel Talavera

Nº 11 Protons stabilize the closed conformation of the gain-of-function mutants of TRPV1 channel Boukalova S. , Teisinger J., Vlachova V.

Nº 12 Novel endogenous N-acyl amides activate TRPV1-4 receptors and are regulated in an acute model of inflammation Heather B. Bradshaw , Siham Raboune, Jordyn M. Stuart

40 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation List of posters Nº 13 TRPV1 and TRPA1 play distinct roles in itch and associated skin inflammation induced by LTB 4 Elizabeth S. Fernandes, Chi-Teng Vong, Samuel Quek, Jessica Cheong, Aisah A. Aubdool, Jennifer V. Bodkin, Richard Heads and Susan D. Brain

Nº 14 Decreased asthmatic responses to Aspergillus and house dust mite in mice deficient in the sensory neuronal ion channel TRPA1 Ana Isabel Caceres , Sven-Eric Jordt

Nº 15 Co-application of TRPV1 and TRPV4 antagonists: a synergistic effect to overcome bladder hyperactivity induced by urinary bladder inflammation Charrua A. , Boudes M., De Ridder D., Cruz C.D., Cruz F.

Nº 16 Pharmacological targeting of recombinant and endogenous TRPM7 channels V. Chubanov , M. Mederos y Schnitzler, M. Meißner, S. Schäfer, K. Abstiens, T. Hofmann, T. Gudermann

Nº 17 Identification and characterization of the interaction between TRPV1 and the PDZ protein Whirlin Ciardo M. Grazia , Cuesta-Garrote N., Andres-Borderia A., Camprubí-Robles M., Ferrer-Montiel A., Planells-Cases R.

Nº 18 Mechanosensation in trigeminal sensory neurons Anna Lucía Conte , Danny Florez and Ana Gomis

Nº 19 Transient Receptor Potential A1 (TRPA1) is not involved in thermoregulation in dogs and humans Rory Curtis , Scott Coleman, Donato Del Camino, Neil J. Hayward, Magdalene M. Moran, Paula Bokesch

Nº 20 Protons activate human TRPA1 Jeanne de la Roche , Mirjam Eberhardt, Nancy Stanslowsky, Florian Wegner, Peter Reeh and Andreas Leffler

Nº 21 Discovery of new hits for TRPV1 blockade by high throughput assays Roberto de la Torre Martínez , Asia Fernández-Carvajal, M. Teresa Aranda, M. Teresa García-López, Rosario González-Muñiz and Antonio Ferrer-Montiel

Nº 22 Effects of blocking TRPA1 in a sheep model of asthma Donato del Camino , Jayhong A. Chong, Neil J. Hayward, Jennifer Monsen, Magdalene M. Moran, Rory Curtis, Christopher Murphy, Lawrence I. Mortin, William M. Abraham

Nº 23 Cold receptor activity in the tongue of TRPM8- and TRPA1-deficient mice Denlinger B.L. , Viana F. and Belmonte C.

Nº 24 Primary sequence analysis of TRPV2 reveals potential conserved key domains implicated in structure/function Doñate Macian P. , Jose-Luis Vázquez-Ibar, Perálvarez-Marín A.

Nº 25 Reduced TRPV1 expression in the ciPTEC Cystinotic cell line PT47.5 Ciara Doran , Katrin Kaschig, Patrick Harrison, Gordon Reid

Nº 26 PLC γ1 and ErbB2 are required for pressure-induced activation of TRPM4 channels in cerebral artery smooth muscle cells Albert L. Gonzales, Ying Yang, Michelle N. Sullivan, Lindsey Sanders and Scott Earley

Nº 27 Neurovascular effects through activation of TRPA1 – how nitroxyl (HNO) works Mirjam Eberhardt , Maria Dux, Barbara Namer, Nada Cordasic, Jan Miljkovic, Jeanne de la Roche, Michael Fischer, Andreas Leffler, Angelika Lampert, Johannes Jacobi, Karl Messlinger, Ivana Ivanovic-Burmazovic, Peter Reeh, Milos R. Filipovic

International Workshop on Transient Receptor Potential (TRP) Channels 41 List of posters Nº 28 Characterization of TRPM8-expressing primary sensory neurons purified by fluorescence-activated cell sorting Enoch Luis , Cruz Morenilla-Palao, Carlos Fernández-Peña and Félix Viana

Nº 29 TRPV1-mediated autophagy in thymocytes is a consequence of proteasome inhibition and unfolded protein response activation Farfariello V. , Amantini C., Nabissi M., Morelli M.B., Liberati S., Eleuteri A.M., Bonfili L., Cecarini V., Sorice M. and Santoni G.

Nº 30 Ligand induced opening of TRPM2 channel requires terminal ribose of ADPR and Arg1433 Ralf Fliegert , Christelle Moreau, Tanja Kirchberger, Anja Schöbel, Mark Thomas, Andreas Bauche, Angelika Harneit, Barry V.L. Potter and Andreas H. Guse

Nº 31 TRPA1 channel is expressed in non-neuronal pulmonary cells and promotes non-neurogenic inflammation Camilla Fusi , Romina Nassini, Pamela Pedretti, Nadia Moretto, Chiara Carnini, Fabrizio Facchinetti, Riccardo Patacchini, Pierangelo Geppetti, and Serena Materazzi

Nº 32 Role of the cytosolic N-terminal tail of TRPV4 in the channel response to hypotonic stimuli Anna Garcia-Elias , Sanela Mrkonjic, Carlos Pardo, Fanny Rubio-Moscardó, Rubén Vicente and Miguel A. Valverde

Nº 33 TRPM8/b1integrin interaction controls vascular endothelial cell migration and adhesion Tullio Genova , Dimitra Gkika, Alexandre Bokhobza, Luca Munaron, Guido Serini, Natalia Prevaskaya and Alessandra Fiorio Pla

Nº 34 Testosterone, the steroid link in TRPM8–mediated cold perception Dimitra Gkika , Alexis Bavencoffe, Jerome Busserolles, Artem Kondratskyi, Alexander Zholos, Eric Chapuy, Monique Etienne, Alain Eschalier, Brigitte Mauroy, Yaroslav Shuba, Roman Skryma and Natalia Prevarskaya

Nº 35 Investigating a role for TRPV4 in the airways Grace, M.S. , Birrell, M.A., Dubuis, E., Ching, Y.M., Bonvini, S., and Belvisi, M.G.

Nº 36 Role of TRP domain in TRPV1 functionality Lucia Gregorio-Teruel , Pierluigi Valente, Gregorio Fernández-Ballester, Feng Qin and Antonio Ferrer-Montiel.

Nº 37 Bee venom modulates TRPV1 signaling Henriques, M.S.T., Munaro-Vieira, D., Melo, P.A. and Guimaraes, M.Z.P.

Nº 38 Phenotype of Drosophila TRPM (dTRPM) and molecular determinants of its magnesium conduction Hofmann T. , Chubanov V., Chen X.D., Dietz A.S.

Nº 39 TRPV1 activation in brain following fatty acid amide hydrolase (FAAH)-mediated bioactivation as a strategy for developing novel analgesics Peter M. Zygmunt, David A. Barrière, Christophe Mallet, Anders Blomgren, Laurence Daulhac, Frédéric Liberta, Eric Chapuya, Monique Etienne, Alain Eschalier and Edward D. Högestätt

Nº 40 Gq-coupled receptors potentiate the osmotic activation of TRPC5 Imane Jemal , Anna Lucia Conte, Sergio Soriano, Ana Gomis

Nº 41 TRPV1 is sensitized by CDK5-dependent phosphorylation Thomas Jendryke , Christian H. Wetzel

Nº 42 Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants Michael Ha, Boyi Liu, Daniel N. Willis, John B. Morris and Sven-Eric Jordt

42 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation List of posters Nº 43 Mustard oil (AITC) activates TRPA1 and TRPV1 but facilitates heat responsiveness by sensitizing unknown transducer(s) Tatjana I. Kichko , Tal Hoffmann and Peter W. Reeh

Nº 44 Structure-activity relationship of TRPM2 and ADP ribose analogues Tanja Kirchberger , Ralf Fliegert, Christelle Moreau, Angelika Harneit, Andreas Bauche, Barry V.L. Potter and Andreas H. Guse

Nº 45 Thermosensitive TRP ion channels in regulation of thermoregulatory and immune functions in homoeothermic organism Kozyreva T.V.

Nº 46 Activation of the human cation channel TRPM8 depends on the interaction between transmembrane segments S3 and S4 Mathis Winking, Cornelia Kühn, Daniel Hoffmann, Andreas Lückhoff and Frank Kühn

Nº 47 A link between TRPV1 and obesity-induced hypertension Lihuan Liang , Nichola Marshall, Jennifer Bodkin, Elizabeth Fernandes and Susan D. Brain

Nº 48 The transcription factor AML1, regulates the Transient Receptor Potential Vanilloid-2 (TRPV2) channel-mediated differentiation of glioblastoma stem cells Liberati S. , Morelli M.B., Nabissi M., Amantini C., Farfariello V., Santoni M., Ricci- Vitiani L., Compieta E. and Santoni G.

Nº 49 Engineering of the TRPC3 pore reveals molecular determinants of cation selectivity and gating Michaela Lichtenegger , Michael Poteser, Thomas Stockner, Hannes Schleifer, Christoph Romanin and Klaus Groschner

Nº 50 Phenytoin, nifedipine and carbamazepine induce gingival enlargement trough TRPA1 activation López-González M.J. , Fajardo O., Meseguer V., Valero M., Pertusa M., Belmonte C., Viana F.

Nº 51 FGF-2, calcium signals and the control of neurite growth in chick developing parasympathetic neurons: involvement of TRPC channels Lovisolo D. , Gilardino A., Ruffinatti F.A., Zamburlin P., Farcito S.

Nº 52 Cold-activated TRPM8 channels are activated by the volatile anaesthetic chloroform J.A. Manenschijn , A. Parra, O. Gonzalez, C. Morenilla, C. Belmonte, F. Viana

Nº 53 Heat activated-ion channels TRPV1 and TRPV3 in thermosensation Irène Marics , Pascale Malapert, Stéphane Gaillard, Aziz Moqrich

Nº 54 Parthenolide, contained in the feverfew herb, selectively activates and desensitizes the Transient Receptor Potential Ankyrin 1 (TRPA1) channel S. Materazzi , C. Fusi, S. Benemei, G. De Siena, E. Rossi, G. Trevisan dos Santos, G. Appendino, P. Geppetti and R. Nassini

Nº 55 Substance-P and α-CGRP silencing reduces inflammatory sensitization of TRPV1 Sakthikumar Mathivanan , Isabel Devesa, Christoph Jakob Wolf, Clotilde Ferrandiz Huertas, Rafael Lujan, Antonio Ferrer-Montiel

Nº 56 Calmodulin and S100A1 bind the N-terminal region of TRPM1 Jirku Michaela , Bumba Ladislav, Teisinger Jan

Nº 57 Overexpression, purification and functional characterization of human TRPA1 Lavanya Moparthi , Sabeen Survery, Mohamed Kreir, Per Kjellbom, Edward D. Högestätt, Urban Johanson and Peter M. Zygmunt

International Workshop on Transient Receptor Potential (TRP) Channels 43 List of posters Nº 58 TRPV2 activation induces cytotoxicity in human multiple myeloma cell lines Nabissi M. , Offidani M., Morelli M.B., Discepoli G., Santoni M., Amantini C., Farfariello V., Liberati S., Santoni G. and Leoni P.

Nº 59 TRPV4 is downregulated in keratynocytes in different human skin tumors R. Nassini , V. Maio, S. Materazzi, T. Oranges, C. Fusi, D. Massi

Nº 60 TRPV1 and GABARAP interaction and their effects on the receptor dynamics Ontoria-Oviedo I. , Estévez-Herrera J., Ferrer-Montiel A. and Planells-Cases R.

Nº 61 Setting up a benchmark for the characterization of TRP channels Alex Perálvarez-Marín

Nº 62 The N-glycosylation of TRPM8 channels modulates the temperature sensitivity of cold-thermoreceptor neurons Pertusa, M. , Madrid, R., Morenilla-Palao, C., Belmonte, C., and Viana, F.

Nº 63 Screening for pharmacological tools to target Ca 2+ activated non-selective cation channels Philippaert K. , Colsoul B., Voets T., Vennekens R.

Nº 64 Pore loop residues and ion permeation through TRPC5 Marcus Semtner, Vera Konieczny, Christina Bütfering, Tim Plant

Nº 65 TRPC3: linking soce and calcineurin/nfat-signaling in mast cells Michael Poteser , Bernhard Doleschal, Michaela Schernthaner, Hannes Schleifer, Katrin Tieber, Irene Frischauf, Christoph Romanin, Klaus Groschner

Nº 66 Activation of transient receptor potential ankyrin 1 induces CGRP release from spinal cord synaptosomes T.E. Quallo , J.L. Sorge, S. Bevan, L.M. Broad, A.J. Mogg

Nº 67 Regulation of TRPC channels by immunophilins in human platelets Lopez E., Berna-Erro A., Salido G.M., Rosado J.A. and Redondo P.C.

Nº 68 TRPC6 confers pH sensitivity to OAG-mediated aggregation in mouse platelets Albarran L., Berna-Erro A., Dionisio N., Redondo P.C. , Salido G.M. and Rosado J.A.

Nº 69 Regulation of lysosomal exocytosis by a TRP channel in the lysosome Mohammad A. Samie , Xiang Wang, and Haoxing Xu

Nº 70 In vitro characterisation of TRPV1 in the inflammatory cardiovascular system Claire Sand , Andrew Grant, Manasi Nandi

Nº 71 Agonist- and Ca 2+ -dependent desensitization of TRPV1 protein targets the receptor to lysosomes for degradation Lucía Sanz-Salvador , Amparo Andrés-Borderia, Antonio Ferrer-Montiel and Rosa Planells-Cases

Nº 72 Are ion channels of the TRP family involved in oligodendrocyte progenitor migration? Nina K. Schwering , Irmgard D. Dietzel, Patrick Happel

Nº 73 Block by BCTC reveals TRPV1-independent responses in rat sensory neurons Selescu T. , Reid G., Babes A.

Nº 74 TRPA1-activation on central afferent terminals by 5,6- (5,6-EET) upon nociceptive stimulation causes mechanical hyperagesia Marco Sisignano , Chul-Kyu Park, Carlo Angioni, Dong Dong Zhang, Andrew Grant, Ruirui Lu, Ru-Rong Ji, Clifford J. Woolf, Gerd Geisslinger, Klaus Scholich and Christian Brenneis

Nº 75 Sub-saturating doses of capsaicin on primary cultured sensory neurons reveals TRPV1 sensitization by inflammatory mediators Jared M. Sprague , Clifford J. Woolf

44 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation List of posters Nº 76 Heating up TRP channel drug discovery Sonja Stoelzle , Alison Obergrussberger, Mohamed Kreir, Michael George, Andrea Brüggemann, Niels Fertig

Nº 77 Identification of the C-terminus as a critical molecular determinant of calcium- sensitivity in human TRPA1 channels Lucie Sura , Vlastimil Zíma, Lenka Marsakova, Anna Hynkova , Ivan Barvík, Viktorie Vlachova

Nº 78 Mutations within the first amino acids of the TRP domain differentially affect the function of TRPM8 channel Francisco J. Taberner , Ainara López, Gregorio Fernández and Antonio Ferrer-Montiel

Nº 79 Bimodal action of cinnamaldehyde and camphor on mouse TRPA1 channels Yeranddy A. Alpizar, Maarten Gees, Alicia Sanchez, Aurelia Apetrei, Thomas Voets, Bernd Nilius and Karel Talavera

Nº 80 A positive feedback loop affecting diffusion and cell-surface expression of TRPM8-containing vesicles Carlos A. Toro , Luis Veliz and Sebastián Brauchi

Nº 81 Adenosine-5`-triphosphate (ATP) and phosphatidylinositol 4,5-bisphosphate (PIP 2) are regulators of transient receptor potential melastatin 3 (TRPM3) channel activity B.I. Toth , J. Vriens and T. Voets

Nº 82 mediated TRPA1 receptor activation on monosodium urate crystals-induced nociceptive and edematogenic responses in rats Gabriela Trevisan , Carin Hoffmeister, Mateus Fortes Rossato, Sara Marchesan Oliveira, Romina Nassini, Serena Materazzi, Camilla Fusi, Pierangelo Geppetti, Juliano Ferreira

Nº 83 Behavioral and electrophysiological study of the effects of thermosensitive TRP channel agonists on touch, temperature and pain sensations Tsagareli M.G. , Iodi Carstens M., Tsiklauri N., Carstens E.

Nº 84 Neuronal networks mediating thermotaxis in the marine annelid Platynereis dumerilii Csaba Verasztó and Gáspár Jékely

Nº 85 Importance of a conserved sequence motif in transmembrane segment S3 for the gating of human TRPM8 and TRPM2 Mathis Winking , Cornelia Kühn, Andreas Lückhoff and Frank Kühn

Nº 86 Knocking down of substance-P and α-CGRP on modulating inflammatory sensitization of TRPV1 Christoph Jakob Wolf Farré , Clotilde Ferrandiz-Huertas, Sakthikumar Mathivanan, Isabel Devesa, Antonio Ferrer-Montiel

Nº 87 Functional characterisation of TRPM8 ionchannel of Mus musculus and Gallus gallus Sven Zielke , Jonas Petersen, Christian Wetzel

Nº 88 Waixenicin a inhibits cell proliferation through magnesium-dependent block of TRPM7 channels Susanna Zierler , Guangmin Yao, Zheng Zhang, W. Cedric Kuo, Peter Pörzgen, Reinhold Penner, F. David Horgen and Andrea Fleig

Nº 89 The interactome of the capsaicin receptor TRPV1 Christina Hanack , Henning Kuich, Jana Rossius and Jan Siemens

Nº 90 Novel gating properties of TRPM3 Joris Vriens and Thomas Voets

International Workshop on Transient Receptor Potential (TRP) Channels 45 Posters Nº 1 Peripheral AMPA receptors differentially modulate TRPV1 function in the context of heat and algogen-mediated nociceptor sensitization

Vijayan Gangadharan 1, Rui Wang 2, Nitin Agarwal 1, Bettina Ulzhofer 1, Gary Lewin 2 and Rohini Kuner 1. 1. Institute for Pharmacology, Heidelberg University, Heidelberg, Germany. 2. Max Delbruck Center for Molecular Medicine, Berlin, Germany.

Primary sensory neurons carry out the important function of sensing environmental stimuli. Noxious stimuli of various modalities are sensed by nociceptors present on unmyelinated C-fibres and thinly-myelinated A δ fibers. Physiochemical properties of noxious stimuli, such as heat, are converted into electrical activity by Transient Receptor Potential–generating channels (TRP channels), amongst others, followed by amplification of these transients by sodium channels that generate action potentials. Nociceptive afferents carrying these peripheral inputs form glutamatergic synapses onto second-order neurons mostly in the superficial laminae (I and II) in the spinal dorsal horn. Among the members of TRP family, TRPV1 has been identified as a capsaicin- and heat activated ion channel, which on activation leads to influx of Ca 2+ . Analysis of TRPV1-deficient mice established that capsaicin acts entirely through TRPV1; although TRPV1 is not the only detector for heat in vivo, but can account for majority of heat-evoked responses in vitro (1, 2). We have obtained evidence for a dichotomy of TRPV1 involvement in mediating sensitivity towards thermal stimuli versus capsaicin-evoked hypersensitivity in nociceptors. Using sensory neuron-specific mouse mutants (3), we investigated the involvement of AMPARs present on central terminals of nociceptive afferent fibres in modulating the strength and duration of algogen-induced excitation of nociceptors. Electrophysiological recordings from skin- nerve preparation and behavioral analysis on knockout mice revealed that capsaicin- evoked excitation is reduced in C-nociceptors that lack GluA1-containing AMPARs; however there was no change in sensitivity to noxious heat. Furthermore, peripheral application of AMPAR antagonists significantly attenuated capsaicin-evoked nocifensive responses, but not heat sensitivity or inflammatory heat hyperalgesia. These and additional pharmacological and behavioral experiments indicate that peripheral AMPAR selectively amplify TRPV1-mediated transient potentials evoked by algogens, but not by heat. These findings are relevant towards therapeutic management of pathological pain.

Reference 1. Caterina, M. J. et al . Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288, 306–313 (2000). 2. Davis, J. B. et al . Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 405, 183–187 (2000). 3. Gangadharan V, et al. Peripheral calcium-permeable AMPA receptors regulate chronic inflammatory pain in mice. J Clin Invest. 2011.

46 Posters Nº 2 Effects of cannabinoids on transient receptor potencial channels (TRP) in Alzheimer disease

Aguirre-Rueda D. , Paredes-Brunet P., Gil-Bisquert A. and Vallés S.L. Department of Physiology, School of Medicine, University of Valencia, Spain.

The hallmark in Alzheimer’s disease (AD) is both accumulation of beta-amyloid (Aβ) plates and the presence of TAU protein inside neurons. Furthermore, glial cell activation, occurs after plates appear in brain damaged. Many studies of experimental models of AD have shown abnormalities in calcium regulation in astrocytes and microglia, suggesting contributions of these alterations to neuronal dysfunction and cell death in AD.Our group has shown these alterations in astrocytes and neurons in primary culture comparing A β with control cells. Accumulating evidence in the literature indicate that both TRPV1 and CB1 have many functions inside the brain, some neuroprotective and others neurotoxic. Here we determined the action of cannabinoids on TRP in astrocytes and neuron in culture. Protein expression levels were detected by western-blot technique in astrocytes and neurons in primary culture treated with A β and/or cannabinoids (Win 55, 212-2). Also we detect interactions between TRPV1 and treatment with cannabinoids contributing to neuroprotection o neurodegeneration.

International Workshop on Transient Receptor Potential (TRP) Channels 47 Posters Nº 3 Characterization of voltage-activated calcium currents in TRPM8-expressing cold thermoreceptors

L. Almaraz , C. Morenilla-Palao, J.A. Manenschijn, F. Viana Instituto de Neurociencias UMH-CSIC. Campus de San Juan, Av Ramón y Cajal s/n, San Juan 03550. Alicante, Spain

Peripheral sensory nerve endings that express TRPM8 (transient receptor potential melastatine type 8) are thought to be key sensors of innocuous cold and several recent studies implicate TRPM8-expressing neurons in cold nociception as well. Besides TRPM8, other ionic conductances will contribute to shape the electrical activity of cold receptors at different environmental temperatures. Although much is known about the function, density and molecular diversity of voltage-gated calcium channels expressed in subpopulations of primary sensory neurons, virtually nothing is known about the molecular identity of calcium channels in peripheral cold thermoreceptors. The aim of the present work is to characterize the calcium channels present these neurons. Cold thermoreceptors were recorded with the patch-clamp technique in short-term cultures of trigeminal ganglia obtained from mice in which the yellow fluorescent protein (YFP) was expressed under the control of the TRPM8 gen promoter. In current clamp recordings, these cells were selectively depolarized by 75-100 µM menthol, a potent stimulant of TRPM8 channel. Macroscopic whole-cell calcium currents of small (diameter ≤ 28 µm), strongly fuorescent neurons, were recorded at room temperature using 5 mM extracelullar barium as charge carrier. In voltage-clamp recordings, standard voltage protocols revealed the presence of high-voltage-activated (HVA) and low-voltage-activated (LVA) calcium currents in TRPM8- expressing cells. Depolarizations from a holding potential (Vh) of -90 mV evoked inward currents that activated around -55 mV, peaked between -20 and -10 mV (-320 pA/pF at -10 mV) and presented an apparent reversal potential of +50 mV. More than 90% of the neurons recorded had a transient, LVA current (mean amplitude 30 pA/pF at test pulse to - 40 mV) that disappeared when cells where depolarized from a holding potential of -60 mV. Application of 0.1 and 1 µM of TTA-P2 reduced T-type current amplitude by 40 and 80% respectively. T-type current present in TRPM8+ neurons was highly sensitive to blockade by nickel (IC50 = 6.5 µM) and zinc (47% inhibition at 1 µM) while its amplitude increased by 32% in the presence of 100 µM L-cysteine. These data indicate that cold thermorreceptors express predominantly Cav 3.2 channels. Preliminary results, using quantitative RT-PCR in YFP- TRPM8(+) neurons isolated by FACS from early postnatal DRGs mice support the above proposal. In current clamp recordings, after application of hyperpolarizing pulses, a rebound depolarization typical of cold receptors was recorded in TRPM8+ neurons. TTA-P2 (1 µM) and nickel (10 µM) decreased the amplitude of the rebound depolarization and therefore the probability of action potential generation. This effect persisted in the presence of 30 µM ivabradine (a selective blocker of HCN channels) indicating that it was specific of T-type current blockade. We also measured the excitability of TRPM8+ neurons during the ADP that follows the action potential spike evoked by injection of brief suprathreshold pulse current to cells held at hyperpolarized potentials (-80 -85 mV). The rheobase for a second action potential during the ADP increased in the presence of TTA-P2 and nickel by 41 and 24% respectively (p ≤0.05). We are currently investigating the subtypes of HVA calcium currents expressed in TRPM8(+) neurons. Preliminary results show that L-type currents are abundant in these neurons since nifedipine blocks 39% of the total calcium current (test pulse to -10 mV from a Vh of -60 mV).

Supported by project SAF2010-14990 to FV and BFU2008-04425 to C

48 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 4 Anesthetic effect of the TRPA1 specific agonist cinnamaldehyde

Yeranddy A. Alpizar 1, Laura van Gerven 2, Wouter Everaerts 1,3 , Brett Boonen 1, François Vermeulen 4, Dirk De Ridder 3, Peter Hellings 2, Thomas Voets 1, Karel Talavera 1 1Lab. of Ion Channel Research, Dept. Molecular and Cellular Medicine. 2Lab. of Clinical Immunology, Dept. of Microbiology and Immunology. 3Lab. of Experimental Urology, Dept. of Development and Regeneration. KU Leuven, Herestraat 49, 3000 Leuven, Belgium

Cinnamaldehyde (CA) and mustard oil (MO) are natural compounds that activate TRPA1, a member of the family of transient receptor potential (TRP) cation channel expressed in nociceptive neurons. In human subjects, they are perceived as pungent, inducing a burning and tingling sensation when orally administered. Thus far, most of the evidence on the pathophysiological role of TRPA1 has been gathered using MO and CA under the assumption that they are specific agonists of this channel. However, we have recently reported that multiple noxious effects of MO, including visceral irritation and acute pain, are partly mediated by the capsaicin receptor TRPV1. These findings leave researchers with CA as the best option to induce specific activation of TRPA1. Accordingly, we used this compound in several models of chemosensation in mice, including oral aversion, visceral irritation and acute pain and inflammation. Cystometry experiments revealed that intravesical infusion of 10 mM CA induces a significantly smaller decrease of the intracontractile interval (ICI) than infusion of MO. Similarly, a forced drinking experiment showed that 10 mM CA induced less oral avoidance than 10 mM MO. Unlike for MO, both CA-induced bladder irritation and avoidance were absent in Trpa1 knockout mice. Finally, CA (10 mM) induced significantly less pain behavior compared to MO when injected in the mouse hind paw. Next, we tested whether the differences in irritation properties of CA and MO found in the mouse are also present in humans. For this, we performed a series of experiments on healthy volunteers to whom CA or MO were applied into the nasal cavity via an aerosol. Irritation and pain sensations were assessed using a Visual Analogue Scale (VAS) and with measurements of nasal mucosal potentials (NMPs). Application of aerosols containing up to 20 mM CA did not induce any irritation or pain responses as determined from the VAS scores nor any significant NMP signals. In contrast, MO induced clear responses at the same concentrations. Thus, in both mice and humans, CA produces only very weak responses when compared to MO, which is in sharp contrast with its powerful agonist action on TRPA1. These data strongly suggests that, at concentrations up to 10 mM, CA fails to trigger neuronal excitatory responses leading to acute irritation and pain. We therefore hypothesized that this compound inhibits neuronal firing. In agreement with this hypothesis, we found that hind paw injection of a mixture of CA and MO (10 mM each) induces significantly less nociceptive behavior than the injection of MO alone. This effect does not seem to be mediated by a mechanism depending on TRPA1, as co-injection of MO with the alcohol analog of CA, which is inactive on TRPA1, also induced a highly attenuated pain response when compared to MO alone. Furthermore, CA was also able to dramatically reduce the nocifensive response elicited by capsaicin, a highly specific TRPV1 agonist. Likewise, we found that, in the presence of CA, MO induced significantly weaker nasal irritation in humans than when applied alone, as determined by VAS scores and NMPs. The structure of CA resembles the general structure of aminoester drugs commonly used + as local anesthetics (LA). LAs potently block voltage-gated Na (Na V) channels, thereby inhibiting the excitability of sensory neurons. In patch-clamp experiments we have found that CA blocks voltage-dependent sodium currents (for details see abstract from Boonen et al .). In conclusion, besides activating TRPA1, CA also exerts a blocking effect on Na V channels, locally impairing action potential initiation and propagation.

International Workshop on Transient Receptor Potential (TRP) Channels 49 Posters Nº 5

Cross-talk between ααα1d -adrenergic receptor ( ααα1d -AR) and Transient Receptor Potential Vanilloid 1 (TRPV1) triggers the proliferation of PC-3 prostate cancer cells

Amantini C. 1* , Farfariello V. 1,3, , Morelli M.B. 1, Nabissi M. 1, Liberati S. 1,2 , Santoni M. 4, Ranzuglia V. 1, Cardinali C. 1, Filosa A. 5, Pieramici T. 5, Ranaldi R. 5, Piergentili L. 6, Quaglia W. 6 and Santoni G. 1 1School of Pharmacy, Section of Experimental Medicine, University of Camerino, Camerino, Italy. 2Dept Molecular Medicine, Sapienza University, Rome, Italy 3Dept Urology and Andrology, University of Perugia, Perugia, Italy 4Dept of Clinic Oncology, Polytechnic University of Marche, Ancona, Italy. 5Dept of Clinical Pathology, Section of Pathological Anatomy, Macerata, Italy. 6School of Pharmacy, Medical Chemistry Unit, University of Camerino, Camerino, Italy. * [email protected]

Growing evidence supports the role of α1-ARs in the direct mitogenic effect of catecholamines on prostate cancer cell growth. We have previously reported the expression of α1D -AR on PC-3 prostate cancer cells and the ability of noradrenalin (NA) to stimulate PC-3 cell proliferation in a α1D -AR-dependent manner (Quaglia et al., 2005). In addition, TRPV1 expression was also found in normal prostate tissue and prostate cancer cells (Sanchez et al., 2006). Aim of this study was to investigate the relationship between α1D -AR and TRPV1 receptors and the involvement of TRPV1 in NA-induced proliferation of PC3 cells. By using confocal microscopy we found a co-localization of α1D -AR and TRPV1 receptors in the membrane and cytosol of PC3. Moreover, a band of 70 kDa likely corresponding to the α1D -AR, in PC3 lysates, immunoprecipitated with an anti-TRPV1 antibody, was found. Treatment of PC3 cells with NA strongly stimulated proton release, calcium influx and cell proliferation that were completely reverted by WS433 and CPZ, respectively α1D -AR and TRPV1 antagonists, when utilized in combination. We also evaluated the ability of NA to induce calcium influx in α1D-AR or TRPV1 silenced PC3 cells. α1D-AR silencing reverted the early (up to 50 sec) calcium overload whereas TRPV1 knock-down resulted in a marked reduction of delayed (50-180 sec) calcium increase. To further address the role of α1D-AR/TRPV1 receptor, PC3 cells were double silenced and then treated with NA. Results demonstrated that the NA- induced increase of survival and proliferation is totally abrogated in α1D-AR/TRPV1 silenced cells. Finally, we investigated the signalling pathways involved in NA stimulation evaluating proton release and cell viability in PC3 cells treated with NA in the presence of Phospholipase C (PLC), Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) inhibitors. Our data showed that PLC and PKC inhibitors markedly reduce (about 50-60%) the NA-induced effects, whereas ERK inhibitor displays a less intensive activity (20-30%). By western blot analysis, we also evidenced that NA stimulates, in a time dependent manner, ERK and PKC substrate phosphorylation that is completely inhibited by WS433 and partially reverted by CPZ. Overall our results demonstrated that a functional and structural cross-talk between α1D-AR and TRPV1 receptors controls the NA-induced proliferation of prostate cancer cells.

Funding: This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) National Grant 2011-2013 (Number 11095), and MIUR National Grant 2010-11.

50 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 6 The diabetic marker methylglyoxal produces painful neuropathy by stimulating TRPA1

David A. Andersson 1, Clive Gentry 1, Angelika Bierhaus 2, Thomas Fleming 2, Peter P. Nawroth 2 and Stuart Bevan 1 1Wolfson CARD, King’s College London, SE1 1UL, London, United Kingdom. 2University Hospital Heidelberg, Heidelberg, Germany

Diabetic neuropathy is a common and severe complication of long-standing diabetes and one of the major etiologies of chronic neuropathic pain. Oxidative stress and an increased production of advanced glycation endproducts (AGE) are prominent among the candidates that have been proposed to be responsible for the development of diabetic neuropathy. Although it is well established that a number of lipid peroxidation products and reactive oxygen species exert a direct pronociceptive effect by stimulating TRPA1, raised levels of these factors are also seen in conditions that are not primarily characterized by pain and hypersensitivities. However, during hyperglycemic episodes in diabetic patients there is a specifically increased production of the electrophilic dicarbonyl compound methylglyoxal (MG). Here we report that MG stimulates TRPA1 channels directly and evokes pain as well as hypersensitivity to heat, cold and mechanical stimulation in a TRPA1 dependent fashion. 2+ Application of MG evoked increases in [Ca ]i in CHO cells expressing mTRPA1 (EC 50 =0.9±0.1mM) or hTRPA1 (EC 50 =0.7±0.2mM), but not in untransfected CHO cells or in the absence of extracellular Ca 2+ . In DRG neurons cultured from Trpa1 +/+ mice, MG 2+ induced [Ca ]i-responses in about two thirds of capsaicin sensitive neurons, but was 2+ -/- without effect on [Ca ]i in DRG neurons from Trpa1 mice. Since methylglyoxal is a shortlived, reactive compound formed intracellularly and electrophilic compounds are thought to stimulate TRPA1 by modifying intracellular cysteine residues in the channel protein, we examined the effect of MG applied to the intracellular face of excised inside-out patches. MG was much more potent in this configuration than when applied extracellularly, stimulating TRPA1 channel activity with an EC 50 of 74 M, consistent with MG acting at intracellular sites in the TRPA1 protein. Several cysteine residues are thought to mediate the effects of electrophilic, thiol-reactive compounds on TRPA1 and we therefore examined 2+ the importance of three of these residues for MG induced [Ca ]i-responses by substituting them for serine (C621S, C641S and C665S). Although each of the amino acid substitutions significantly reduced the agonist potency of mustard oil, none of the mutations affected MG 2+ evoked [Ca ]i-responses. Local administration of TRPA1 agonists evokes pain acutely and we next examined whether this was also the case with MG. Trpa1 +/+ and Trpa1 -/- mice were given intraplantar injections of MG (250 nmoles) and the duration of pain-related behaviours was measured during 5 min following injection. In these experiments, MeGly induced a significant nociceptive response in Trpa1 +/+ mice, but was without effect in Trpa1 -/- mice, confirming that TRPA1 is required for the acute pronociceptive effect of MeGly in vivo . To model the long-lasting, widely increased MG levels associated with hyperglycemia in poorly controlled diabetes more accurately, we used a selective glyoxalase-1 (GLO-1) inhibitor. GLO-1 converts MG in sensory neurons to lactate and its inhibition thereby leads to increased intracellular levels of MG. Mice treated with the GLO-1 inhibitor for 2 weeks developed a marked hypersensitivity to heat, cold and mechanical stimulation that was completely absent in Trpa1 -/- mice. Our results demonstrate that the glucose metabolite MG stimulates TRPA1 and produces a TRPA1 dependent hypersensitivity to stimulation with several sensory modalities. These findings suggest that elevated levels of MG contribute to the development of painful diabetic sensory neuropathy by stimulating TRPA1.

International Workshop on Transient Receptor Potential (TRP) Channels 51 Posters Nº 7 Role of TRPA1 in the peripheral vasculature in vivo : Increasing evidence for a CGRP and nitric oxide-sensitive mechanism

Aisah A. Aubdool 1, Bodkin J.V. 1, Xenia Kodji 1, Ross King 1, Clive Gentry 2, Elizabeth S. Fernandes 1, Stuart Bevan 2 and Susan Brain 1 1Cardiovascular Division and Centre for Integrative Biomedicine & 2Wolfson CARD Centre, King’s College London, London SE1 9NH, UK.

Transient receptor potential ankyrin 1 (TRPA1) is expressed by primary afferent neurons and has polymodal activation, including cold temperature, pungent products from vegetables, reactive oxygen species and mechanical stimuli (Aubdool & Brain, 2011; Ryckmans et al., 2011). Our recent findings provided evidence indicating the potential of TRPA1 as an oxidant sensor for vasodilator responses in vivo (Graepel et al., 2011). Furthermore, TRPA1 can also influence changes in blood pressure of possible relevance to autonomic system reflexes (Pozsgai et al., 2010). The current study explores the in vivo effects of the proposed TRPA1 agonist cinnamaldehyde using various pharmacological inhibitors and genetically modified mice. TRPA1 WT and KO mice were inplanted with radiotelemetry devices (PA-C10 probe catheterising the left carotid artery) for the measurement of conscious blood pressure at baseline. Cutaneous blood flow was measured in mice (20-30g) anaesthetised with ketamine (75mg/kg) and medetomidine (25mg/kg) i.p. using non-invasive laser Doppler techniques and images were captured using a Full-Field laser perfusion imager. 20 l of cinnamaldehyde (10%) or vehicle (10% DMSO in ethanol) was applied topically to the ipsilateral and contralateral ears, respectively and blood flow was recorded for 30 min. All animals were randomly assigned to drug-treated or their respective control groups. Results were expressed as mean + s.e.m. in arbitrary flux units (x10 3 flux units), and analysed by 2-way ANOVA + Bonferroni’s test. Our studies show that cinnamaldehyde significantly increased ear blood flow, as compared to vehicle ears (176.2 + 27.9 vs 35.0 + 0.7 (x10 3 flux units), p<0.01, n=5) and this response was significantly attenuated in TRPA1 KO mice (n=3-4, p<0.01). A similar trend was observed in mice pre-treated i.p. 30 min with the TRPA1 antagonists HC030031 (100mg/kg, n=5) or TCS5861528 (10mg/kg, n=5). Telemetry data suggests that both TRPA1 WT and KO mice have similar systolic, diastolic and mean blood pressure (BP), as well as heart rate at basal physiological conditions (daytime systolic BP WT 113 + 16mmHg, KO 117 + 8mmHg, n=10). The selectivity of cinnamaldehyde for other TRP channels such as TRPV1 and TRPM8 was also tested. Neither TRPV1 deletion nor pharmacological blockade of TRPM8 caused any change in cinnamaldehyde-induced vasodilatation, as shown in TRPV1 KO mice, on a C57BL/6 background (n=5) and CD1 mice pre-treated with AMTB (10mg/kg, n=5), respectively. We conclude that cinnamaldehyde causes vasodilatation by selectively activating TRPA1 but not TRPV1 or TRPM8 in vivo. Our data also showed that cinnamaldehyde- induced vasodilatation involves a neurogenic component where CGRP KO (n=3) and WT mice pre-treated with CGRP receptor antagonist CGRP 8-37 (400nmol/kg, i.v. , n=5) display significantly lower vasodilatation than their respective control pre-treated groups (p<0.01, p<0.0001 respectively). We also observed a significant decrease in responses with WT pretreated with a combination of CGRP 8-37 and substance P NK1 receptor antagonist SR140333 (480nmol/kg, i.v , n=5, p<0.001) ). The cyclo-oxygenase inhibitor indomethacin (5mg/kg, s.c. 30 min, n=3) had no effects on cinnamaldehyde- induced vasodilatation, Further work shows that neuronal nitric oxide synthase (nNOS) inhibition by SMTC (10mg/kg, i.v. n=6) caused a significant decrease in cinnamaldehyde-induced blood flow responses (p<0.001). A cocktail treatment of

52 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters

CGRP 8-37 , SR140333 and SMTC ( i.v. n=5-6) significantly attenuated cinnamaldehyde- induced vasodilatation in WT mice (p<0.0001). This study highlights the potent ability of TRPA1 to mediate peripheral sensory vasodilatation via cutaneous stimulation. We present novel evidence that the mechanisms involve NO derived from a neuronal isoform, in addition to the release of the potent vasodilator neuropeptide CGRP. By comparison, TRPA1 does not appear to contribute to the regulation of baseline blood pressure in studies with TRPA1 KO mice.

Aubdool .A., Brain S.D. (2011). JID Symp Proc 15: 33-9. Graepel R., Fernandes E.S., Aubdool A.A. et al. (2011). J Pharmacol Exp Ther 337, 117-24. Pozsgai, G., Bodkin, J.V., Graepel, R., et al. (2008). Cardiovasc Res 87 , 760-8. Ryckmans T., Aubdool A.A., Bodkin J.V., et al. (2011). Bioorg Med Chem Lett 21, 4857-9.

This study was supported by a BBSRC-led IMB capacity building award.

International Workshop on Transient Receptor Potential (TRP) Channels 53 Posters Nº 8 D-series resolvins potently suppressed sensory TRP activities leading to multiple anti-nociception

Sangsu Bang , Sungjae Yoo, Tae-Jin Yang, Ji Yeon Lim, Sun Wook Hwang Korea University Graduate School of Medicine, Seoul, Korea

Transient receptor potential ion channels expressed in primary afferents and skin keratinocytes (sensory TRPs) play a central role for peripheral detection of noxious insults. While natural and synthetic ligands have been found to act on sensory TRPs, little is known about endogenous substances that negatively regulate these TRP activities. Lipidergic nature of a number of TRP-activating endogenous ligands has been known. We focused on D-series resolvins (RvDs) that are anti-inflammatory and pro-resolving lipid molecules naturally generated during inflammatory processes. We asked whether RvDs are able to affect the sensory TRP channel activation. We examined the effects of 17(S)-RvD1 and 17(R)-RvD1 on the six sensory TRPs using calcium imaging and whole cell electrophysiology experiments with a HEK cell heterologous expression system, cultured sensory neurons and HaCaT keratinocytes. We also checked changes in agonist-specific acute nociceptive behaviors (licks, flicks and flinches) and TRP-related mechanical and thermal pain behaviors using Hargreaves, Randall-Selitto and von Frey assay systems with or without inflammation. As a result, 17(S)-RvD1 acted on multiple TRP channels and on the other hand, 17(R)- RvD1 specifically affected TRPV3 activity. 17(S)-RvD1 inhibited the activities of TRPA1, TRPV3 and TRPV4 at nanomolar and micromolar levels. Consistent attenuations in agonist-specific acute pain behaviors by immediate peripheral administration with 17(S)-RvD1 were also observed. Local treatment with 17(S)-RvD1 significantly prevented mechanical and thermal hypersensitivity in inflamed tissues. For 17(R)-RvD1, only TRPV3-mediated in vitro responses were suppressed upon its application at nanomolar and micromolar concentrations. TRPV3-specific pain behaviors were also attenuated by locally injected 17(R)-RvD1. The administration with 17(R)-RvD1 significantly reversed the thermal hypersensitivity that occurred under inflammation. The inhibitory mechanism appears due to a shift in voltage-dependence of TRP channels. In conclusion, RvDs are potent endogenous sensory TRP channel inhibitors, which is a rarely found in nature. The results of our studies implicate that our body has an internal antinociceptive potential with endogenous TRP-regulating lipids and also that RvDs may help understand pain mechanisms involving interactions of TRP with pro-resolving substances.

54 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 9 TRPV1 receptor potentiation contributes to pruritogenesis and thermal hypersensitivity in a rat model of liver disease

Majedeline Belghiti *, Judith Estévez-Herrera*, Carla Giménez-Garzó*, Alba González- Usano*, Carmina Montoliu ‡, Antonio Ferrer-Montiel §, Vicente Felipo*, Rosa Planells- Cases* *Centro de Investigación Príncipe Felipe. Valencia. Spain ‡Fundación Investigación Hospital Clínico de Valencia. INCLIVA. Valencia. Spain §Instituto de Biología Molecular y Celular. Universidad Miguel Hernández de Elche. Spain

Background & aims: Persistent pruritus associated to chronic liver disease seems to arise from the interplay of disease-specific itch-mediators between skin and peripheral and central nervous systems. Because the unknown pathogenesis, including mediators and the underlying neuronal mechanism(s), therapeutic intervention often leads to limited or null symptoms relief. We hypothesized that, similar to patients with primary cholestasis, a standard rat model with biliar obstruction by bile duct ligation (BDL) will display increased scratching bouts allowing validation as a model of cholestatic pruritus. We addressed whether a concomitant peripheral neuronal sensitization arise with enhanced scratching. Specific signalling pathways and/or subpopulations of itch- responsive neurons were further analyzed.

Methods: Time-course of spontaneous scratching and peripheral sensitization behaviours were explored in BDL or control (sham) rats. Contribution of signalling pathways was analyzed by pharmacological intervention. Functional analysis of BDL and sham nociceptor primary neurons was assessed by intracellular Ca2+ fluorometry. Western blot and inmunocytochemistry were also performed.

Results: BDL rats display enhanced scratching, thermal hyperalgesia and mechanical allodynia, evidencing neuroinflammation. While antihistaminics were ineffective, the Protease Activated Receptor-2 (PAR-2) pathway was engaged in itching and peripheral sensitization of BDL rats. Furthermore, transient receptor potencial vanilloid 1 (TRPV1) played a pivotal role mediating both noxious and pruritogenic signalling. Sensitized TRPV1 activity in BDL was associated to receptor overexpression in DRG neurons, concomitant with an increased proportion of small-diameter peptidergic neurons along with a shifted-expression to medium-sized DRG nociceptors.

Conclusion: Chronic pruritus in liver disease is mediated by TRPV1 sensitization, most likely through PAR-2 signalling. Accordingly, pharmacological TRPV1 intervention appears to be a valuable therapeutic approach to itching associated to hepatic disease.

International Workshop on Transient Receptor Potential (TRP) Channels 55 Posters Nº 10 Modulation of voltage-dependent sodium currents by the TRPA1 agonist cinnamaldehyde

Brett Boonen 1, Bristol Denlinger 2, Yeranddy A. Alpizar 1, Thomas Voets 1, Victor M. Meseguer 2, Carlos Belmonte 2, Karel Talavera 1 1Laboratory of Ion Channel Research, KU Leuven, Herestraat 49, 3000 Leuven, Belgium and 2Unit of Cellular and Systems Neurobiology, Institute of Neuroscience of Alicante, Sant Joan d’Alacant, Spain.

Cinnamaldehyde (CA) is a highly reactive compound that has been widely used in experimental models of neurogenic inflammation and has been identified as a potent specific agonist of the broadly tuned chemonociceptor TRPA1. We have recently used CA to study the role of TRPA1 in several models of chemonociception in mice and humans, and surprisingly, we have found that CA only produces very weak avoidance, pain or visceral irritation responses. Furthermore, we have found that co-injection of CA with mustard oil (MO) or capsaicin induced pain responses that are significantly smaller than those evoked by these compounds alone (see accompanying Abstract Alpizar et al. ). Action potentials in sensory neurons are generated and sustained by the voltage-gated Na + channels. Inhibition of these channels leads to abrogation of action potential generation and conduction, which is perceived in vivo as an anesthetic effect. We thus hypothesized that CA inhibits the trigger of action potential firing underlying pain sensation. To test this possibility we performed extracellular electrophysiological recordings in trigeminal afferent fibers innervating the tongue using an ex vivo mouse preparation. We found that application of 200 µM MO induced an increase in the firing frequency in a number of fibers and did not have a significant effect on the response to cooling from 35 to 12 °C. In contrast, application of 200 µM CA induced dramatic decrease of the basal firing and the response to cooling, which indicates that, indeed, this compound inhibits afferent firing. Noting the structural similarities between CA and classical local anesthetics, we investigated whether CA blocks voltage-gated Na + channels in sensory neurons. Voltage-dependent Na + currents were recorded in trigeminal neurons of Trpa1 knockout mice using whole-cell patch-clamp. Currents were routinely elicited by 30 ms- lasting voltage steps from a holding potential of -75 mV to 10 mV, every 2 s. Application of CA induced a concentration-dependent decrease of the Na + current. This inhibitory effect was largely and quickly reversible, suggesting that the underlying mechanism is not based on covalent modification of cysteine residues as described for the activation of TRPA1. To determine whether CA has differential effects on the various Na + channel types expressed in sensory neurons, we performed a series of experiments in which cells were exposed to 0.5 µM tetrodotoxin (TTX). The remaining Na + currents (TTX- resistant) were not sensitive to CA, indicating that only TTX-sensitive Na + channels are inhibited by this compound. In another series of experiments we characterized the effects of CA on the quasi steady-state (activation and inactivation curves) and kinetic properties of Na + currents. In conclusion, our data demonstrate that besides activating TRPA1, CA induces an inhibitory effect on voltage-gated TTX-sensitive Na+ channels, which may lead to the inhibition of action potential firing. The identification of this anesthetic-like property of CA is essential for a full understanding of the effects of this compound when used to study the pathophysiological roles of TRPA1.

56 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 11 Protons stabilize the closed conformation of the gain-of- function mutants of TRPV1 channel

Boukalova S .1,2 , Teisinger J. 2, Vlachova V. 2 1. Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague 2, Czech Republic 2. Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic

The vanilloid transient receptor potential channel TRPV1 is a molecular integrator of noxious stimuli, including capsaicin, heat and protons. Despite clear similarities between the overall architecture of TRPV1 and voltage-dependent potassium (Kv) channels, the extent of conservation in the molecular logic for gating is unknown. In Kv channels, a small contact surface between S1 and the pore-helix is required for channel functioning. To explore the function of S1 in TRPV1, we used tryptophan- scanning mutagenesis and characterized the responses to capsaicin and protons. Wild-type-like currents were generated in 6 out of 11 mutants; three mutants (M445W, A452W, R455W) were non-functional. The conservative mutation R455K in the extracellular extent of S1 slowed down the capsaicin-induced activation and prevented normal channel closure. This mutant was neither activated nor potentiated by protons, on the contrary, protons promoted rapid deactivation of the currents. Similar phenotypes were found in two other gain-of-function mutants and also in the pore-helix mutant T633A known to uncouple proton activation. We propose that the S1-pore interface might serve to stabilize conformations associated with TRPV1 channel gating.

This work was supported by Czech Science Foundation Grant 305/09/0081, P301/10/1159 and GAUK 500512 (Charles University in Prague).

International Workshop on Transient Receptor Potential (TRP) Channels 57 Posters Nº 12 Novel endogenous N-acyl amides activate TRPV1-4 receptors and are regulated in an acute model of inflammation

Heather B. Bradshaw *, Siham Raboune, Jordyn M. Stuart Department of Psychological and Brain Sciences at Indiana University, Bloomington Indiana 47405

During the last decade, a novel family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (), called N- acyl amides has emerged as a family of biologically active compounds. Examples are N-arachidonoyl dopamine and N-oleoyl dopamine, structurally similar to the exogenous TRPV1 agonist capsaicin that are potent endogenous ligand at TRPV1 receptors; however, their distribution is limited to only a few brain areas (striatum and hippocampus) and they are not produced where TRPV1 receptors are more highly expressed in the periphery. Similarly, other endogenous TRPV agonists such as Anandamide and the N-acyl taurines have either relatively low abundance or lower efficacy at the receptors, suggesting that the full complement of endogenous TRPV activation is not fully recognized. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, we have screened over 80 N-acyl amide molecules for activity at TRPV1-4 in HEK 293 expression systems using Fura2AM calcium imaging and have identified 21 novel N-acyl amides that collectively activate (as an agonist or antagonist) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we show that levels of at least 8 of these N-acyl amides that activate TRPVs are regulated in paw skin and brain after intraplantar vehicle or carrageenan injection. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at TRPV receptors in the context of inflammation.

Acknowledgement: This work was supported by NIH DA032150.

58 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 13 TRPV1 and TRPA1 play distinct roles in itch and associated skin inflammation induced by LTB 4

Elizabeth S. Fernandes 1, Chi-Teng Vong 1, Samuel Quek 1, Jessica Cheong 1, Aisah A. Aubdool 1, Jennifer V. Bodkin 1, Richard Heads 1 and Susan D. Brain 1,2 1Vascular Biology Group and 2Centre for Integrative Biomedicine; Cardiovascular Division, King’s College London, London, England

Itch is a common symptom of many systemic and dermatological diseases and it is a sensation that causes the desire to scratch. Many physiological and psychogenic factors are involved in itch and a fine interaction between cutaneous and neuronal effectors accounts for the complexity of this sensation (Davidson and Giesler, 2010). Transient receptor potential (TRP) channels such as TRP vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1); are located in neurons and keratinocytes and are now recognized as important regulators of itch transmission (Costa et al., 2008; Gooding et al., 2010; Wilson et al., 2011). We now seek to increase the understanding of the role of TRP channels in itch by using LTB 4, a potent polymorphonuclear (PMN) leukocyte chemoattractant, as a triggering agent. For this purpose, female CD1 mice (20-25 g) were used. Briefly, one day before the experiments, the hair at the back of the mouse neck was shaved. On the day of the experiments, the animals were placed individually in a clear acrylic cage for at least 1 h, to acclimatize. After this period, mice received an intradermal (i.d.) injection of LTB 4 (0.1 nmol/site; 50 µl) or vehicle (0.1% BSA in saline; 50 µl) and the total number of scratches over 40 min was quantified. Superoxide and myeloperoxidase activity (an indicator of PMN accumulation) were quantified in LTB 4- treated skin. Vehicle-treated samples were used as control. Here, we demonstrate that itch induced by the i.d. injection of LTB 4 (0.1 nmol/site) is blocked by intraperitoneal (i.p) pre-treatment with the TRPA1 antagonist HC-030031 (100 mg/kg, -30 min). Pre- treatment with the selective TRPV1 antagonist SB366791 (0.5 mg/kg, i.p., -30 min) had a similar effect on LTB 4-induced itch. These results suggest the involvement of TRPA1 and TRPV1 channels. Itch was also reduced by intravenous injection with the leukocyte migration inhibitor fucoidin (10 mg/kg, - 15 min). LTB 4 caused the release of superoxide which was inhibited by the pre-treatment with HC-030031, SOD or fucoidin. The TRPV1 antagonist SB366791 did not exhibited any effects on superoxide release. Interestingly, LTB 4 increased skin myeloperoxidase that was reduced by SB366791 and fucoidin but not by HC-030031 pre-treatment. Our results show that LTB 4-induced itch and skin inflammation requires leukocyte accumulation which is dependent on TRPV1 activation whilst releasing superoxide in a TRPA1-dependent mechanism. We suggest that TRPA1 and TRPV1 contribute to skin inflammation and itch via distinct mechanisms which are relevant to skin disease.

This work was supported by the Arthritis Research UK, the BHF and the BBSRC.

Costa et al. (2008) Br. J. Pharmacol., 154: 1094-1103. Davidson, S., Giesler, G.J. (2010) Trends in Neurosci., 33:550-558. Gooding et al. (2010) Int. J. Dermatol., 49: 858-865. Wilson et al. (2011) Nat Neurosci. , 14: 595-602.

International Workshop on Transient Receptor Potential (TRP) Channels 59 Posters Nº 14 Decreased asthmatic responses to Aspergillus and house dust mite in mice deficient in the sensory neuronal ion channel TRPA1

Ana Isabel Caceres , Sven-Eric Jordt Dept. of Pharmacology, Yale University School of Medicine. New Haven, CT (USA), [email protected]

Asthma is an inflammatory disorder caused by airway exposures to allergens or chemical irritants. Studies focusing on immune, smooth muscle and airway epithelial function revealed many aspects of the disease mechanism of asthma. However, the limited efficacies of immune-directed therapies suggest the involvement of additional mechanisms in asthmatic airway inflammation. The airways are densely innervated by peripheral sensory neurons. Activation of chemosensory C-fibers by chemical irritants triggers defensive reflexes such as cough and sneezing, and initiates airway constriction and glandular fluid secretion. Local neuronal release of pro-inflammatory peptides promotes edema formation and vascular leakage. TRPA1 is an irritant-sensing ion channel expressed in airway chemosensory nerves. We recently demonstrated that TRPA1 is essential for airway inflammation and hyperreactivity in ovalbumin-induced asthma in mice. TRPA1 deficient mice, or mice treated with the TRPA1 antagonist, HC-030031, showed strongly diminished pulmonary inflammation and airway hyperreactivity (1). Here, we demonstrate that TRPA1 is also contributing to the induction of asthma following respiratory exposures of mice to the fungal allergen, Aspergillus fumigatus , or to house dust mite extract. We utilized TRPA1 -/- mice and two different TRPA1 inhibitors, HC-030031 and A-967079, to probe the role of TRPA1 in Aspergillus and house dust mite induced asthma models. Animals were sensitized and challenge with the allergens intranasally, without any previous systemic administration. Allergen- challenged TRPA1 -/- mice displayed a significant reduction in bronchoalveolar lavage (BAL) eosinophils compared to wild-type mice. Animals pretreated with TRPA1 antagonists also showed diminished cell infiltration in the lungs. Mice exposed to Aspergillus developed a mild airway hyper-reactivity (AHR) that was diminished in mice treated with HC-030031. Similar to HC-030031, the orally bioavailable TRPA1 antagonist, A-967079, inhibited asthmatic inflammation in the ovalbumin induced mouse model. These results suggest that TRPA1 is a key integrator of the interaction between the immune and nervous systems in the airways, and confirm that the sensory channel drives the asthmatic airway inflammation following inhaled challenge by diverse allergens. We assert that TRPA1 may represent a promising pharmacological target for the treatment of asthma and other allergic inflammatory conditions.

1: Caceres et al. A sensory neuronal ion channel essential for airway inflammation and hyperreactivity in asthma. Proc Natl Acad Sci U S A. 2009 Jun 2; 106(22):9099-104.

60 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 15 Co-application of TRPV1 and TRPV4 antagonists: A synergistic effect to overcome bladder hyperactivity induced by urinary bladder inflammation

Charrua A .1,2,3 , Boudes M.4, De Ridder D.5, Cruz C.D.1,2 , Cruz F.1,3 1 IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal 2 Department of Experimental Biology, Faculty of Medicine of University of Porto, Portugal 3 Department of renal, urologic and infectious disease, Hospital S. João, Porto, Portugal 4 Department of surgery, KU Leuven, Louvain, Belgium 5 Department of molecular cell biology, KU Leuven, Louvain, Belgium

It is known that TRPV1 and TRPV4 are expressed in the urinary bladder, being expressed in the urothelium. Although TRPV1 expression in bladder nerve fibres is widely accepted, only now it was demonstrated that bladder nerve fibres express TRPV4. Although 30% of the entire population of L6 dorsal root ganglia (DRG) neurons co-express TRPV1 and TRPV4, it is still unknown if that neuronal population project to the urinary bladder or if remains constant upon inflammation. TRPV1 and TRPV4 don’t seem to play a role in the normal urinary bladder micturition reflex. However, it was shown that the application of TRPV1 or TRPV4 antagonist is able to reduce cystitis-induced bladder hyperreflexia. Hence, our aim was to study: (1) the effect of cystitis on the expression of TRPV1-TRPV4 in L6-S1 DRG population that projects to the urinary bladder (2) the effect of TRPV1 and TRPV4 antagonists co-application in the bladder reflex activity For that, the bladder of female Wistar rats was injected with fluorogold (FG). Five days after FG injection, animals were divided in two groups: lipopolysaccharide (LPS)- inflamed group and control group. LPS-treated group was intravesical instilled with 2 mg/ml of LPS, for 1 h. Control group was instilled with saline for 1 h. Twenty-four hours later, animals from both groups were perfused and L6 DRG harvested, sectioned and immunoreacted against TRPV1-TRPV4. Images were acquired and analysed. Furthermore, adult female rats were divided in 6 groups: three groups of LPS-inflamed and three groups of vehicle-treated animals. For experimental procedure, animals were anaesthetised and a catheter was inserted in inferior cava vein for antagonist injection. Cystometries were performed before and after animals were treated with saline and increasing doses (0.01, 0.1, 1, 10 and 100 µM) of one or two of the antagonists, as follow: Group 1 vehicle treated + SB366791 (SB, TRPV1 antagonist); Group 2 vehicle treated + RN1734 (RN, TRPV4 antagonist); Group 3 vehicle treated + (SB+RN); Group 4 LPS-treated + SB; Group 5 LPS-treated + RN; Group 6 LPS-treated + (SB+RN). In the L6 DRG of control animals, 36% of TRPV1 immunopositive cells and 31% of TRPV4 immunopositive cells presented TRPV1 and TRPV4 co-localization. Upon inflammation the percentage of cells that co-localize the two receptors drastically decreased. This result supports the idea that two different bladder afferents subtypes may have different bladder activity. The low co-localization during inflammation may be partially due to an increase in total TRPV1 and TRPV4 expressing cells. SB, RN or SB+RN treatments had no effect on bladder reflex activity of control animals. SB and RN were unable to reverse LPS-induce bladder hyperactivity. However, co- application of SB+RN reversed LPS-induced hyperflexia at doses of 0.01 µM, demonstrating that TRPV1 and TRPV4 have a synergistic activity upon pathological conditions. Despite the drastic reduction on TRPV1 and TRPV4 co-localization during inflammation these receptors present a synergistic activity. In a therapeutic perspective this data seem to be promising in order to overcome potential side effects of each antagonist.

International Workshop on Transient Receptor Potential (TRP) Channels 61 Posters Nº 16 Pharmacological targeting of recombinant and endogenous TRPM7 channels

V. Chubanov 1, M. Mederos y Schnitzler 1, M. Meißner 1, S. Schäfer 1, K. Abstiens 1, T. Hofmann 2, T. Gudermann 1 1Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians University of Munich, Goethestrasse 33, 80336 Munich, Germany 2Institute for Pharmacology and Toxicology, Philipps-University of Marburg, Karl-von-Frisch- Straße 1, 35043 Marburg, Germany

TRPM7 is a ubiquitously expressed kinase-coupled cation channel which regulates many essential cellular functions such as cellular Mg 2+ homeostasis, cell spreading and mechanosensitivity. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. We found that the recombinant TRPM7 channel is inhibited by the known modulators of SK1-3 channels such as antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31, UCL 1684. The most potent of these compounds, NS8593 (IC 50 1.6 M), interferes with the regulation of TRPM7 by cytosolic Mg 2+ . NS8593 (10 M) fully and reversibly inhibits native TRPM7-like currents in HEK 293 cells, rat basophil leukemia (RBL) cells, mouse trophoblast stem (TS) cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. Furthermore, we examined whether targeting of the native TRPM7 currents by NS8593 would impact cellular processes known to be affected by a genetic inactivation of TRPM7. We found that NS8593 (10-30 M) suppressed motility of HEK 293 cells, reduced the proliferation rate of TS cells and induced striking morphological changes of RBL cells. Taken together, our results indicate that NS8593 is a potent inhibitor of recombinant and endogenous TRPM7 currents and may be very instrumental for revealing of the cellular roles of TRPM7.

62 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 17 Identification and characterization of the interaction between TRPV1 and the PDZ protein Whirlin

Ciardo M. Grazia , Cuesta-Garrote N., Andres-Borderia A., Camprubí-Robles M., Ferrer-Montiel A., Planells-Cases R. Instituto de Biología Molecular y celular. Universidad Miguel Hernandez, Elche, Alicante, Spain.

TRP channels associate with auxiliary proteins forming macromolecular protein complexes known as transducisomes or signalplexes that modulate the receptors function and regulation. These highly organized signaling complexes are thought to provide highly efficient and specific signal transduction units, localize TRP in specific membrane subdomains and determine the receptor function in different physiological states. Such TRP signaling complexes, besides the cation channel, contain trafficking, scaffolding, adaptor and cytoskeletal proteins, kinases and various signaling molecules. TRPV1 has been reported to interact with different intracellular proteins that regulate the receptor function, and consequently TRPV1-mediated responses of nociceptive primary neurons. Given the pivotal role of TRPV1 in inflammatory pain, we aimed to identify other auxiliary members of the TRPV1 signalplex. To identify new potential components of the TRPV1 transducisome, a yeast two-hybrid (Y2H) library from rat brain was screened using the intracellular regions of the TRPV1 receptor as baits. Whirlin, which forms part of a macromolecular PDZ protein scaffold in photoreceptor and hair cell synapses, was identified. The interaction between TRPV1 and Whirlin was validated by pull-down and co-inmunoprecipitation assays. Here, we report that Whirlin modulates TRPV1 receptor expression and stability at the plasma membrane and intracellularly, both in heterologous and native expression systems. Given that TRPV1 activity potentiation leads to nociceptor sensitization under inflammatory conditions, these findings provide new insights about the role of an accessory PDZ protein controlling TRPV1 plasma membrane expression and function.

International Workshop on Transient Receptor Potential (TRP) Channels 63 Posters Nº 18 Mechanosensation in trigeminal sensory neurons

Anna Lucía Conte , Danny Florez and Ana Gomis Instituto de Neurociencias de Alicante. Universidad Miguel Hernández-CSIC. 03550 Alicante, Spain.

Detection of mechanical forces by the somatosensory system is performed by terminals primary afferent neurons whose cell bodies are located in the dorsal root ganglia (DRG) and the trigeminal ganglia (TG). Recent work has uncovered specific properties of mechanotransducer currents in different subsets of mechanosensory DRG neurons from rodents. These results suggest that mechanical stimulation could activate certain types of ionic channels that differ in mechanical sensitivity. TG neurons innervate deep and superficial tissues of the head and face, however their mechanical sensitivity has not been reported. In the present study, we use fura2-based fluorimetric calcium imaging in combination with whole-cell patch-clamp recordings to characterise different populations of newborn and adult mice TG neurons in response to a hypoosomotic solution (210 mosm Kg -1), which causes membrane stretch, and to static indentation using a glass pipette driven by a micromanipulator system. We also classified the neurons according to a number of parameters that are specific for nociceptive and non-nociceptive cells. The responses to mechanical stimulation of a specific population of low threshold neurons (parvalbumin expressing (PV)) were also investigated. We identified three mechanically sensitive populations of neurons responding to hypoosmotic solution (14%), indentation (43%) or both stimuli (31%). The percentage of mechanosensitive neurons was the same in newborn and adult animals. However, there was an increase in the responses of noxious mechanical neurons in adult animals. Mechanical stimulation activated three different currents based on the inactivation kinetics: rapidly, intermediate and slowly adaptive currents, with the slower currents found in putative noxious neurons. 75% of PV neurons responded to hypoosmotic stimulation and none of them responded to noxious stimuli. Mechanical stimulation only induced RA currents in PV positive neurons. It has been shown that TRPA1 channels mediate SA and IA currents in a subpopulation of adult DRG neurons. Our results suggest a different molecular candidate underlying these currents in newborn animals that do not express TRPA1 significantly. In summary, we characterised specific subpopulations of TG neurons with low and high threshold mechanosensitivity. Further characterisation could be useful in identifying stretch and mechanical channels.

This work is funded by grants BFU2009-07835, Consolider-Ingenio 2010 CSD2007-00023 and FSE programa JAE-CSIC.

64 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 19 Transient Receptor Potential A1 (TRPA1) is not involved in thermoregulation in dogs and humans

Rory Curtis *, Scott Coleman*, Donato Del Camino^, Neil J. Hayward^, Magdalene M. Moran^, Paula Bokesch* * Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, Massachusetts, 02421, U.S.A. ^ Hydra Biosciences, Inc., 790 Memorial Drive, Cambridge, Massachusetts 02139, U.S.A.

Transient Receptor Potential (TRP) channels are non-selective cation channels that respond to various physiological stimuli as well as endogenous and exogenous ligands. TRPV1 is activated by heat (>43 oC), acid and capsaicin (the ‘hot’ ingredient in chili peppers). TRPA1 is activated by numerous reactive compounds including inflammatory mediators, via covalent interactions with intracellular cysteine residues. Cold also modulates TRPA1 activity. TRPV1 and TRPA1 are expressed by sensory neurons and represent important analgesic targets. Genetic deletion and pharmacological inhibition show that reducing the activity of TRPV1 and TRPA1 blocks pain responses in a number of animal models. However, development of TRPV1 inhibitors has been problematic due to hyperthermia lasting several days in humans and also observed in non-clinical species. TRPV1 inhibitors also raise the threshold for humans to recognize noxious heat. These results suggest that TRPV1 serves to sense noxious heat and regulate body temperature in humans. A gain of function mutation in TRPA1 in humans leads to episodes of debilitating upper body pain triggered by fasting, cold and physical stress (Familial Episodic Pain Syndrome), suggesting that TRPA1 plays a role in human pain. We examined whether TRPA1 plays a role in body temperature regulation or the ability to sense heat and cold We determined if TRPA1 inhibitors affect regulation of body temperature in continuously-monitored radiotelemetry-implanted female Beagle dogs. Two out of three TRPA1 inhibitors had no effect on body temperature up to 48 hours post-dose, while 1 TRPA1 inhibitor showed modest elevation of body temperature several hours after peak plasma exposure (0.4-0.5 o C, 9-17 hours post-dose). These results suggest that TRPA1 inhibition in dogs has little or no direct effect on the regulation of body temperature. We also determined whether TRPA1 inhibitor CB-625 can affect body temperature regulation or the ability to sense heat and cold in a Phase 1 human clinical trial. In healthy human male volunteers (n=6), single ascending doses of CB-189,625 from 200 mg to 1600 mg had no effect on body temperature at any time up to 48 hours post- dose. CB-189,625 also did not affect blinded self-reporting of cold sensation (alcohol wipe on forearm) or heat sensation (hand immersion in a warm water bath) up to 24 hours post-dose. These results suggest that single doses of CB-189,625 at anticipated clinically relevant exposures have little or no effect on the regulation of body temperature or the ability to sense heat and cold in normal healthy volunteers.

International Workshop on Transient Receptor Potential (TRP) Channels 65 Posters Nº 20 Protons activate human TRPA1

Jeanne de la Roche 1*, Mirjam Eberhardt 2, Nancy Stanslowsky 3, Florian Wegner 3, Peter Reeh 2 and Andreas Leffler 1 1Clinic for Anaesthesia and Critical Care Medicine, Hannover Medical School, Carl-Neurberg- Str.1, 30625 Hannover, Germany. 2 Institute of Physiology and Pathophysiology, Friedrich- Alexander-University Erlangen-Nuremberg, 91054 Erlangen, Germany. 3Clinic for Neurology, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany.

Drop of extracellular pH following ischemia and inflammation is known to cause pain. Protons are known to modulate the nociceptor ion channels ASIC and TRPV1 from the extracellular site (1, 2) and weak acids like lactate were recently shown to activate TRPA1 by inducing an intracellular acidosis (3). However, conflicting reports have been published in regard to the effects of extracellular acidosis on TRPA1. While rodent TRPA1 does not seem to be activated by extracellular applied protons (3), extracellular acidification was reported to gate human TRPA1 (4). As several other substances differentially interact with hTRPA1 and rodent TRPA1, the scope of this study was to further explore the effects extracellular acidosis on human and rodent TRPA1. Recombinant human and mouse TRPA1 were expressed in HEK293t or CHO cells and investigated by means of whole cell patch clamp recordings and calcium-imaging experiments. Furthermore, dorsal root ganglion neurons (DRG) from wild type and TRPV1-knockout mice were examined by calcium-imaging experiments. While lactic acid at pH 6.0 activates both hTRPA1 and mTRPA1, severe extracellular acidosis only active hTRPA1. Protons act as classical TRPA1-agonists on hTRPA1, i.e. evoking outwardly-rectifying membrane currents which are potently blocked by the TRPA1-agonist HC-030031. Furthermore, proton-evoked inward currents are potentiated by external Ca 2+ . Our data suggest that TRPA1 is likely to be an important molecular mechanism mediating pain in humans in states of acidosis with or without accumulation of lactic acid.

References: 1. Jordt SE, Tominaga M, Julius D. Acid potentiation of the capsaicin receptor determined by a key extracelular site. Proc Natl Acad SciUSA 2000 Jul 5; 97 (14):8134-9. 2. Immke DC, McCleskey EW. Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade. Neuron 2003 Jan 9; 37 (1):75-84. 3. Wang YY, Chang RB, Allgood SD, Silver WL, Liman ER. A TRPA1-dependent mechanism for the pungent sensation of weak acids. J Gen Physiol. 2011 Jun; 137(6):493-505. 4. Takahashi N, Mizuno Y, Kozai D, Yamamoto S, Kiyonaka S, Shibata T, Uchida K, Mori Y.Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators. Channels (Austin) . 2008 Jul-Aug 2(4):287-98.

66 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 21 Discovery of new hits for TRPV1 blockade by high throughput assays

Roberto de la Torre Martínez 1, Asia Fernández-Carvajal 1, M. Teresa Aranda 2, M. Teresa García-López 2, Rosario González-Muñiz 2 and Antonio Ferrer-Montiel 1 1Instituto de Biología Molecular y Celular. Universidad Miguel Hernández. 03202 Elche (Alicante) Spain. 2Instituto de Química Médica, CSIC. Juan de la Cierva, 3, 28006 Madrid, Spain.

Although pain is a normal and necessary alarm/defense mechanism for life, chronic pain, either inflammatory or neuropathic, is a pathologic process for which there is not still an adequate treatment for avoid it. On the recent years an ion channel called TRPV1 belonging to the family of the Transient Receptor Potential (TRP) channel has been related with chronic pain. Pharmacological blockade and genetic deletion experiments, has validated TRPV1 as a therapeutic target, generating intensive drug discovery programs aimed at developing orally active antagonists. Consequentially, numerous TRPV1 antagonists have been identified that block the receptor with high efficacy and potency. However, and rather disappointingly, very few candidates have progressed into clinical trials because of unpredicted side effects such as hyperthermia. In this study was evaluated the biological activity of a new chemical library, through high throughput screening. We report here the identification of compounds that presented a high blockade activity on TRPV1. This new pharmacophoric scaffold can be used as a hit for analgesic drug development targeting TRPV1.

Funded by MICINN (BFU2009-08346), MICCIN (BES-2010-037112), the Consolider-Ingenio 2010 and La Fundacion La Marato de TV3, Prometeo-GVA.

International Workshop on Transient Receptor Potential (TRP) Channels 67 Posters Nº 22 Effects of blocking TRPA1 in a sheep model of asthma

Donato del Camino 1, Jayhong A. Chong 1, Neil J. Hayward 1, Jennifer Monsen 1, Magdalene M. Moran 1, Rory Curtis 2, Christopher Murphy 2, Lawrence I. Mortin 2, William M. Abraham 3 1 Hydra Biosciences, Inc., Cambridge, MA, U.S.A. 2 Cubist Pharmaceuticals, Inc., Lexington, MA, U.S.A. 3 Mount Sinai Medical Center, Miami Beach, FL, U.S.A.

Exposure to multiple respiratory irritants can result in heightened activation of dorsal root, jugular and nodose ganglion neurons innervating the lung. Such activation can lead to many of the symptoms characterizing both allergic and chronic obstructive pulmonary diseases including, sneezing, coughing, mucus production and bronchoconstriction. Recently, TRPA1,a non-selective cation channel of the Transient Receptor Potential Superfamily, has been identified as a key player in hyperactivity of pulmonary sensory nerves. TRPA1 serves as a broad irritancy receptor. Rather than being activated by a few specific ligands in a lock-and-key fashion, covalent modification of N-terminal cysteine residues by any of thousands of reactive chemicals causes increased channel activity. These reactive chemicals include many known pulmonary irritants such as acrolein (a key component of smoke), , and chlorine. Whereas acute exposure to these agents activates neurons causing cough, chronic exposure is associated with the development of asthma and chronic obstructive pulmonary disease. We recently showed that reducing TRPA1 function through pharmacological inhibition or genetic deletion significantly attenuated asthma severity in a mouse ovalbumin model of allergic asthma. To further investigate the role of TRPA1 in asthma, we tested TRPA1 antagonists in a sheep model of allergic asthma. We found that oral administration of TRPA1 antagonists reduced the late asthmatic response and blocked antigen-induced airway hyperresponsiveness. We conclude that TRPA1 continues to be an exciting target for the treatment of asthma and other pulmonary diseases.

68 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 23 Cold receptor activity in the tongue of TRPM8- and TRPA1- deficient mice

Denlinger B.L ., Viana F., and Belmonte C. 1Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, 03550 San Juan de Alicante, Spain.

The tongue is richly innervated by cold-sensitive nerve afferents. The molecular basis of cold transduction in sensory fibers is not fully understood; TRP channels and leak K+ channels are thought to play important roles. We developed an ‘in vitro’ preparation of the superfused mouse tongue to record impulse activity in single sensory fibers of the lingual nerve in wild-type, TRPM8 (Dhaka et al., 2007), TRPA1 (Kwan et al., 2006) and TRPM8/TRPA1 null mice and analyzed the effect of controlled thermal stimuli on this activity. Sustained nerve impulse discharges were evoked in a fraction of sensory fibers when temperature of the perfusing solution was decreased from a basal temperature of 34-35º down to 10ºC. Based on their thermal threshold, cold-sensitive fibers were subdivided into low ( ≥ 26ºC, LT) and high threshold (< 26ºC, HT) fibers. Deletion of TRPM8 did not completely abrogate the population of cold-sensitive fibers with those still responding to cold exhibiting thresholds in the low and high ranges. The vast majority of cold-sensitive fibers in TRPA1 -/- were LT fibers, supporting the contention that cold transduction in the population of HT fibers depends largely on TRPA1. In TRPM8/TRPA1 -/- mice, cold-sensitive fibers were still found, all being LT fibers. Chloroform (20 mM), an activator of thermosensitive TREK-1 potassium channels, attenuates or eliminates responses of these fibers to cold. Altogether, these results indicate that activation of lingual sensory fibers by cold depends on several transduction mechanisms. TRPM8 appears to be important but not unique for the detection of discrete temperature reductions. Other channels, presumably background potassium channels also appear to be involved, while TRPA1 seems to mediate transduction of more intense cooling. We also explored the contribution of cold thermoreceptors in the oral mucosa to drinking preference and overall water intake in mice using an experimental cage in which the water temperature of two drinking bottles could be cooled and heated from a control temperature of 22ºC. After water deprivation during 24 hours, drinking time and volume were measured over a 1 hour period. Wild-type mice drastically preferred water at 22ºC over cooler water, while TRPM8 -/- mice had no preference and TRPM8/TRPA1 -/- mice even preferred the cooler water. TRPA1 -/- mice avoided the coldest water at 5ºC. All knockouts also spend a longer total time drinking at each temperature than wild-type mice. These data suggest that cold thermoreceptor signaling, in particular mediated by TRPM8 channels is an important cue in thirst and in preabsorptive signaling of satiety.

International Workshop on Transient Receptor Potential (TRP) Channels 69 Posters Nº 24 Primary sequence analysis of TRPV2 reveals potential conserved key domains implicated in structure/function

Doñate Macian P. , Jose-Luis Vázquez-Ibar , Perálvarez-Marín A. Center for Biophysical Studies, Department of Biochemistry and Molecular Biology Department, Universitat Autònoma de Barcelona. 08193, Bellaterra.

TRPV2 is an orphan ion channel which role remains elusive. TRPV2 is present in many tissues and it has been related to some syndromes, such as muscular dystrophy and primary immune response, evidencing its potential as a drug target. To better understand this channel, better knowledge of the structural and functional aspects is required. As a preliminary approach, bioinformatics and evolutionary approaches can be used to get some specific hints about relevant domains for the structure and function of the protein. Here, we have used ancestor sequence reconstruction (ASR) and sequence divergence analysis to study the conservation of the primary sequence of TRPV2 channel. Based on species-dependent evolutionary differences we have identified specific domains that may be interesting candidates to be further studied through biochemical and biophysical approaches.

70 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 25 Reduced TRPV1 expression in the ciPTEC Cystinotic cell line PT47.5

Ciara Doran , Katrin Kaschig, Patrick Harrison, Gordon Reid Department of Physiology, University College Cork, Ireland.

Cystinosis is a rare lysosomal storage disorder associated with the widespread accumulation of cystine. It is caused by mutations in the CTNS gene, which is located adjacent to TRPV1 on chromosome 17p13. The most common and severe form of the disease is infantile cystinosis which results in end stage renal disease (ESRD). The pathogenesis of the renal failure is poorly understood. Renal insufficiency has been shown to be strain-dependent in CTNS -/- mice, with no strain developing ESRD. A common CTNS mutation, termed the 57 kb deletion, has been shown to extend into intron 2 of TRPV1 (Freed et al., 2011). TRPV1 could potentially act as a modifier gene in the pathogenesis of cystinosis. It is expressed in the kidney and may be subject to tight regulation. Elevated TRPV1 expression has been linked to N-arachidonoyl- dopamine (NADA)-induced cell death in mononuclear cells of ESRD patients (Saunders et al., 2009). Conversely, TRPV1 null mice have elevated glomerular sclerosis, albuminuria and tubulointerstitial fibrosis in response to DOCA-salt loading compared with wild-type mice; consistent with a protective role for TRPV1 (Wang, 2011). We have investigated whether TRPV1 is expressed in ciPTEC proximal tubule cell lines. Analysis by SYBR green based qPCR suggests that TRPV1 is down-regulated more than 80 % in the cell line PT47.5, derived from a nephropathic, cystinotic patient heterozygous for the 57 kb deletion, as compared with the control healthy cell line PT33.3. Preliminary functional analysis of TRPV1 in PT33.3 by calcium microfluorimetry indicates that the channel retains its thermal sensitivity in the proximal tubule. Responses to heating were partially blocked by the TRPV1 antagonist, SB366791, but not by capsazepine. Furthermore, SB366791 also attenuated response amplitudes to pH 5.5. We did not observe any capsaicin-induced activation, however the cells did respond to the TRPV agonist, 2APB. The pharmacological profile of PT33.3 may suggest the presence of a TRPV1 splice variant. Reduced expression of this isoform could potentially impact on proximal tubule function and by extension contribute to the renal phenotype of cystinosis. Acknowledgments: We wish to thank Elena Levtchenko and Martijn Wilmer for their generous donation of the ciPTEC cell lines.

FREED, K. A., BLANGERO, J., HOWARD, T., JOHNSON, M. P., CURRAN, J. E., GARCIA, Y. R., LAN, H.-C., ABBOUD, H. E. & MOSES, E. K. 2011. The 57 kb deletion in cystinosis patients extends into TRPV1 causing dysregulation of transcription in peripheral blood mononuclear cells. Journal of Medical Genetics, 48, 563-566. SAUNDERS, C. I., FASSETT, R. G. & GERAGHTY, D. P. 2009. Up-regulation of TRPV1 in mononuclear cells of end-stage kidney disease patients increases susceptibility to N- arachidonoyl-dopamine (NADA)-induced cell death. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1792, 1019-1026. WANG, Y. W. D. H. 2011. Protective Effect of TRPV1 against Renal Fibrosis via Inhibition of TGF-β/Smad Signaling in DOCA-Salt Hypertension. Molecular medicine.

International Workshop on Transient Receptor Potential (TRP) Channels 71 Posters Nº 26 PLC γ1 and ErbB2 are required for pressure-induced activation of TRPM4 channels in cerebral artery smooth muscle cells

Albert L. Gonzales, Ying Yang, Michelle N. Sullivan, Lindsey Sanders, and Scott Earley Vascular Physiology Research Group, Department of Biomedical Science, Colorado State University, Fort Collins, CO 80523 USA

Activity of the melastatin (M) transient receptor potential (TRP) channel TRPM4 is essential for pressure-induced membrane depolarization of cerebral arterial myocytes and myogenic vasoconstriction, suggesting that elevated intraluminal pressure activates TRPM4. As mechanisms underlying this response have not been characterized, we tested the hypothesis that TRPM4 is inherently mechanosensitive. Consistent with our hypothesis, we found that in perforated patch clamp recordings obtained from native vascular myocytes, transient inward cation current (TICC) activity is reversibly increased when negative pressure is applied to the patch pipette (half- maximal pressure for activation (P50) = 12.8 mmHg). This response is blocked by the selective TRPM4 antagonist 9-phenanthrol, indicating that mechanical deformation of the plasma membrane stimulates TRPM4 activity. However, single channel activity of TRPM4 expressed in HEK 293 cells did not increase when negative pressure was applied, indicating that TRPM4 is not inherently mechanosensitive. We therefore examined indirect mechanosensing mechanisms. Our prior work demonstrates that TRPM4 is activated by Ca 2+ release from inositol trisphosphate receptors (IP3R) located on the sarcoplasmic reticulum (SR) in native vascular myocytes. IP3R are activated by inositol trisphosphate (IP3), generated by hydrolysis of phosphotidylinositol (4,5)-bisphosphate by phospholipase C (PLC). We hypothesized that mechanical stress indirectly activates TRPM4 by increasing PLC activity. We found that pressure-induced increases in TRPM4 activity were attenuated by the PLC inhibitor U-73122, and restored by Bt-IP3, a membrane-permeable IP3 analog. Together, these data indicate that PLC activity contributes to stretch-induced activation of TRPM4. Since multiple isoforms of three PLC families ( β, γ, and δ) are present in vascular myocytes, we investigated the specific PLC isoform(s) involved in TRPM4 activation using siRNA knockdown in intact cerebral arteries. PLC γ1 knockdown attenuated TRPM4 activity as well as pressure-induced smooth muscle cell depolarization and myogenic vasoconstriction, demonstrating that PLC γ1 is required for pressure-dependent responses in vascular myocytes. PLC γ isoforms often act downstream from receptor tyrosine kinases (RTKs) present on the plasma membrane. Therefore, we investigated the role of RTKs in pressure activation of TRPM4. We found that all four members of the ErbB RTK family of epidermal growth factor receptors (ErbB1-4) are present in native cerebral artery myocytes and that the orphan receptor ErbB2 is highly localized to the plasma membrane. In patch clamp experiments, we found that the selective ErbB2 antagonist AG 825 blocked negative pressure-induced activation of TRPM4, suggesting a critical role for ErbB2. Together, these data indicate that pressure-induced activation of TRPM4 in vascular smooth muscle cells requires both PLC γ1 and ErbB2. R01HL091905 (SE); F31HL094145 (ALG).

72 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 27 Neurovascular effects through activation of TRPA1 – how nitroxyl (HNO) works

Mirjam Eberhardt 1,5 , Maria Dux 1,4 , Barbara Namer 1, Nada Cordasic 3, Jan Miljkovic 2, Jeanne de la Roche 5, Michael Fischer 1, Andreas Leffler 5, Angelika Lampert 1, Johannes Jacobi 3, Karl Messlinger 1, Ivana Ivanovic-Burmazovic 2, Peter Reeh 1, Milos R Filipovic 2 1Institute of Physiology and Pathophysiology and 2Department of Chemistry and Pharmacy, and 3Department of Nephrology and Hypertension, Friedrich-Alexander University Erlangen- Nuremberg, Germany 4Department of Physiology, University of Szeged, Hungary 5Department of Anesthesiology and Intensive Care, Medical School Hannover , Germany

The role of the universal chemosensory receptor-channel TRPA1 in nociception and inflammatory diseases 1 has recently been elucidated by identifying several endogenous activators emerging under pathological conditions. Activation of TRPA1 receptors leads to release of calcitonin gene-related peptide (CGRP) a potent vasodilator which also has positive ionotropic effects on the heart. Up to now a constitutively produced intra- or intercellular signal that would activate TRPA1 and release CGRP has not been encountered up to now. Thus, a potential role of TRPA1 in cardiovascular physiology remained hypothesis. Emerging as a potential mediator in regulating cardiovascular functions nitroxyl² (HNO) is known to lower blood pressure and enforce cardiac functions by releasing CGRP³. As a reduced congener of nitric oxide (NO) and coproduced by nitric oxide synthase 4, HNO employs an entirely distinct signaling pathway. However, the mechanism by which HNO stimulates CGPR release was unknown. By combining calcium imaging, classical ion channel and biochemical techniques we provide evidence that HNO activates TRPA1 by modifying critical cysteines 5 leading to formation of disulfide bonds, which represents a novel mechanism for a remarkably sustained activation of the channel. Furthermore in rat and knockout mice studies we show that HNO induces CGRP release in a TRPA1-dependent manner from ubiquitous sensory nerve fibers and that HNO-induced activation of the TRPA1/CGRP pathway results in increase of meningeal blood flow and reduction of blood pressure in vivo . Finally, in a psychophysiological study we provide evidence for this new signaling cascade to be functional in humans. The neuroendocrine HNO/TRPA1/CGRP pathway may constitute a previously unknown physiological control element of cardiovascular functions. Furthermore, combining vasodilation with positive inotropic effects 6, HNO donors could be considered a great promise for the treatment of heart failure, without inducing nitrate tolerance as with NO· donors.

1. D. M. Bautista , et al., Cell . 124 , 1269-1282 (2006). 2. Bullen, M.L., Antioxid. Redox Signal. 14 , 1675-1686 (2011). 3. Paolocci, N. et al., Proc. Natl. Acad. Sci. U.S.A. 98 , 10463 (2001). 4. Schmidt, H. H, Proc. Natl. Acad. Sci. U.S.A. 93 , 14492-11197 (1996). 5. A. Hinman, H., Proc. Natl. Acad. Sci. U.S.A . 103 , 19564-19568 (2006). 6. Irvine, J.C. et al., Trends Pharmacol. Sci . 29 , 601-608 (2008).

International Workshop on Transient Receptor Potential (TRP) Channels 73 Posters Nº 28 Characterization of TRPM8-expressing primary sensory neurons purified by fluorescence-activated cell sorting

Enoch Luis , Cruz Morenilla-Palao, Carlos Fernández-Peña and Félix Viana Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas. San Juan de Alicante, 03550 Alicante, Spain.

Primary sensory neurons are morphologically and functionally diverse. A major problem when studying the function of a particular subpopulation is their identification. This is usually based on the response pattern to the application of a battery of thermal, mechanical and chemical stimuli, or by the labeling with specific antibodies. This problem is specially limiting in the study of cold thermoreceptors because they comprise only about 10% of all sensory neurons identified in culture or in histological sections. To facilitate the characterization of TRPM8-expressing cold thermoreceptors, we generated a transgenic mouse line expressing the yellow fluorescent protein (YFP) under the control of the TRPM8 promoter, using homologous recombination techniques to modify a bacterial artificial chromosome (BAC) harboring the TRPM8 genomic sequence. In this study, we developed a simple method using fluorescent activated cell sorting (FACS) to purify TRPM8-expressing neurons from neonatal mice (P6-P13). Dissociated cultures of primary sensory neurons were obtained from dorsal root ganglia. The effects of different drugs on individual neurons were assessed by fura-2 calcium imaging and whole-cell patch-clamp. Cold sensitivity was investigated with a temperature drop of a continuously flowing external solution from 32-34Cº to 20ºC. Microscopic observation confirmed that FACS-sorted neurons expressed YFP and samples of FACS-purified cells were found to be >85% pure. Using quantitative RT- PCR we verified an 800-fold enrichment of TRPM8 RNA in sorted samples compared to unlabeled cells. During rapid reductions in bath temperature, 69% (47/68) of neurons responded with an elevation in their [Ca2+]i. A very similar percentage, 68% (46/68) of sorted neurons were also activated by menthol. The percentage of responding neurons increased to 90% (61/68) when menthol was applied in conjunction with a cold ramp. Furthermore, 48.5% (33/68) of the neurons responded to capsaicin and just 1/68 responded to cinnamaldehyde, a specific TRPA1 agonist. The threshold of cold-sensitive sorted sensory neurons in culture, identified with intracellular calcium imaging, varies over a wide range from 21.5 to 32.6ºC, with a mean value of 28ºC. BCTC (10 µM), a blocker of TRPM8 channels, produced a significant inhibition (P< 0.001) of cold- and menthol-responses that recovered during wash. In conclusion, these results show that the TRPM8-YFP transgenic mouse is a useful tool in the study of neurons involved in cold transduction, facilitating the identification, purification and characterization of primary somatosensory neurons expressing the TRPM8 channel.

Supported by project SAF2010-14990 (Spanish Ministry of Science and Innovation) to F.V.

74 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 29 TRPV1-mediated autophagy in thymocytes is a consequence of proteasome inhibition and unfolded protein response activation

Farfariello V. 1,2* , Amantini C. 1, Nabissi M. 1, Morelli M.B. 1, Liberati S. 1,3 , Eleuteri A.M. 4, Bonfili L. 4, Cecarini V. 4, Sorice M. 5 and Santoni G. 1 1School of Pharmacy, Experimental Medicine Section, University of Camerino, Camerino, Italy. 2Dept. Andrology and Urology, University of Perugia Perugia, Italy. 3Dept. Molecular Medicine, Sapienza University of Rome, Rome, Italy. 4School of Biosciences and Biotechnologies, University of Camerino, Camerino, Italy. 5Dept. Experimental Medicine, Sapienza University of Rome, Rome, Italy. *[email protected]

Macroautophagy and the ubiquitin–proteasome system are two complementary pathways for protein degradation. Recent findings indicate that suppression of the ubiquitin–proteasome system induces macroautophagy through multiple pathways (Wu et al., 2010). We have previously shown that TRPV1 activation by capsaicin (CPS) (Farfariello et al., 2012 in revision) induces autophagy of thymocytes at 2 hrs after treatment through a ROS-mediated signalling pathway and that this event ensures thymocyte survival. Interestingly, we found that in the early phases, probably through the ROS signals generation, TRPV1 activation by CPS induces inhibition of 20S and 26S proteasomes, as shown by reduced chimotrypsin- and trypsin-like activity at different times. Accumulation of misfolded proteins as consequence of proteasome inhibition induces endoplasmic reticulum stress and activates the unfolded protein response (UPR) in order to restore cellular homeostasis (Obeng et al., 2006; Ding et al., 2007). To elucidate the molecular mechanisms involved in TRPV1-activated-UPR, we performed a mouse Autophagy RT-PCR array in CPS-treated thymocytes. Interestingly, at 1h after CPS treatment, we found a significative upregulation of calreticulin, a multifunctional protein that binds to misfolded proteins and prevents them from being exported from the endoplasmic reticulum to the Golgi apparatus. Moreover, we evidenced upregulation of both Derlin-1 and Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 that are part of a complex that mediates UPR and endoplamic reticulum-associated degradation (ERAD). We also found increased levels of Insulin induced gene 2, the endoplasmic reticulum proteins that block the processing of sterol regulatory element binding proteins (SREBPs) by binding to SREBP cleavage-activating protein (SCAP), thus preventing SCAP from escorting SREBPs to the Golgi. Overall these preliminary data suggest that activation of TRPV1 in thymocytes is responsible for the impairment of ubiquitin proteasome degradation pathway. As consequence, in response to UPR signalling, autophagy occurs in order to eliminate unfolded proteins and rescue cells from apoptotic death.

Funding: This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) National Grant 2011-2013 (Number 11095).

International Workshop on Transient Receptor Potential (TRP) Channels 75 Posters Nº 30 Ligand induced opening of TRPM2 channel requires terminal ribose of ADPR and Arg1433

Ralf Fliegert 1, Christelle Moreau 2, Tanja Kirchberger 1, Anja Schöbel 1, Mark Thomas 2, Andreas Bauche 1, Angelika Harneit 1, Barry V.L. Potter 2, and Andreas H. Guse 1,3 1The Calcium Signalling Group, Department of Biochemistry and Signal Transduction and 3Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg- Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; 2Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, UK

Adenosine diphosphoribose (ADPR) has long been considered a mere product of the breakdown of NAD +, cADPR or mono- or poly-ADP-ribosylated proteins. The discovery that TRPM2, a member of the melastatin subfamily of TRP channels, is activated by intracellular ADPR has stimulated interest in ADPR as a second messenger. However, investigation of the physiological role of ADPR/TRPM2 is largely hampered by the lack of specific inhibitors. Rational design of ADPR/TRPM2 antagonists might greatly benefit from a better understanding of the structure-activity relationship. Binding of ADPR to TRPM2 has been shown to be mediated by its C-terminal NUDT9H domain involving amino acid residues of the Nudix box. To obtain further insight into the structure-activity relationship of ADPR/TRPM2, we chemically synthesized ADPR analogues replacing the terminal ribose with smaller structures. In patch clamp experiments these analogues and additional compounds (ADP, ADP-glucose) were tested for potential agonist activity. At a pipette concentration of 100 µmol/L none of the tested compounds exerted agonist activity when compared to ADPR. When tested for antagonist activity two of the compounds resulted in a significant reduction of current when applied at 9-fold excess over ADPR. These results indicate that the antagonist compounds, while not able to induce opening of the channel, were at least able to bind to the NUDT9H domain. To understand which amino acid residues might be involved in interactions with the terminal ribose of ADPR we built a homology model of the NUDT9H domain of TRPM2 based on the crystal structure of human NUDT9 and computationally docked ADPR. Based on these studies we chose amino acid residues potentially interacting with the terminal ribose and introduced respective point mutations into an expression vector for hTRPM2. The impact of the mutations was tested in calcium imaging experiments in transiently transfected HEK293 cells stimulated with hydrogen peroxide. While some mutants were without effect on the calcium signal, the fraction of responding cells was diminished in T1347V and Y1349F. Cells transfected with R1433M did not respond to hydrogen peroxide at all. In summary our findings suggest that both the terminal ribose of ADPR and the amino acid side chain of R1433 are necessary for the ligand induced opening of TRPM2.

76 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 31 TRPA1 channel is expressed in non-neuronal pulmonary cells and promotes non-neurogenic inflammation

Camilla Fusi ,1 Romina Nassini, 1 Pamela Pedretti, 1,2 Nadia Moretto, 2 Chiara Carnini, 2 Fabrizio Facchinetti, 2 Riccardo Patacchini, 2 Pierangelo Geppetti, 1 and Serena Materazzi 1 1Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy; 2Pharmacology Department, Chiesi Farmaceutici SpA, Parma, Italy

The transient receptor potential ankyrin 1 (TRPA1) channel is localized to airway sensory nerves and has been recently proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via neurogenic mechanisms. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1. Here, we report that TRPA1 channel is functionally expressed in non-neuronal districts of human and mouse airway, where it produces inflammatory responses. In this study C57BL/6 mice, wild-type or TRPA1-deficient mice generated by heterozygous mice on a C57BL/6 background were used. By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts (IMR90 and NHLF), epithelial (A549 and SAEC) and smooth muscle (HBSMC) cells express functional TRPA1 channels. For functional characterization we used selective exogenous and endogenous TRPA1 agonists, cinnamaldehyde and acrolein respectively, and cigarette smoke extract (CSE). By using immunohistochemistry TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung and from airways and lung of wild-type, but not TRPA1-deficient mice. In addition, acrolein and CSE exposure (16 h) evoked IL-8 release in cultured human airway epithelial and smooth muscle cells and fibroblasts in a concentration dependent manner, an effect selectively reduced by TRPA1 antagonists, HC-030031 and AP18 (both 10 µM). Stimulation of TRPV1 (by capsaicin, 100 µM/30 µl i.t.), co-expressed with TRPA1 in the same airway sensory nerves, and TRPA1 (by acrolein 5 mM/30 µl i.t or CS 1 OD/30 µl i.t ), or the neuropeptide, substance P (SP, 25 nM/30 µl i.t ), which is released from sensory nerve terminals stimulation, provoked neurogenic inflammation in mouse airways. However, TRPA1 activation but not TRPV1 or SP activation, was able to increase release of keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in BAL of wild-type mice, and this effect was attenuated by TRPA1 antagonism or in TRPA1-deficient mice. In addition, pretreatment with NK1 receptor antagonist (2µmol/kg i.v.) failed to reduce the increase of KC evoked by either acrolein or CSE, supporting the role non-neuronal TRPA1 in this phenomenon. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni’s post hoc analysis for comparison of multiple groups and the unpaired 2- tailed Student’s t-test between 2 groups. P < 0.05 was considered significant. For in vivo studies at least of 6 mice for group have been used. Thus, present data suggest that while both TRPV1 or TRPA1 activation causes airway neurogenic inflammation, TRPA1, in this non-neuronal localization, produces an additional, prominent non-neurogenic inflammatory response, which may contribute to inflammatory airway diseases. These data offer a novel interpretation of the role of TRPA1 that could be a target for the treatment of inflammatory respiratory diseases.

International Workshop on Transient Receptor Potential (TRP) Channels 77 Posters Nº 32 Role of the cytosolic N-terminal tail of TRPV4 in the channel response to hypotonic stimuli

Anna Garcia-Elias , Sanela Mrkonjic, Carlos Pardo, Fanny Rubio-Moscardó, Rubén Vicente and Miguel A. Valverde Laboratory of Molecular Physiology and Channelopathies, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, C/ Dr. Aiguader 88, Barcelona 08003, Spain.

The TRPV4 channel was initially described as an osmosensor, activated by changes in environmental osmolarity. Although it is now known that the channel activation occurs down-stream of the activation of phospholipase A2, it is still not known what domains of the channel are necessary for its gating. Here we show that the N-terminal tail of TRPV4 is important for the hypotonic response as a deletion of the first 130 amino acids leads to a channel that is less sensitive to hypotonic stimuli. Besides, we narrowed the relevant region to four positively charged amino acids within a proposed plekstrin-homology (PH)-like domain located just before the proline-rich domain (PRD). Neutralization of these 4 charges prevented TRPV4 activation by hypotonic stimuli. The gating defect observed is stimulus-specific since currents recorded after application of channel-agonist 4 α-PDD are equal to the wild-type channel. The PRD upstream the PH-like domain is important for the binding of PACSIN3, a regulatory protein that affects trafficking and activity of TRPV4. In fact, the N-terminus deletions mimicked the modulatory effect of PACSIN, reducing TRPV4 response to hypotonic stimuli, without affecting TRPV4 trafficking or 4 -PDD responses. We also showed that TRPV4 modulation by PACSIN3 requires the F-BAR domain, as a PACSIN3 mutant without this domain did not affect channel activity. Together our data confirmed that PACSIN3 and N-terminal TRPV4 deletions equally affected hypotonic-dependent channel gating, and suggested an important role of the N-terminus in the channel response to hypotonic cell swelling.

Funded by the Spanish Ministry of Science and Innovation, Red HERACLES (Fondo de Investigación Sanitaria) and Generalitat de Catalunya.

78 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 33 TRPM8/b1integrin interaction controls vascular endothelial cell migration and adhesion

Tullio Genova 1, Dimitra Gkika 2, Alexandre Bokhobza 2, Luca Munaron 1, Guido Serini 3, Natalia Prevaskaya 2 and Alessandra Fiorio Pla 1,2 1Lab. of Cellular and Molecular Angiogenesis DBIOS, University of Torino, Italy 2Lab. of Cell Physiology INSERM U 1003, Université de Lille 1, France 3Laboratory of Cell Adhesion Dynamics, IRCC, Dept. of Oncological Sciences, University of Torino School of Medicine, Candiolo (TO), Italy

Changes in intracellular calcium [Ca 2+ ]i levels control critical cytosolic and nuclear events that are involved in the initiation and progression of tumor angiogenesis in endothelial cells (ECs). Therefore, the mechanisms involved in agonist-induced [Ca 2+ ]i signaling are potentially important molecular target for controlling angiogenesis and tumor growth. Several studies have shown that blood vessels in tumors differ from normal ones in their morphology, blood flow and permeability. We recently reported a key role for (AA)- activated TRPV4 channel in tumor angiogenesis in vitro . Here we report an opposing effect of TRPM8 channel: as TRPV4, TRPM8 is differentially expressed in EC derived from human breast carcinomas (BTEC) as compared with ‘normal’ EC (HMVEC). However, in contrast with TRPV4 expression, TRPM8 is highly downregulated in BTEC compared with HMEC. Wound healing assays revealed a key role of TRPM8 in inhibiting cell migration of HMEC but not of BTEC. Interestingly overexpression of TRPM8 in BTEC significantly reduces cell migration while its downregulation in HMEC restores cell migration to similar levels as BTEC. TRPM8-mediated inhibition of endothelial cell migration closely correlates with its role on b1-intergin-mediated EC adhesion: again TRPM8 activation inhibits EC adhesion while downregulating the channel reverts the effect. Moreover activation of b1-integrin completely revert TRPM8-mediated effect on EC adhesion. On the other hand TRPM8 immunoprecipitate with b1-integrin in different primary EC types as well as in overexpression HEK stably trasfected with TRPM8. Moreover TRPM8 activation significantly inhibits b1-integrin activated pathway as showed by P-b1-integrin and P- FAK inhibition. Although the complete molecular mechanism remains to be clarified, the data presented clearly show that TRPM8 inhibits EC migration and adhesion by interfering with b1-integrin pathway, suggesting a balance between TRP channels in EC.

International Workshop on Transient Receptor Potential (TRP) Channels 79 Posters Nº 34 Testosterone, the steroid link in TRPM8–mediated cold perception

Dimitra Gkika 1, Alexis Bavencoffe 1, Jerome Busserolles 2, Artem Kondratskyi 1, Alexander Zholos 1, Eric Chapuy 2, Monique Etienne 2, Alain Eschalier 2, Brigitte Mauroy 1, Yaroslav Shuba 1, Roman Skryma 1 and Natalia Prevarskaya 1 1Inserm U1003, Equipe labellisée par la Ligue Nationale Contre le Cancer, F59655 Villeneuve d’Ascq, France; Université des Sciences et Technologies de Lille (USTL), F59655 Villeneuve d’Ascq, France 2 Laboratoire de Pharmacologie Médicale, Clermont Université, Université d’Auvergne, Faculté de Médecine/CHU, Clermont-Ferrand, France; Inserm, UMR 766, Pharmacologie Fondamentale et Clinique, Faculté de Médecine, Clermont-Ferrand, France It is now largely accepted that a number of sensory modalities including cold and warmth perception are affected by changes of the individual hormonal status. However, the mechanisms by which steroids influence sensory functions are yet to be determined. Cold/menthol-activated member of Transient Receptor Potential channels family, TRPM8, is mainly expressed in the sensory neurons where it represents the primary in vivo sensor of external innocuous cold temperatures. Despite extensive studies on TRPM8 activation by cold and chemical cooling compounds, the physiological mechanisms modulating TRPM8 activity remain unknown. Here we show that the male sex hormone, testosterone, specifically inhibits TRPM8 channel via a non-genomic pathway and reduces male sensitivity to non-noxious cold temperatures. Thus, testosterone constitutes a novel physiological modulator of TRPM8 inhibiting innocuous cold perception in vivo and variation of which could account for the gender as well as inter- and intra-individual disparity in thermosensation.

80 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 35 Investigating a role for TRPV4 in the airways

Grace, M.S. , Birrell, M.A., Dubuis, E., Ching, Y.M., Bonvini, S., and Belvisi, M.G. Respiratory Pharmacology, Airway Disease Section, National Heart and Lung Institute, Faculty of Medicine and Centre for Integrative Physiology and Pharmacology, Imperial College London, SW7 2AZ, UK

Transient Receptor Potential (TRP) ion channels are expressed in the airways, and modulate a variety of functions such as inflammation, airway smooth muscle (ASM) tone, and activation of sensory afferents. 1,2 TRPV4 is widely expressed in mammalian tissues, including ASM, the alveolar wall, lung tissue, lung vessels and inflammatory cells. It is activated by moderate temperatures (>30°C), hypoton ic solutions and mechanical stress. 3-8 TRPV4 has also been implicated in the activation of colonic sensory nerves, both directly 9 with selective agonists, and indirectly via the G protein-coupled receptor PAR2 . The aim of this study was to investigate whether TRPV4 modulates airway sensory nerve activation. Methods: the expression profile of TRPV4 mRNA was investigated in guinea pig tissue using real-time PCR. A potential role for TRPV4 in activating sensory afferents was explored using isolated murine, guinea pig and human vagal nerves and the selective TRPV4 agonists GSK1016790A and 4 α-PDD. Selectivity of these compounds was assessed by both antagonist (HC-067047) and genetic modulation ( Trpv4 -/- mice) studies. The ability of GSK1016790A to activate airway-specific primary sensory neurons was assessed using calcium (Fura-2) and membrane voltage (Di8-ANNEPS) imaging of nodose and jugular neurons retrogradelly labelled with the fluorescent tracer DiI (dosed i.n.). Results and Discussion: TRPV4 mRNA was widely expressed in guinea pig tissues; including the cell bodies for the airway afferents (nodose and jugular ganglia). Airway sensory nerve fibres are carried from the lungs to the central nervous system via the vagus nerve, thus we subsequently investigated the ability of TRPV4 agonists to activate isolated vagal tissue in vitro . Both GSK1016790A and 4 α-PDD caused concentration-dependent activation of murine, guinea pig and human vagus nerves. Agonist-induced depolarisation was inhibited by the TRPV4 selective antagonist HC067047; and vagal tissue from Trpv4 -/- mice failed to respond, indicating that the agonists were causing activation via the TRPV4 ion channel. Because vagus nerves also house fibres innervating visceral organs other than the airway, we went on to analyse calcium and membrane voltage changes in DiI stained primary neurons isolated from the nodose and jugular ganglia. GSK1016790A caused robust activation of airway primary ganglia cells. Conclusion: The TRP superfamily of ion channels appears to play an important role in airway function, both in the healthy and disease states. In this study, TRPV4 mRNA was found to be expressed in the guinea pig lung, and cell bodies of airway sensory nerves. Furthermore, stimulation of the TRPV4 ion channel caused activation of the vagus nerve and primary airway neurons. These results highlight TRPV4 as a potentially interesting novel target, as activation of sensory afferents can lead to adverse events such as cough and bronchoconstriction.

References: 1. Bessac B. & Jordt S., Physiology. 2008; 23: 360-370. 2. Nassini R. et al ., Curr. Opin. Invest. Drugs. 2010; 11(5): 535-542. 3. Alvarez D. et al ., Circ Res. 2006; 99:988-995. 4. Dietrich A. et al. , Pharmacol Ther. 2006; 112:744-760. 5. Hamanaka K. et al ., Am J Physiol Lung Cell Mol Physiol. 2010; 299:L353-362. 6. Jia Y. et al ., Am J Physiol Lung Cell Mol Physiol. 2004; 287:L272-278. 7. Liedtke W. & Simon S., Am J Physiol Lung Cell Mol Physiol. 2004; 287:L269-271. 8. Yang X. et al ., Am. J. Physiol. Lung Cell Mol. Physiol; 290: L1267-L1276. 9. Sipe W. et al ., Am. J. Physiol. Gastrointest. Liver Physiol.; 294: G1288-G1298.

International Workshop on Transient Receptor Potential (TRP) Channels 81 Posters Nº 36 Role of TRP domain in TRPV1 functionality

Lucia Gregorio-Teruel 1, Pierluigi Valente 1,2 , Gregorio Fernández-Ballester 1, Feng Qin 3 and Antonio Ferrer-Montiel 1.

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. 3 Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences. University of Buffalo. Buffalo, NY 14214. USA

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 α-helical transmembrane segments with a pore region between the fifth and sixth segment. The cytoplasmatic N- and C-termini contains several residues involved in the modulation of channel activity. 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. We carried out the study of the mutated chimeras in transitory transfected HEK cells. First of all, we examined the expression level and the presence in plasmatic membrane for the mutated channels. Afterwards, the response of the mutated chimeras to capsaicin, depolarizing voltages and high temperature was also studied using the Patch Clamp technique. Taking together, our data suggest that the TRP domain region is critical for the functional coupling of the activating stimuli. Particularly, we found that alterations in the TRP domain affected the energetic of channel opening. These results demonstrate that the preservation of this region is essential for the correct functionality of TRPV1.

Funded by MICINN, CONSOLIDER-INGENIO 2010, ISCIII, Fundació La Marató de TV3.

82 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 37 Bee venom modulates TRPV1 signaling

Henriques, M.S.T., Munaro-Vieira, D., Melo, P.A. and Guimaraes, M.Z.P. Programa de Farmacologia e Inflamação, Universidade Federal do Rio de Janeiro, Brazil

Bee envenomations are known to cause pain described as a burning sensation. Because of this, other authors have proposed that components in the crude venom, in particular apamin, elicit nociception through the activation of the TRPV1 channel. However, there has not been definite proof of direct activation of TRPV1 by bee venom (BV). Therefore, the goal of this study was to examine the BV effects on TRPV1 activity in animal and heterologous systems models. Methods and Results: First, young mice were treated with high doses of capsaicin with the purpose of knocking down nociceptor signaling. These animals then received BV and were compared to untreated animals in various aspects. For instance, paw edema caused by BV was greatly reduced in capsaicin-pretreated animals. On the other hand, myotoxicity elicited by BV was not affected by nociceptor silencing. Then TRPV1-expressing Xenopus oocytes were exposed to BV while performing TEVC. Even at concentrations as high as 500 ng/mL, BV was unable to trigger currents in these cells. However, when we co applied 1 µM capsaicin and BV at this high concentration, the currents were augmented by approximately 50% when compared to capsaicin alone. Finally, TRPV1-transfected HEK293 cells were assayed for YO-PRO-1 uptake elicited by BV and/or capsaicin. At high concentrations, BV caused dye uptake even in untransfected cells. We chose then a low concentration, 1 ng/mL, which did not promote dye uptake in untransfected cells. When this low concentration of BV was applied to TRPV1-transfected cells, there was no change in dye uptake. Conversely, when the same concentration was co applied with 20 µM capsaicin, there was an approximate 100% increase in dye uptake, compared to capsaicin alone. This increased dye uptake was blocked by ruthenium red. Discussion: These data suggest that crude BV is capable of increasing TRPV1 activity in the presence of another agonist, in this case capsaicin. However, it does not activate TRPV1 by itself, as seen in the heterologous models. It remains to be establish which component or components of BV has this TRPV1-modulating ability, as well as the nature of this modulation.

International Workshop on Transient Receptor Potential (TRP) Channels 83 Posters Nº 38 Phenotype of Drosophila TRPM (dTRPM) and molecular determinants of its magnesium conduction

Hofmann T.* , Chubanov V. #, Chen X.D.*, Dietz A.S.* *Pharmakologisches Institut, BPC Marburg, Philipps-Universität Marburg, 35043 Marburg, Germany # Walther-Straub-Institut für Pharmakologie, Ludwig-Maximilians-Universität, 80336 München, Germany

In insects, the TRPM family of cation channels is respresented by one single gene, whose Drosophila null allele results in two developmental phenotypes: (1) larval growth retardation due to severe hypermagnesemia that results from a dysfunction of the Malpighian “kidney” tubules and (2) a failure to undergo larval-pupal transition (pupation). We present (1) new findings that address the importance of magnesium homoiostasis for both of these phenotypes as well as (2) results of in vitro mutageneses delineating the molecular determinants for divalent cation selectivity, single channel amplitude and magnesium regulation of the dTRPM channel. It remains unclear if the two facets of the dTRPM developmental phenotype are connected, and controversial whether magnesium homoeostasis or rather the handling of other ions is responsible for it. We previously found a negative correlation of food magnesium supply with larval growth rate but paradoxically, magnesium restriction could not rescue the metamorphosis arrest. Having observed major defects in fat body function in magnesium- deprived larvae, we hypothesized that, like in mammalian TRPM7 cells, an actual failure to of magnesium uptake in peripheral organs might cause the metamorphosis arrest. In order to test this, we induced hypermagnesemia only in mature larvae that were grown beforehand on low magnesium food. With this paradigm we observed animals passing beyond the larval-pupal transition point, a few of them to the point of mature pupae that initiate eclosure. This rescue demonstrates that magnesium transport by (or regulated by) dTRPM is necessary and sufficient to explain the developmental phenotype of the trpm gene. Wild type dTRPM is highly permeable to magnesium and a variety of other divalent cations, 2+ if intracellular Mg is considerably buffered (IC 50 =22 µM). dTRPM is not voltage- dependent, and its single channel conductance (as determined by fluctuation analysis at +80 mV) is 2 pS. In order to identify domains responsible for the striking differences compared with mammalian TRPMs, we have introduced mutations in the pore-forming region, TM6 and the TRP domain of dTRPM. In summary, we could find that (1) G1006- V1008 are a pore loop signature extremely susceptible to minor variation of the amino acid side chain such that E1007D is divalent-nonpermeable and E1007Q nonfunctional. (2) Variations in the C-terminal part of the pore helix have profound effects on the single channel conductance. For instance, changing invertebrate L1004 into an Isoleucin, a residue conserved in mammalian TRPMs, raises single channel conductance to 36 pS, whereas the reciprocal mutation introduced in mouse TRPM3 shows dramatically reduced current amplitudes. (3) Another set of mutations, located in the TRP domain, affects the sensitivity to intracellular magnesium, with the IC50 of being shifted to 306 µM in the mutant S1067E, for example, making this mutant channel constitutively active. Taken together, our findings prove that the currents observed are carried by the dTRPM pore and demonstrate that minor variations in the pore and TRP domain account for most functional differences observed between dTRPM and its mammalian counterparts. The ease by which dTRPM can be converted into mammalian-like TRPM channels and a well-defined and easily- scorable dTRPM phenotype makes Drosophila an excellent platform for the study of function-phenotype relationships of the TRPM family.

84 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 39 TRPV1 activation in brain following fatty acid amide hydrolase (FAAH)-mediated bioactivation as a strategy for developing novel analgesics

Peter M. Zygmunt 1, David A. Barrière 2, Christophe Mallet 2, Anders Blomgren 1, Laurence Daulhac 2, Frédéric Liberta 2, Eric Chapuya 2, Monique Etienne 2, Alain Eschalier 2 and Edward D. Högestätt 1 1Department of Clinical Chemistry and Pharmacology, Lund University, Lund, Sweden 2Clermont Université, Université d'Auvergne, Pharmacologie Fondamentale et Clinique de la Douleur, Clermont-Ferrand, France

We have previously demonstrated that paracetamol is rapidly converted to the potent TRPV1 activator AM404 (1) and that activation of TRPV1 in brain contributes to the antinociceptive activity following oral administration of this common analgesic in rodents (2). Here we examined whether systemic administration of p-aminophenol, the deacetylated form of paracetamol, and the primary amine 4-hydroxy-3- methoxybenzylamine (HMBA) also generates TRPV1 active metabolites in brain and produces antinociception in various pain tests (formalin, von Frey and tail immersion tests) in mice. Intraperitoneal injection of p-aminophenol and HMBA produced both antinociception and a dosedependent formation of the potent TRPV1 activators AM404 and arvanil in brain. The antinociceptive effect disappeared and the brain levels of AM404 and arvanil were substantially reduced in mice subjected to genetic deletion of TRPV1 and FAAH, respectively. Intracerebroventricular injection of the TRPV1 blocker capsazepine prevented the antinociceptive effect of both p-aminophenol and HMBA in the formalin test. These findings highlight a strategy for developing novel analgesics targeting TRPV1 in the brain based on the administration of a predrug undergoing FAAH-dependent metabolic conversion to TRPV1 active metabolites in the central nervous system.

1. Högestätt et al. , J Biol Chem 280 , 31405, 2005. 2. Mallet et al. , PLoS One 5, e12748 (2010).

International Workshop on Transient Receptor Potential (TRP) Channels 85 Posters Nº 40 Gq-coupled receptors potentiate the osmotic activation of TRPC5

Imane Jemal , Anna Lucia Conte, Sergio Soriano, Ana Gomis Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas. Sant Joan d’Alacant, 03550 Alicante, Spain.

Mammalian transient receptor potential canonical (TRPC) genes encode a family of nonselective cation channels that are activated following stimulation of G-protein- coupled membrane receptors linked to phospholipase C. TRPCs are candidates for Ca 2+ entry channels in brain since they are highly expressed in the nervous system, form Ca 2+ -permeable channels in vitro and are activated by agonists that induce intracellular Ca 2+ release. 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. Our laboratory showed that hypoosmotic and pressure-induced membrane stretch activated TRPC5 channel. Moreover we showed that the response to osmotically induced membrane stretch is blocked by GsMTx-4, an inhibitor of stretch activated ion channels. Here we show that this activation is further potentiated by osmotic modulation of G- protein-coupled receptors via PLC-coupled and Ca 2+ -dependent signalling pathways. When TRPC5 is co-expressed with the Gq/ 11 -coupled type1 histamine-receptor (H 1), 2+ the osmotically-evoked [Ca ]i response results from a dual mechanism: calcium entry from the extracellular medium and calcium release from intracellular stores. Other 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 hypoosmotic 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 and CONSOLIDER_INGENIO 2010 (CS2007- 00023).

86 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 41 TRPV1 is sensitized by CDK5-dependent phosphorylation

Thomas Jendryke , Christian H. Wetzel Department of Cell Physiology, Ruhr-University Bochum, 44780 Bochum, Germany

Temperature and chemical compounds are perceived by a variety of ion channels and receptors, in particular members of the transient receptor potential (TRP) ion channel family. TRPV1 as well as TRPA1 are well established nociceptors and it is supposed that, expressed in dorsal root ganglia neurons, these receptors initiate nociception. During inflammatory conditions, receptor activating and modulating factors are released. They sensitize primary afferent neurons of dorsal root ganglia that lead to hyperalgesia and allodynia. The inflammatory factors activate intracellular signaling pathways resulting in the sensitization of TRPV1 ion channels. Predominantly, the sensitization process depends on phosphorylation of TRPV1 by kinases like PKA, PKC and CAMKII. Moreover, Pareek et al. (2007) demonstrated in a mouse model that TRPV1 can also be phosphorylated by the cyclin-dependent kinase 5 (CDK5). Therefore, we developed a strategy to characterize the CDK5 dependent phosphorylation of TRPV1. To investigate the modulation more directly we performed electrophysiological experiments in a heterologous cell system. CHO cells transfected with cDNA coding for TRPV1 or co-transfected with TRPV1, CDK5 and the CDK5 activator P35, were challenged by application of capsaicin. Under Ca 2+ -free conditions, the presence of CDK5 and P35 induced a left-shift of the concentration/response- relationship, indicating sensitization of TRPV1 that is dependent on CDK5. Also TRPV1 single-channel events that were induced by depolarizing voltage-steps in outside out patches, point to a CDK5-dependent effect on single-channel kinetics. In presence of 2 mM CaCl 2, the repetitive application of 3.3 M capsaicin induced a strong tachyphylaxis of TRPV1 that is delayed and reduced by co-expression of TRPV1, CDK5 and P35. In addition, we generated a TRPV1 T407A mutant to inhibit phosphorylation of TRPV1 at the CDK5 consensus site, and generated a T407D mutant to mimic the phosphorylation. A pharmacological and electrophysiological approach using these mutants will help to further characterize the effect of phosphorylation at position T407 and its effect on sensitization and tachyphylaxis. Taken together, these first data support the observation that TRPV1 is phosphorylated and sensitized by CDK5.

International Workshop on Transient Receptor Potential (TRP) Channels 87 Posters Nº 42 Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants

Michael Ha 1, Boyi Liu 2, Daniel N. Willis 1, John B. Morris 1 and Sven-Eric Jordt 2 1: Toxicology Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06029 2: Department of Pharmacology, Yale School of Medicine, New Haven, CT 06520, [email protected]

Menthol, the cooling agent in peppermint, is added to almost all commercially available cigarettes. Menthol stimulates olfactory sensations, and interacts with Transient Receptor Potential Melastatin 8 (TRPM8) ion channels in cold-sensitive sensory neurons, and TRPA1, an irritant-sensing channel. It is highly controversial whether menthol in cigarette smoke exerts pharmacological actions affecting smoking behavior. Using barometric plethysmography, we investigated the effects of menthol on the respiratory sensory irritation response in mice elicited by the smoke irritants, acrolein, acetic acid or cyclohexanone. Menthol, at a concentration (16 ppm) lower than in smoke of mentholated cigarettes, immediately abolished the irritation response to acrolein, an agonist of TRPA1, as did (460 ppm), a ligand of TRPM8 and other TRP ion channels. Menthol's effects were partially reversed by a TRPM8 antagonist, AMTB and by a novel high-affinity TRPM8 receptor antagonist. Menthol's effects were not specific to acrolein, as menthol also attenuated irritation responses to acetic acid, and cyclohexanone, an agonist of the capsaicin receptor, TRPV1. Menthol was efficiently absorbed in the respiratory tract, reaching local concentrations sufficient for activation of sensory TRP channels. These experiments demonstrate that menthol and eucalyptol, through activation of TRPM8, act as potent counter-irritants against a broad spectrum of smoke constituents. Through suppression of respiratory irritation, menthol may facilitate smoke inhalation and promote addiction and smoking- related morbidities.

88 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 43 Mustard oil (AITC) activates TRPA1 and TRPV1 but facilitates heat responsiveness by sensitizing unknown transducer(s)

Tatjana I. Kichko , Tal Hoffmann and Peter W. Reeh Department of Physiology and Pathophysiology, University Erlangen-Nuremberg, Germany

The pungent constituent AITC of mustard oil excites rat cutaneous nociceptors and sensitizes to heat (Reeh et al.1986); in humans it induces burning pain, relieved by colling and heat hyperalgesia (Koltzenburg et al.1994), suggesting mediation by the heat transducer channel TRPV1. Indeed, higher concentrations of AITC (>100µM) have recently been shown to activate recombinant TRPV1, whereas lower ones act selectively through TRPA1 (Everaerts et al.2011). Apart from coexpression in primary nociceptive neurons, there are indications for molecular interactions between both TRP channels (Akopian 2011). Findings from cellular models may require validation using peripheral innervated tissues, and one way is to measure stimulated CGRP release in vitro employing wildtype and knockout mice, here TRPV1 -/-, TRPA1 -/- and double knockouts (dKOs). Isolated hairy skin, buccal mucosa and trachea were used, tissues with less, more and maximal coexpression of TRPA1 in TRPV1 positive sensory nerves, respectively. AITC 1mM was required to stimulate robust CGRP release from skin and mucosa which in both tissues was substantially reduced in TRPV1 -/-, but not TRPA1 -/- and further abolished or reduced in dKOs, suggesting a possible master-slave relationship between TRPV1 and TRPA1, respectively. In contrast, 100µM AITC was sufficient to induce comparable CGRP release from the trachea which was linearly concentration-dependent (10-1000µM) and abolished in TRPA1 -/- and dKOs (at 100µM) but fully retained in TRPV1 -/-. In all three tissues the noxious heat-induced CGRP release depends to 50-70% on TRPV1, the retained response in TRPV1 -/- and dKOs being due to unknown heat transducer (s). Chemical and inflammatory sensitization to heat usually depends on TRPV1, however, upon AITC application TRPV1 played no facilitating role in the trachea, and heat sensitization was even more pronounced in skin and mucosa of TRPV1 -/- as well as in all three tissues of TRPA1 -/-. Most consistent was the proportional enhancement by AITC of the heat responses in all three tissues of the dKOs, strongly suggesting a compensatory, functional or transcriptional, upregulation of the yet unknown other heat transducer (s) that is (are) obviously sensitized by AITC. Possible candidates currently are TRPV4 and /or TRPM3 (Vriens et al.2011). Thus, sensitization to heat is possible without TRPV1, which insight may be extended to inflammatory hyperalgesia and pain.

International Workshop on Transient Receptor Potential (TRP) Channels 89 Posters Nº 44 Structure-activity relationship of TRPM2 and ADP ribose analogues

Tanja Kirchberger *, Ralf Fliegert*, Christelle Moreau §, Angelika Harneit*, Andreas Bauche*, Barry V.L. Potter §, and Andreas H. Guse* *Calcium Signalling Group, Department of Biochemistry and Signal Transduction, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany §Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom

TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a non-selective cation channel located in the plasma membrane and permeable for calcium and sodium [1, 2]. Channel opening is mediated by intracellular adenosine diphosphoribose (ADPR) and modulated by cytosolic calcium [3]. Activation of TRPM2 by ADPR plays an important role in induction of apoptosis [Gasser et al., 2006] and in chemotaxis [Partida-Sanchez, 2007/8]. Here, the structure-activity relationship of ADPR at TRPM2 was studied. HEK293 cells were stably transfected with TRPM2 and activation of TRPM2 was analysed by infusion of a range of different ADPR analogues modified either at the adenine moiety, the central ribose or the pyrophosphate bridge. Simpler monophosphate such as AMP and analogues were also synthesized and characterized. Interestingly, out of the twenty-six ADPR analogues only 2 were active as agonists. One of these compounds was modified on C2’ of the central ribose (agonist A), whereas the other compound possess an alteration on C2 of adenine nucleobase (agonist B). While agonist B only showed a weak agonist activity, agonist A was active as strong agonist, potentially more potent than ADPR itself. To check for specificity, the agonists were infused to a TRPM2 negative control clone. In summary, interaction of ADPR and TRPM2 are very specific since almost all modifications of ADPR resulted in loss of agonist activity. For example IDP-ribose that differs from ADPR only by the replacement of the imino–group at C6 by an oxo–group did not activate TRPM2. Thus far, our results appear to show that modifications at the C2’ of the central ribose or the C2 of adenine nucleobase may be key to agonist activity.

[1] Perraud AL (2001) Nature, 411, 595-599; [2] Sano Y (2001) Science, 293, 1327-1330; [3] McHugh D (2003) J Biol Chem, 278, 11002-11006; [4] Gasser A (2006) J Biol Chem, 281, 2489-2496; [5] Partida Sanchez P (2007) J Immunol, 179, 7827-7839.

90 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 45 Thermosensitive TRP ion channels in regulation of thermoregulatory and immune functions in homoeothermic organism

Kozyreva T.V. Institute of Physiology, Russian Academy of Medical Sciences, Timakov str., 4, Novosibirsk, Russia

Results of recent studies indicate that temperature-sensitive ion channels, such as TRP channels are in the basis of thermoreception, but their possible regulatory role in maintenance of the whole body functions is not clear. In the present work we would like to present some data concerning the following questions: 1) how a preliminary activation of TRPM8 ion channel by its agonist menthol influences on thermoregulatory and immune parameters in thermoneutral conditions and under cold and heat exposure; 2) whether the genetic level of the thermosensitive TRP ion channel regulation is involved in thermoregulatory adaptive changes; 3) whether any polymorphisms in TRPM8 gene are related to changes in thermosensitivity. In thermoneutral conditions a preliminary activation of TRPM8 ion channel: 1) increased oxygen consumption and decreased respiratory exchange ratio, which may be evidence of enhanced fat oxidation; 2) enhanced the antigen binding and inhibited the antibody production in the spleen, significantly reduced the amount of IgG in blood, and 3) increased the level of IL-1β in blood two times. At deep cooling (rapid and slow) on the background of activated TRPM8 ion channel there were: 1) the decrease in temperature thresholds for all cold-defense responses; 2) the enhancement of metabolic component of emergency thermogenesis at rapid cooling, and the increase in the skin blood vessel constrictor response at slow cooling. All these lead to improved maintenance of core temperature in the cold. For the immune parameters, preliminary activation of TRPM8 eliminated the inhibitory effect of deep cooling on antigen binding and antibody production in spleen. At heating , the activation of TRPM8 caused: 1) a decrease in temperature thresholds for heat-defense skin blood vessel response; 2) earlier increase in metabolic response; 3) elimination of inhibitory effect of heating on antibody production; and 4) stimulation of the antigen binding in spleen due to heating inversed to suppression at heating on the background of TRPM8 activation. Thus, the activation of TRPM8 ion channel may significantly change the functional responses of thermoregulatory and immune systems confirming their interrelation. The data on the influence of long-term adaptation to cold on the expression of genes of thermosensitive TRP ion channel in brain structures of rat demonstrated involving the genomic level of regulation in adaptive changes of thermoregulation. For example, the reduction of mRNA TRPV3-channel after adaptation to cold was specific to the hypothalamus. As shown to date, there are a large number of gene polymorphisms of thermosensitive TRP ion channels. It may indicate the possibility of molecular genetic diversity of temperature sensitivity. We have shown in human the functional differences, associated with sensitivity to cold and menthol in subjects with different genotypes of the single-nucleotide polymorphism rs11562975 (GG and GC) in TRPM8 gene. Subjects with heterozygous genotype GC were characterized by increased sensitivity to cold and reduced sensitivity to menthol, agonist of the ion channel TRPM8, compared with subjects with homozygous genotype GG. These data are evidence of the possibility to identify the genetic predictors defining features of thermosensitivity.

International Workshop on Transient Receptor Potential (TRP) Channels 91 Posters Nº 46 Activation of the human cation channel TRPM8 depends on the interaction between transmembrane segments S3 and S4

Mathis Winking, Cornelia Kühn, Daniel Hoffmann, Andreas Lückhoff and Frank Kühn Institute of Physiology, Medical Faculty, RWTH Aachen, D-52057 Aachen, Germany

For TRPM8 (M8) a gating mechanism has been suggested that is closely related to the well described mechanism of voltage-dependent cation channels (Voets et al., 2004). A central element within the gating structure of M8 is represented by a few positively charged amino acid residues located in transmembrane segment S4 and the S4-S5 linker, which make the channel sensitive to voltage changes across the cell membrane (Voets et al., 2004, 2007). This voltage-sensing element seems to be quite primitive, if compared to the perfectly tuned S4-voltage sensors of the classical voltage-dependent cation channels. In contrast to these, M8 is not only gated by voltage changes but additionally stimulated by cold temperatures and various natural compounds from plants (e.g. menthol, eucalyptol). It has been shown that these stimuli shift the current- voltage relation of M8 to physiological ranges, and therefore most probably interact with the voltage sensor element in S4-S5, either in a direct or indirect manner (Voets et al., 2004). The hypothesis of an indirect mechanism is supported by the finding that also transmembrane segments S2 and S3 bear sites critical for the sensitivity of M8 to menthol and the synthetic agonist icilin, respectively (Bandell et al., 2006; Chuang et al., 2004). Hitherto, an interaction between S2, S3 and S4 during the process of channel activation has not been experimentally demonstrated. In an attempt to identify charge pairing in M8, formed between D802 in S3 and R842 in S4, we functionally characterized a series of charge-reversal mutations by whole-cell patch-clamp analysis and by Ca 2+ -imaging experiments. The single charge-reversal mutations R842D or R842E in the putative voltage sensor fully abrogated the function of M8. We reasoned that, if the loss-of-function phenotype of these S4 mutants possibly is caused by the disruption of specific electrostatic interactions with D802, the function might be rescued by the second reversal mutation D802R in S3. Indeed, the functional analysis of the double charge-reversal mutants R842D+D802R and R842E+D802R demonstrated that both M8 variants largely regain the sensitivity to voltage, cold temperatures and to menthol. No sensitivity to icilin was restored. The mutant R842E+D802R exhibited more recovery of the sensitivity to cold than to menthol, whereas the opposite was observed for the mutant R842D+D802R. Both double charge-reversal mutants were significantly less sensitive to menthol and to cold than the wt-M8 channel. The single charge-reversal mutation D802R was found to disrupt the icilin sensitivity while retaining the sensitivity to menthol and a reduced sensitivity to cold. We conclude that the effects of icilin, menthol and cold on M8 require a well-balanced assembly of charged residues within S3 and S4 and that the total amount of negative or positive charges is of critical importance.

This study was supported by the Deutsche Forschungsgemeinschaft (DFG KU-2271/1-1).

92 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 47 A link between TRPV1 and obesity-induced hypertension

Lihuan Liang, Nichola Marshall, Jennifer Bodkin, Elizabeth Fernandes and Susan D. Brain Centre of Integrative Biomedicine and Cardiovascular Division, Franklin-Wilkins Building, Waterloo Campus, King’s College London, British Heart Foundation Centre, London SE1 9NH, UK

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel and can be activated by a range of stimuli, such as capsaicin, heat (>43ºC), protons (pH<6) and certain mediators. Traditionally TRPV1 is associated with pain and the release of vasodilator neuropeptides, that can have protective roles in cardiovascular disease. However, a role for TRPV1 has been suggested also in association with type 2 diabetes development. We have investigated the role of TRPV1 in a mouse model of obesity-associated hypertension induced by high fat diet (HFD, 35% of fat from lard). In this study, 3-week old female (n= 8-13) and male (n=4) TRPV1 wild type (WT) and TRPV1 knockout (TRPV1 KO) mice 1 (age and sex matched) were fed with either normal (4% of fat) or HFD (what percentage) for 12 weeks. At the end of the study, both WT and TRPV1 KO mice became similarly obese after HFD feeding. But the histological results showed that the percentage of enlarged adipocytes is significantly higher in WT mice than in TRPV1 KO mice. On the other hand, analysis of mesenteric vessel responsiveness by wire myography, showed no significant changes to a vascular constrictor (phenylephrine), a vasodilator (CGRP) and an endothelial dependent vasodilator (carbachol), irrespective of genotype and diet. However, a significant increase (p<0.01) in systolic blood pressure and mean arterial pressure was observed in the HFD-fed WT but not KO mice, when compared to the normal diet group. This difference was observed by both tail cuff and radiotelemetry techniques. Furthermore, significant increases in aortic medial wall width (p<0.01) and collagen wall width (p<0.001) were observed in HFD-fed WT compared to normal diet group. The same effect was not observed in TRPV1 KO mice. These findings reveal that TRPV1 is involved in the development of high blood pressure and vascular hypertrophy associated with obesity.

References: 1. Fernandes ES, Liang L, Smillie SJ, Kaiser F, Purcell R, Rivett DW, Alam S, Howat S, Collins H, Thompson SJ, Keeble JE, Riffo-Vasquez Y, Bruce KD, Brain SD. TRPV1 Deletion Enhances Local Inflammation and Accelerates the Onset of Systemic Inflammatory Response Syndrome. J Immunol 2012; 188: 5741-5751.

This work is supported by British Heart Foundation (NJM), a Capacity Building Award in Integrative Mammalian Biology funded by the British Biotechnology Science Research Council, British Pharmacological Society, Higher Education Funding Council, Knowledge Transfer Network, Medical Research Council and Scottish Funding Council (LL and SDB), Arthritis Research UK (ESF)

International Workshop on Transient Receptor Potential (TRP) Channels 93 Posters Nº 48 The transcription factor AML1, regulates the Transient Receptor Potential Vanilloid-2 (TRPV2) channel-mediated differentiation of glioblastoma stem cells

Liberati S.1,2* , Morelli M.B. 1, Nabissi M. 1, Amantini C. 1, Farfariello V. 1,3 , Santoni M. 4 , Ricci-Vitiani L. 5, Compieta E. 1 and Santoni G. 1 1 School of Pharmacy, Section of Experimental Medicine, University of Camerino, Camerino; Italy 2 Dept Molecular Medicine, Sapienza University, Rome; Italy 3 Dept Urology and Andrology, University of Perugia; Italy 4 Dept of Clinic Oncology, Polytechnic University of Marche, Ancona, Italy. 5 Istituto Superiore di Sanità, Rome; Italy. * [email protected]

Astrocyte differentiation occurs through the regulation of a variety of transcription factors. Among these, AML1/RUNX1 has been suggested to play a pivotal role not only in definitive hematopoiesis or in embryonic stem cells but also in the development of stem/neural progenitor cells. Microglia, the resident macrophages of the SNC, are associated with the pathogenesis of many stem cell-derived brain tumours. Ontogenetically they derive from primitive myeloid progenitors, and represent a distinct haematopoietic population in the mononuclear phagocyte system. In this regard, we have recently reported that TRPV2 channel promotes both in vitro and in vivo Glioblastoma Stem Cell (GSC) differentiation and inhibits their proliferation (Morelli et al., Int J Cancer 2012), however no findings on transcriptional regulation of TRPV channels during astroglial differentiation of GSCs have been provided so far. AML1/RUNX1 coordinates the proliferation and differentiation of olfactory neuron stem/neural progenitor cells, but to date its involvement on GSC progression have not been elucidated. Herein, we firstly evaluated the relationship between the expression of AML1 splice variants in five different GSC cell lines. We found that all the GSC cell lines express high levels of AML1a, AML1b and AML1c splice variants. We found that astroglial differentiation, as well as Phorbol 12-myristate 13-acetate (PMA)-induced proliferation or overexpression of TRPV2 in GSCs, were accompanied to changes in AML1 splice variants, and these effects seem to be regulated in an ERK1/2-dependent manner. Finally, changes in AML1 splice variant espression and differentiation levels in GSC cell lines were associated with increased susceptibility of these cells to chemotherapeutic treatment. Better understanding of the molecular mechanisms and transcriptional factors that regulate the proliferation-differentiation balance in GSCs would lead to a more specific targeting of pharmacological agents.

Funding: This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) National Grant 2011-2013 (Number 11095).

94 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 49 Engineering of the TRPC3 pore reveals molecular determinants of cation selectivity and gating

Michaela Lichtenegger 1, Michael Poteser 2, Thomas Stockner 3, Hannes Schleifer 2, Christoph Romanin 4, and Klaus Groschner 2 1) Institute of Pharmaceutical Sciences - Pharmacology and Toxicology, University of Graz 2) Institute of Biophysics, Medical University of Graz 3) Institute of Pharmacology, Medical University of Vienna 4) Institute of Biophysics, University of Linz

Transient receptor potential canonical 3 (TRPC3) represents one of the first identified mammalian relatives of the Drosophila TRP channel. TRPC3 function was shown to be involved in various physiological processes such as development of neuronal and cardiac jtissues, immune cell maturation and blood vessel constriction as well as in several pathophysiological events such as cardiovascular hypertrophy and hypertension. Upon activation of GPCR linked to phospholipase C, TRPC3 mediates a nonselective cation current being potentially involved in a wide spectrum of Ca 2+ signalling mechanisms. To date, little is known about the architecture of the channel protein and the coherences between structure and function. Guided by a molecular structure- modelling, we combined site directed mutagenesis with patch-clamp measurements in order to identify residues essential for selectivity and gating of TRPC3 channels. We have recently identified a central negatively charged glutamate residue (E630) that is essential for divalent permeability representing a key element of the channel’s selectivity filter (Poteser et al., PNAS 2011). Cystein scanning mutagenesis revealed that E630 is located within the narrowest region of the permeation pathway, formed by the putative pore loop. An amine-based sizing approach demonstrated a decreased pore diameter resulting from exchange of E630 to neutral glutamine. Mutational neutralization of the two negatively charged residues E615 and E616 and a positively charged lysine residue K619, located at the entrance of the permeation pathway, resulted in loss of sensitivity of the channel to GPCR/PLC-signalling. Surprisingly, basal channel activity remained fully preserved, indicating a structurally intact permeation pathway. We conclude that three charged residues situated at the outer pore-vestibule are pivotal for the channel’s activation machinery, most likely by enabling sensitivity to lipid mediators. These results provide insight into the molecular basis of TRPC3 channel function and may represent an important step towards understanding the channel’s role in native tissues.

Supported by the DK+ Metabolic and Cardiovascular Disease Grant W2126-B18

International Workshop on Transient Receptor Potential (TRP) Channels 95 Posters Nº 50 Phenytoin, nifedipine and carbamazepine induce gingival enlargement trough TRPA1 activation

López-González M.J. , Fajardo O., Meseguer V., Valero M., Pertusa M., Belmonte C., Viana F. Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Alicante, Spain.

Gingival enlargement is a common side effect observed in patients treated with antiepileptic (phenytoin and carbamazepine), inmunosuppresant (cyclosporin) and some antihypertensive (nifedipine) drugs. The molecular mechanisms producing gingival overgrowth by these agents are unknown. Recently, we discovered that 1,4 dihydropyridines, including nifedipine, can activate TRPA1 channels, a cationic channel expressed in nociceptors that is activated by many irritant compounds. We found that phenytoin also activates TRPA1 expressing neurons. Therefore, we hypothesized that TRPA1 could be the molecular target leading to drug-induced gingival overgrowth. Phenytoin, carbamazepine and nifedipine increased intracellular calcium in CHO cells expressing mouse TRPA1 and HEK cells expressing human TRPA1. These responses were not observed in cells lacking TRPA1 or cells expressing TRPM8 or TRPV1. No such effect was observed with cyclosporin. By RT-PCR we demonstrated that TRPA1 is expressed in cultured human gingival fibroblasts (HGF). Calcium imaging showed that HGF cells responded to TRPA1 agonists (e.g. mustard oil) and drugs producing gingival enlargement. Moreover, activation of TRPA1 by phenytoin was blocked by HC030031 a specific blocker for this channel. The use of shRNA against hTRPA1 in HGF cells, reduced TRPA1 expression and phenytoin activation. Ascorbic acid and α- tocopherol, two antioxidants, are reduced in blood samples from patients with gingival enlargement. We found that in the presence of ascorbic acid and α-tocopherol TRPA1 activation by phenytoin was markedly reduced. Gingival enlargement may be caused by fibroblast proliferation or a higher synthesis and secretion of collagen by the fibroblasts. Using MTT cell proliferation assay, we demonstrate that none of these drugs induce proliferation of human gingival fibroblasts. These results indicated that phenytoin, nifedipine and carbamazepine can activate TRPA1 in heterologous systems, primary sensory neurons and native gingival fibroblasts. Currently, our working hypothesis is that TRPA1 leads to enhanced collagen synthesis by fibroblasts, leading to gingival enlargement.

Supported by grant SAF2010-14990 to F.V. and a predoctoral fellowship to MJ.LG.

96 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 51 FGF-2, calcium signals and the control of neurite growth in chick developing parasympathetic neurons: involvement of TRPC channels

Lovisolo D. 1,2,3 , Gilardino A. 1,2 , Ruffinatti F.A. 1, Zamburlin P. 1, Farcito S. 1 1 Department of Life Sciences and Systems Biology, University of Torino, Italy 2 NIS Centre of Excellence for Nanostructured Interfaces and Surfaces, University of Torino, Italy 3 NIT Neuroscience Institute of Torino

Basic Fibroblast Growth Factor (bFGF or FGF-2) is a well established and multifunctional neurotrophic factor for peripheral as well as central neurons. Previous studies have shown that in embryonic chick ciliary ganglion neurons FGF-2 promotes neuronal survival and neurite growth in both dissociated and organotypic cultures. Moreover, the former effect has been shown to be dependent on calcium influx through voltage-independent channels. The aim of the present study has been to analyze the role of calcium influx neurite growth promoted by FGF-2, the different pathways involved and their spatial localization. Experiments conducted on ciliary ganglion neurons with the intracellular calcium chelator BAPTA-AM point to a specific involvement of calcium signals in this FGF- mediated neurotrophic effect. Furthermore, inhibitors of L- and N-type voltage dependent calcium channels, but not of voltage dependent sodium channels, significantly decreased neurite growth sustained by the factor. A similar reduction in neurite growth was observed in the presence of the non specific TRPC inhibitor SKF96365 (5 M), pointing to an involvement of this channel family. A further sets of experiments was performed by taking advantage of compartmentalized cultures (Campenot chambers). The results point at a cooperative effect of the FGF-2 induced calcium signals at the soma and at the growth cones of the neurites; voltage dependent calcium channels and TRPCs are involved at both compartments Accordingly, calcium imaging experiments have shown that the FGF-2 induced signals are partially reduced by blockers of the two classes of calcium permeable channels both at the cell soma and at the growth cone.

International Workshop on Transient Receptor Potential (TRP) Channels 97 Posters Nº 52 Cold-activated TRPM8 channels are activated by the volatile anaesthetic chloroform

J.A. Manenschijn , A. Parra, O. Gonzalez, C. Morenilla, C. Belmonte, F. Viana Instituto de Neurociencias de Alicante (UMH-CSIC), San Juan de Alicante, Alicante, Spain

TRPM8 is a member of the transient receptor potential family of non-selective cationic channels. TRPM8 is activated by cold temperature, cooling compounds and voltage. Recent studies have implicated TRPM8 in the pathophysiology of cold nociception and cold allodynia, leading to a strong interest in the search for novel modulators of TRPM8 activity. Here we describe the effects of chloroform (CLF), a volatile aneasthetic. Using patch-clamp techniques, we studied CLF-induced responses in a HEK293 cell line stably expressing rat TRPM8. CLF activated whole-cell currents with a typical TRPM8 signature (strong outward rectification and near zero mV reversal). Using ratiometric fluorescence calcium imaging, we found that the effect of CLF was concentration-dependent and reversibly blocked by BCTC, a TRPM8 antagonist. CLF activated HEK293-cells transfected with the menthol-insensitive Y745H-TRPM8 mutant normally. We also investigated effects of CLF on cold-sensitive neurons from the dorsal root ganglia (DRG) of adult TRPA1-KO mice, and found that CLF activates these neurons, an effect which is reversibly blocked by BCTC. Similar effects were found in TRPM8-YFP(+) cold-sensitive DRG neurons driven by the TRPM8 promoter. Our results suggest that a low concentration of CLF is able to decrease the threshold of TRPM8 by cold. We recorded spontaneous nerve terminal impulse (NTI) activity from cold thermoreceptors innervating the mouse cornea in vitro . CLF produced an increase in the NTI resting activity as well as in the mean frequency during sustained cooling pulses in the presence of CLF. In brief, we show robust activation of heterologously and natively expressed TRPM8 channels by CLF. The mechanism of activation is yet unexplained. However, the absence of the menthol binding site did not affect the effect of CLF on TRPM8, suggesting that this site is not critical for its activation by chloroform.

Supported by grant SAF2010-14990 to F.V. and a predoctoral fellowship to J.A.M.

98 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 53 Heat activated-ion channels TRPV1 and TRPV3 in thermosensation

Irène Marics , Pascale Malapert, Stéphane Gaillard, Aziz Moqrich Developmental Biology Institute of Marseilles-Luminy, UMR6216, France

Thermosensation is one of the sensory modalities of the skin. It provides (1) a thermoregulatory afferent signal for homeostatic mechanisms which keep the body at an optimal working temperature, (2) the capability to detect potentially noxious thermal stimuli that pose an immediate threat to the integrity of the integument (noxious heat and cold stimuli). A specific sensory transduction mechanism is needed, including as a key element- a molecular sensor- transforming physical parameters (temperature) into a biologically significant signal. Over the last decade, a number of transient receptor potential (TRP) ion channels have been identified whose activity depends on the temperature of their environment. Each of the receptors operates over a specific temperature range, thereby providing a potential molecular basis for thermosensation (figure 1) . We are interested in several thermo-TRPs, namely TRPV1 and TRPV3 whose activation overlaps over a wide range of heat temperature. The aim of this project is to generate mice deficient for both TRPV1 and TRPV3 channels. The simultaneous inactivation of the TRPV1 and TrpV3 genes will allow us to understand their function in temperature transduction through a communication between skin cells and dorsal root ganglia (DRG) fibers.

International Workshop on Transient Receptor Potential (TRP) Channels 99 Posters Nº 54 Parthenolide, contained in the feverfew herb, selectively activates and desensitizes the Transient Receptor Potential Ankyrin 1 (TRPA1) channel

S. Materazzi 1, C. Fusi 1, S. Benemei 1, G. De Siena 1, E. Rossi 1, G. Trevisan dos Santos 1, G. Appendino 2, P. Geppetti 1 and R. Nassini 1 1Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy; 2Department of Chemical, Alimentary, Pharmaceutical and Pharmacological Sciences, University of Eastern Piedmont, Novara, Italy.

Parthenolide, the main bioactive component extracted from the leaves of Tanacetum parthenium, or feverfew herb, exhibits anti-inflammatory properties and is used for the treatment of migraine and arthritis. However, the mechanism(s) of action of these possible beneficial effects of feverfew is still not unclear. Parthenolide is a sesquiterpene lactone with a α-methylene-γ-lactone ring and an epoxide moiety that can interact with nucleophilic sites of several molecules. The Transient Receptor Potential Ankyrin 1 (TRPA1) channel, co-localized on primary sensory nerves with the “capsaicin receptor” (TRPV1), is activated by a wide variety of noxious and irritant reactive molecules that covalently modify cysteine or lysine residues of the protein. We hypothesized that parthenolide, which can trap thiol groups in an irreversible complex, activates and desensitizes TRPA1 channel expressed on primary sensory nerves, producing, through this mechanism, anti-inflammatory and analgesic effects. Calcium imaging experiments were performed in primary neurons isolated from rat or mouse dorsal root ganglion (DRG) and in human recombinant TRPA1 expressing HEK293 cells. Parthenolide (1-1000 µM) produced a concentration-dependent increase in intracellular calcium mobilization both in rat DRG neurons and in human TRPA1- HEK293 cells. The response was selectively abated by the TRPA1 antagonist, HC- 030031 (10 µM) or in DRG neurons taken from TRPA1-deficient mice. Contractile responses, modulated by TRPA1 activation, have been studied in isolated strips of rat urinary bladder. Parthenolide (1-300 µM), similarly to the selective TRPA1 agonist, mustard oil (MO), produced a contractile response via a neurogenic mechanism and TRPA1 activation. Further, exposure to the highest concentration of parthenolide (300 µM) or MO (300 µM) generated desensitization to their contractile effects (100 µM) and cross-desensitization to capsaicin (0.3 µM). In addition, we evaluated the ability of parthenolide (100 µM) to elicit CGRP-release from rat spinal cord and meninges/trigeminal ganglia. CGRP-release induced by parthenolide was significantly reduced by HC-030031 (10 µM), in a calcium-free medium and after tissue desensitization with capsaicin (10 µM, 20 min). Similar results have been obtained in preparations exposed to a high concentration of parthenolide (300 µM, 20 min), suggesting desensitization. Finally, ocular instillation of parthenolide evoked a concentration-dependent acute nociceptive behavior (eye wiping) in wild-type mice, an effect that was absent in TRPA1-deficient mice. Repeated exposures/treatments to/with parthenolide caused desensitization to parthenolide itself but also to MO, and reduced the response induced by capsaicin, indicating self- and cross-desensitization. Taken together, these findings indicate that parthenolide selectively activates TRPA1 channel expressed on sensory neurons and following exposure to sufficient amounts causes desensitization. The ability of parthenolide to desensitize sensory nerve terminals may be responsible for the anti-inflammatory and analgesic effects of the compound, including the reported antimigraine effect.

100 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 55 Substance-P and α-CGRP silencing reduces inflammatory sensitization of TRPV1

Sakthikumar Mathivanan 1, Isabel Devesa 1, Christoph Jakob Wolf 1, Clotilde Ferrandiz Huertas 1, Rafael Lujan 2, Antonio Ferrer-Montiel 1 1Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, Elche, Spain 2 Facultad de Medicina, Universidad de Castilla la Mancha, Albacete, Spain

TRPV1, a polymodal non selective cation channel, acts as a major integrator of painful stimuli. During inflammation, the release of inflammatory mediators act on TRPV1 leading to enhanced nociceptor excitability and thermal hyperalgesia. Acute inflammatory sensitization of TRPV1 involves both the modification of channel gating properties by phosphorylation and the recruitment of channels to the neuronal surface. Mobilization of new channels to the plasma membrane by some pro-inflammatory mediators occurs through SNARE-dependent exocytosis, but the exact mechanism involved remains to be elucidated. We propose that inflammatory recruitment of channels occurs in the neuronal subpopulation that contains the neuropeptides substance P (SP) and α-CGRP. Therefore, we have investigated the contribution of SP and α-CGRP on TRPV1 sensitization on cultured neurons isolated from neonatal rat dorsal root ganglions. Silencing of Tachykinin 1, which encodes SP, and α-CGRP mRNA expression resulted in decreased inflammatory sensitization of TRPV1, which was assessed by functional analysis using calcium-imaging influxes and patch clamp electrophysiology. These results show that SP and α-CGRP contributes to inflammatory sensitization of TRPV1 where membrane recruitment of the channel is essential, and suggest a potential role of these neuropeptides on TRPV1 mobilization to the vesicles and the neuronal surface.

International Workshop on Transient Receptor Potential (TRP) Channels 101 Posters Nº 56 Calmodulin and S100A1 bind the N-terminal region of TRPM1

Jirku Michaela 1, Bumba Ladislav 2, Teisinger Jan 1 1 Institute of Physiology, Academy of Sciences of the Czech Republic 2 Institute of Microbiology, Academy of Sciences of the Czech Republic Melastatin channel (MLSN) or TRPM1 (transient receptor potential melastatin 1) is the founding member of the subfamily of TRPM ion channels belonging to the superfamily of TRP channels. Intracellularly located N- and C-tails are responsible for regulation of TRP channels, which carry binding sites for signal molecules like calmodulin (CaM) or S100A1. [1, 2] TRPM1 is present in human melanocytes and retina. It seems that loss of TRPM1 correlates with increased aggressiveness in melanoma. [1, 3 ] TRPM1 is localized in bipolar cells in retina and participates in processes connected to vision. Mutations of TRPM1 gene are associated with congenital stationary night blindness in humans. [4, 5] There is currently a scarcity of structural / functional data on TRPM1 channel. In this study we identified CaM and S100A1 binding site on the N-terminus (NT) of rat TRPM1 channel. The domain L 242 -E344 on NT was cloned into the pET32b vector and verified by sequencing. This construct and appropriate mutants were amplified in E. coli Rosetta cells and purified in two-step purification process. Amino acid sequence was checked by MS MALDI-TOF. Surface plasmon resonance and steady-state fluorescence anisotropy measurements were used to test the binding of CaM and S100A1 to L242 -E344 . We determined several positive and hydrophobic residues to be responsible for binding of TRPM1-NT L 242 -E344 to CaM and S100A1. The results of the experiments also suggest that CaM and S100A1 bind to the same or overlapping binding site.

This project was supported by Grants GACR 301/10/1159, GACR 207/11/0717.

References: [1] L. M. Duncan, J. Deeds, J. Hunter, J. Shao, L. M. Holmgren, E. A. Woolf, R. I. Tepper, A. W. Shyjan, Cancer Res 58 (1998), 1515-1520 [2] M. X. Zhu, Pflugers Arch 451 (2005), 105-115 [3] L. Hammock, C. Cohen, G. Carlson, D. Murray, J. S. Ross, C. Sheehan, T. M. Nazir, J. A. Carlson, J Cutan Pathol 33 (2006), 599-607 [4] Z. Li, P. I. Sergouniotis, M. Michaelides, D. S. Mackay, G. A. Wright, S. Devery, A. T. Moore, G. E. Holder, A. G. Robson, A. R. Webster, Am J Hum Genet 85 (2009), 711-719 [5] M. Nakamura, R. Sanuki, T. R. Yasuma, A. Onishi, K. M. Nishiguchi, C. Koike, M. Kadowaki, M. Kondo, Y. Miyake, T. Furukawa, Mol Vis 16 (2010), 425-437

102 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 57 Overexpression, purification and functional characterization of human TRPA1

Lavanya Moparthi 1, Sabeen Survery 1, Mohamed Kreir 2, Per Kjellbom 1, Edward D. 3 1 3 Högestätt , Urban Johanson and Peter M. Zygmunt 1Dept of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, Lund, Sweden 2Nanion Technologies GmbH, Gabrielenstrasse 9, D-80636 Munich, Germany 3Clinical Chemistry & Pharmacology, Department of Laboratory Medicine, Lund University, Lund, Sweden

Transient receptor potential (TRP) ion channels are non-selective cation channels, which can be divided into seven subfamilies (1). Several TRP channels are involved in sensory perception, including chemo-, thermo-, and mechanosensation (2). The present study focused on TRPA1, which differs from the other TRP channels by having a large number of ankyrin repeats in the N- terminal domain. TRPA1 is expressed in a subset of nociceptive neurons of dorsal root and trigeminal ganglia. It has been shown to act as a chemosensor of various plant-derived reactive compounds, environmental pollutants and endogenous inflammatory mediators (3, 4, 5). However, its role as a noxious cold- and mechanosensor is still unclear. In this study, we overexpressed human TRPA1 in the yeast Pichia pastoris , using a similar procedure as that successfully applied to eukaryotic aquaporins, another superfamily of integral membrane proteins (6, 7). The coding sequence of the full length human gene and two truncated constructs were amplified and cloned into a modified pPICZB-vector encoding an N-terminal deca-histidine tag and transformed into E. coli . After confirming the sequence, the constructs were transformed into the Pichia X-33 strain. A zeocin selection was employed to isolate high copy clones, followed by small scale expression and Western blot to identify high expression clones. A feed-batch fermentor was used to get optimal production and the protein purified by Ni-affinity chromatography. The functional characteristics of purified human TRPA1 were studied after its incorporation into planar bilayers (8). Electrophilic (allyl , cinnamaldehyde, N-acetyl-pbenzoquinoneimine) and non-electrophilic (menthol and 9- tetrahydrocannabiorcol) compounds known to activate TRPA1 induced membrane currents of different conductance levels at holding potentials of -60 mV and +60 mV, using both full length TRPA1 and TRPA1 lacking the N-terminal domain.

1. Clapham. Nature 426 517-524 (2003). 2. Nilius and Voets. Pflugers Arch Eur J Physiol 451 1–10 (2005). 3. Jordt et al Nature 427 260-265 (2004). 4. Bautista et al PNAS 102 12248-12252 (2005). 5. Andersson et al Nat Commun 2 551 (2011). 6. Törnroth-Horsefield et al Nature 439 688-694(2006). 7. Horsefield et al PNAS 105 13327-13332 (2008). 8. Kreir et al Lab-on-a-Chip 8 587-595 (2008).

International Workshop on Transient Receptor Potential (TRP) Channels 103 Posters Nº 58 TRPV2 activation induces cytotoxicity in human multiple myeloma cell lines

Nabissi M. 1* , Offidani M. 2, Morelli M.B. 1, Discepoli G. 3, Santoni M. 4, Amantini C. 1, Farfariello V. 1, Liberati S. 1,5 Santoni G. 1 and Leoni P. 2 1 School of Pharmacy, Section of Experimental Medicine, University of Camerino, Italy. 2 Clinic of Haematology, Azienda Ospedaliero-Universitaria Ospedali Riuniti Ancona, Italy. 3 Centre of Genetic Medicine and Prenatal Diagnosis, Az.Osp, G.Salesi, Ancona, Italy. 4 Dept of Clinic Oncology, Polytechnic University of Marche, Ancona, Italy. 5 Dept Molecular Medicine, Sapienza University, Rome, Italy. *[email protected]

Multiple myeloma (MM) is a relative common polyclonal B-cell malignancy, where normal and malignant B-cell differentiation stages have been described using cell surface phenotype B-cell restricted and associated antigens as CD138 (syndecan-1). Interphase fluorescent in situ hybridization (FISH) analysis in CD138 + plasma cells (PCs) have demonstrated that karyotypic abnormalities are present at least in 60% of stage III patients, as t(4;14), 1 gain and del (17p), with strong implication with worst clinical outcome. Recently, oncogenomic studies have advanced understanding of the molecular pathogenesis of MM, revealing that several signaling pathways play a pivotal role in CD138 + PCs proliferation (Ras-Raf-MEK-ERK and NF κB pathways), resistance to apoptosis (BCL-XL over-expression) and drug resistance (PI3K and NF-κB pathways). Since, the need for new prognostic classification and the identification of new therapeutic targets is strongly required. Transient Receptor Potential Channel type-2 (TRPV2), located in chromosome 17p11.2, is a non-selective cation channel showing non selective Ca 2+ permeability triggered by agonists as (CBD). In human cancer, TRPV2 activation or over-expression has been demonstrated to reduce proliferation and increase chemosensitivity and apoptosis by regulating ERK-phosphorilation and Fas-induced apoptosis (Nabissi et al., 2010). Moreover, TRPV2 down-regulation has been associated to a high proliferative and chemoresistant cell phenotype. Although TRPV2 functionality has been evaluated in different cancer cell and tissue, its role in MM has not been investigate. So, firstly we isolated CD138 + PCs from 15 bone marrow aspirations, analysed for 17p11.2 aberration by FISH analysis and for TRPV2 expression by FACS analysis. The results show the absence of del(17p11.2) in all samples and the presence of two distinct subpopulations (TRPV2 + and TRPV2 -) expressed in the CD138 + PCs. Thus, to evaluate the role of in vitro CBD-induced TRPV2 activation, we utilized the RPMI8226 and U266 human MM cell lines, firstly characterized for 17p11.2 by FISH analysis. We found that both cell lines were del(17p). Moreover, by FACS analysis we showed that both cell line were >90% CD138 + and totally TRPV2 -. So, to analyse the effects of CBD-induced TRPV2 activation in CD138 +TRPV2 + cells we create, by TRPV2 gene transfection, a TRPV2-expressing MM cell line model. CBD-TRPV2-dependent effects on cell viability and proliferation was determined in CD138 +TRPV2 +-transfected RPMI8226 and U266 cell lines. As controls CD138 +TRPV2 - RPMI8226 and U266 cells were utilized. The results showed that CBD treatments significantly reduced cell viability and proliferation of both CD138 +TRPV2 +-transfected RPMI8226 and U266 cell lines respect to wild type cell lines.

Funding: This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) National Grant 2011-2013 (Number 11095).

104 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 59 TRPV4 is downregulated in keratynocytes in different human skin tumors

R. Nassini 1, V. Maio 2, S. Materazzi 1, T. Oranges 2, C. Fusi 1, D. Massi 2 1Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy 2Department of Critical Care Medicine and Surgery, Italy Division of Pathological Anatomy University of Florence, Florence, Italy.

The transient receptor potential (TRP) family of channels encompasses 28 proteins expressed in a variety of cell types where they mediate a large series of physiological functions and play major pathophysiological roles. The TRP vanilloid 4 (TRPV4) has been found highly expressed in a subset of somatosensory neurons which also express the capsaicin receptor, TRPV1, and by releasing neuropeptides mediate neurogenic inflammation. It is gated by small reductions in tonicity and by temperatures >27 °C. The expression of TRPV4 by neurosensory str uctures, including circumventricular organs, which detect changes in systemic osmolality, inner ear hair cells, Merkel cells and sensory neurons, and its activation by hypotonic stimuli, suggests that it functions to detect osmotic and mechanical stimuli. TRPV4 immunoreactivity was differentially identified on basal and suprabasal keratinocytes of healthy human skin and their functions have been related to cell survival after skin exposure to noxious heat. However, the expression and function of TRPV4 in skin cancer is poorly understood. The TRP family of proteins exhibits differential expression in cancer tissues. Rather than mutations, changes in expression of TRP proteins seem to be related to alterations in wild type protein level, which might be associated with specific stages of cancer. As the possible role of TRPV4 in skin cancer has been poorly explored. The present project will focus on the identification, function and plasticity of TRPV4 in keratinocytes under normal or pathophysiological circumstances. In normal skin, TRPV4 was diffusely expressed in basal and suprabasal epidermal keratinocytes, and was consistently observed in adnexal structures. Intense immunostaining was detectable in the epidermal and dermal part of the eccrine sweat gland ducts. The secretory portion of sweat glands showed staining of single secretory and myoepithelial cells. Endothelial cells decorating dermal blood vessels were also TRPV4 positive. In solar keratoses and Bowen’s disease, atypical keratinocytes showed a partial to complete loss of TRPV4 expression. In UV-induced SCC on sun- exposed skin and in SCC on protected sites TRPV4 was strongly downregulated while BCC, irrespective of different histotypes, were TRPV4 negative. The TRPV4 agonist, 4αPDD evoked a calcium response in NHEK and in NCTC cells. These responses were inhibited by the TRPV4 selective antagonist, HC-067047.

Present results suggest that TRPV4 is significantly downregulated in skin cancer tissues compared with normal skin tissues. Whether downregulation of TRPV4 in skin cancer is required for or is a consequence of cancer progression remains to be investigated. TRPV4 activation could be involved in the release of some protective cytokines which might be associated with specific stages of skin cancer.

Keywords: TRPV4; keratinocytes, human skin cancer

International Workshop on Transient Receptor Potential (TRP) Channels 105 Posters Nº 60 TRPV1 and GABARAP interaction and their effects on the receptor dynamics

Ontoria-Oviedo I. 1, Estévez-Herrera J. 1,3 , Ferrer-Montiel A. 2, and Planells-Cases R. 1,2 1 Centro de Investigación Príncipe Felipe, Valencia, Spain. 2 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández de Elche, Spain. 3 Present address: Universidad de La Laguna, La Laguna, Tenerife, Spain.

Transient receptor potential vanilloid 1 (TRPV1) has a pivotal role in the physiopathology of pain transduction. We previously found that TRPV1 associates with gamma-amino butyric acid A-type (GABA A) receptor associated protein (GABARAP) both in vitro and ex-vivo. Notably, GABARAP increased TRPV1 increased receptor expression and receptor clustering at the plasma membrane in HEK293 expressing cells while showing altered channel gating and desensitization (Laínez et al . 2010). We further characterized GABARAP-induced membrane TRPV1 clustering by Total Internal Reflection Fluorescence (TIRF) microscopy. The nature of TRPV1 cluster formation was investigated by either varying membrane properties by cholesterol removal or by cytoskeleton alteration. We found that while cholesterol sequestration and removal from the plasma membrane did not affect GABARAP-induced TRPV1 protein increase nor altered its receptor kinetics, tubulin stabilization (Weissmann et al . 2009) lead to increased TRPV1 surface expression and receptor clustering thus mimicking the interaction with the cytosolic adaptor protein GABARAP. Interestingly, pull down assays demonstrate an enhanced binding of tubulin to TRPV1 in the presence of GABARAP. All together, our findings are consistent with a model in which TRPV1 expression and clustering is strongly influenced by interaction with cytoskeleton and suggests that -associated acute pain syndrome is associated with increased surface TRPV1 expression.

Laínez, S., Valente, P., Ontoria-Oviedo, I., Estévez-Herrera, J., Camprubí-Robles, M., Ferrer- Montiel, A., and Planells-Cases, R. (2010). GABA A receptor associated protein (GABARAP) modulates TRPV1 expression and channel function and desensitization. The FASEB Journal (24) 6:1958-1970 Weissmann, C., Reyher, H.J., Gauthier, A., Steinhoff, H.J., Junge, W., and Brandt, R (2009). Microtubule binding and trapping at the tip of neurites regulate tau motion in living neurons. Traffic (10) 11:1655-1668.

This work was supported by grants from el Ministerio de Ciencia e Innovación (MICINN; SAF2010-17045 to R.P.-C) and the Consolider-Ingenio 2010 (MICINN; CSD2008-00005 to A.F.- M and R.P.-C). I. Ontoria-Oviedo was supported by a predoctoral fellowship FPI-MEC (BOE 22 agosto 2008).

106 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 61 Setting up a benchmark for the characterization of TRP channels

Alex Perálvarez-Marín Centre d’Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, Spain.

TRPV1 and TRPV2 are members of the superfamily of the Transient Receptor Potential (TRP) ion channels. They are assembled into homotetramers and allow cations across the membrane in response to stimuli such as heat (TRPV1 and TRPV2) and capsaicin (TRPV1). TRPV1 and TRPV2 share ~50% sequence identity, however the pharmacology difference profiles for TRPV1 and TRPV2 are not so well understood, in fact TRPV2 is an orphan receptor, since no specific endogenous ligand has been identified yet. We propose a multidisciplinary approach to study these channels. To better understand TRPV1 and TRPV2 roles and to go further into their structure, large-scale protein expression of the active forms is required. Here we present our advances in the protein expression and purification of active TRPV1 and TRPV2. In addition, to infer significant hints about the role of TRPV2 and to go further into its function, sequence analysis of orthologs of TRPV2 has been carried out to define common and differential functional/architectural regions. Preliminary biophysical characterization such as thermal stability, and secondary structure composition analysis has been carried out on TRPV2 to identify key structural points in the TRPV2 topology. In addition, chemoinformatics analysis has been performed to identify relevant pharmacological seeds to develop a drug design approach.

International Workshop on Transient Receptor Potential (TRP) Channels 107 Posters Nº 62 The N-glycosylation of TRPM8 channels modulates the temperature sensitivity of cold-thermoreceptor neurons

Pertusa, M. #, ψ, Madrid, R. ψ, Morenilla-Palao, C. #, Belmonte, C. #, and Viana, F. #. #Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, San Juan de Alicante, Spain. ψDepartamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile.

TRPM8 is a member of the transient receptor potential (TRP) ion channel family, which is expressed in sensory neurons and is activated by cold, by voltage and by cooling compounds, such as menthol. TRPM8 is the principal transducer for cold temperature in the somatosensory system. Activation of TRPM8 by cold and menthol takes place through shifts in its voltage-activation curve, allowing channel opening at physiological membrane potentials. Here we studied the N-glycosylation occurring at the pore loop of TRPM8 and its impact on TRPM8 function. This post-translational modification occurs in several ion channels, affecting aspects like their trafficking and gating. We found that the unglycosylated TRPM8 mutant (N934Q) displays marked functional differences compared with the wild-type channel. Principal among these differences is a shift in the threshold of activation towards colder temperatures and a reduced response to menthol and cold stimuli. These changes are not related to modifications in the number of channels expressed in the plasma membrane, but to changes in their biophysical properties. Electrophysiological experiments confirmed that these functional effects are due to a shift in the voltage dependence of TRPM8 activation towards more positive potentials. Treatment with tunicamycin, a compound that prevents N-glycosylation of proteins, induced a similar reduction in the responses of TRPM8 to cold and menthol, mimicking the behaviour of the unglycosylated mutant N934Q. Furthermore, by using tunicamycin we evaluated the effect of the N- glycosylation on the responses of trigeminal sensory neurons expressing TRPM8. We found that the lack of glycosylation affects the function of native TRPM8 ion channels in a similar way to heterologously expressed ones, causing an important shift of the temperature threshold of cold-sensitive thermoreceptor neurons. Altogether, these results suggest that post-translational modification of TRPM8 is an important cellular event, modulating mammalian cold-thermoreceptor function.

Supported by the following projects: SAF2010-14990 to F.V. and CONSOLIDER-INGENIO CSD2007-00023 to C.B. M.P. was supported by predoctoral fellowships from the Spanish Ministry of Education and Science, and FONDECYT grant #3110128. R.M. was supported by a postdoctoral fellowship of the Spanish Fundación Marcelino Botín, and FONDECYT grant #1100983.

108 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 63 Screening for pharmacological tools to target Ca 2+ activated non-selective cation channels

Philippaert K ., Colsoul B., Voets T., Vennekens R. Laboratory of Ion Channel Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgie

TRP proteins form cation channels that are regulated through strikingly diverse mechanisms. Recently TRPM4 and TRPM5 were identified as monovalent cation selective ion channels, which are activated by an increase of the intracellular Ca2+ concentration. Physiologically, TRPM5 is a key player in the transduction of signals from taste buds in the tongue towards the central nervous system. Furthermore the channel plays a role in the release of insulin from pancreatic ß-cells. TRPM4 on the other hand is important for tuning the activation state of mast cells, and might be a novel drug target for allergic diseases. Moreover, data is accumulating that TRPM4 also plays an important role in the regulation of cardiac contraction strength and the development of arrhythmic diseases. To date no pharmacological tools are available to evaluate their potential as drug targets. In this study we present a novel screening method for identifying compounds, which target these channels. We used a fluorescence based high throughput device to visualize intracellular [Na +] dynamics to screen an extensive library of compounds (> 10.000). Furthermore we have characterized a novel activator of TRPM5. This compound is selective for the TRPM5 channel, and has a direct effect on the channel. We present data that this compound influences specifically the Ca 2+ sensitivity of the channel. Potentially it could play an important role in the development of new taste modulators, and novel treatments for type 2 diabetes.

International Workshop on Transient Receptor Potential (TRP) Channels 109 Posters Nº 64 Pore loop residues and ion permeation through TRPC5

Marcus Semtner, Vera Konieczny, Christina Bütfering, Tim Plant Pharmakologisches Institut, BPC Marburg, FB-Medizin, Philipps-Universität Marburg, 35043 Marburg, Germany.

Like many other TRP channel family members, channel subunits of the TRPC family form nonselective cation channels with a permeability to Ca 2+ that is similar to, or slightly higher than that to Na +. Based on sequence similarity, TRPC subunits can be divided into 4 groups: (1) TRPC1, (2) TRPC2, (3) TRPC3, 6 and 7, and (4) TRPC4 and TRPC5. When expressed as homomultimers, group 3 and group 4 channels display distinctive current-voltage (IV) relationships. Unlike for other major TRP subfamilies, relatively few studies have looked at the pore properties and have tried to identify the pore region of TRPC channels (Owsianik et al., 2006). This is further complicated by the relatively long pore loop between TM5 and TM6 with a lack of similarity to K + channels for which the crystal structure is known. In this study, we looked at the cation permeability of TRPC5 and compared this to TRPC3 and TRPC6. We also investigated the effect of point mutations within the pore loop on the cation permeability of TRPC5. In the putative selectivity filter region of the group 4 channels TRPC4 and TRPC5, between the pore helix and TM6, there are no negatively-charged amino acids except two Glu residues just before TM6. However, group 3 TRP channels have a conserved Glu at a position in the selectivity filter region where TRPC4 and 5 have an Asn residue. To test the effect of a negative charge at this position in TRPC5, we replaced Asn by Asp. This resulted in a significant increase in P Ca /P Na and the modification of the IV relationship to a shape more typical for group 3 TRPCs. Removal of the negative charge in TRPC3 had the opposite effect; a decrease in P Ca /P Na , and a change to a group 4-like IV relationship. To further study the effect of negatively-charged residues in the pore loop of TRPC5 on Ca 2+ permeation, we generated a series of mutants in which the residues were neutralized (Glu →Gln or Asp →Asn). Two mutations in the pore loop preceding the putative pore helix, and another within the pore helix significantly reduced P Ca /P Na . The permeabilities of TRPC5 to the alkali metal cations Li +, K +, Rb + and Cs + relative to that of Na + were close to unity and followed the sequence Li +

Owsianik G, Talavera K, Voets T, Nilius B (2006) Permeation and selectivity of TRP channels. Annu Rev Physiol 68:685-717.

110 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 65 TRPC3: linking soce and calcineurin/nfat-signaling in mast cells

Michael Poteser 2, Bernhard Doleschal 2, Michaela Schernthaner 2, Hannes Schleifer 2, Katrin Tieber 2, Irene Frischauf 3, Christoph Romanin 3, Klaus Groschner 1 1Institute for Biophysics, Medical University Graz, 8010 Graz, Austria 2Department of Pharmacology and Toxicology, Institute of Pharmaceutical Sciences, University of Graz, 8010 Graz, Austria 3Institute of Biophysics, Johannes Kepler University Linz, 4040 Linz, Austria

ORAI1 and STIM1 have been identified as the central molecular constituents of store operated calcium entry (SOCE) in immune cells, mediating the highly calcium selective calcium release-activated calcium current (Icrac). This pathway has been shown to provide the transmembrane calcium signal required for calcineurin/NFAT mediated initiation of gene transcription and subsequent activation of immune cells. However, the molecular machinery that links Icrac to NFAT activation has not been resolved completely so far. Channels of the canonical transient receptor potential channel (TRPC) family have been implicated in several SOCE triggered cellular signals, including calcineurin/NFAT signaling, while there is only little evidence for a direct store operated activation mechanism, especially within in the TRPC3/6/7 subfamily. We investigated the interaction of TRPC3 with several molecular elements of SOCE and NFAT mediated gene activation in RBL-2H3 mast cells using patch-clamp, fura2- Ca 2+ -imaging, epifluorescence- and TIRF-microscopy. Orai1-mediated CRAC currents, activated by passive store depletion, were found significantly reduced by over- expression of TRPC3. This negative impact of TRPC3 on I CRAC was independent of channel function as a TRPC3 pore dead mutant (E630K) inhibited I CRAC to a similar extent as wild type TRPC3. Importantly, despite a reduction in I CRAC , NFAT translocation in TRPC3 overexpressing RBL cells remained unchanged, or even slightly promoted. Store depletion-induced NFAT translocation in RBL cells was unaffected by TRPC3 E630K but substantially reduced by TRPC3 mutants which lacked either FKBP12/calcineurin binding (P704Q) or a PKC phosphorylation essential for binding of FKBPs (S712A), Moreover, inhibition of PKC phosphorylation by (GFX109203X; 3 µM) strongly suppressed NFAT signaling. TRPC3 was found colocalized with ORAI1 and calcineurin in specific membrane microdomains. Store depletion induced a rearrangement of membrane-associated calcineurin and short- hairpin RNA induced silencing of TRPC3 was found to impair SOCE induced translocation of labeled calcineurin in RBL-2H3 cells. Our study identifies TRPC3 as a signaling molecule essential for efficient Ca 2+ transcription coupling in mast cells. We suggest that TRPC3 enables efficient linkage of Orai-mediated Ca 2+ -entry to calcineurin activation by targeting calcineurin into Orai1- containing plasma membrane microdomains.

International Workshop on Transient Receptor Potential (TRP) Channels 111 Posters Nº 66 Activation of transient receptor potential ankyrin 1 induces CGRP release from spinal cord synaptosomes

T. E. Quallo 1, J. L. Sorge 2, S. Bevan 1, L. M. Broad 2, A. J. Mogg 2 1King's College London, Wolfson CARD, London, United Kingdom 2Eli Lilly & Company Limited, Lilly Research Centre, Erl Wood Manor, Windlesham, United Kingdom

TRPA1 is an excitatory ion channel expressed in a subset of nociceptive fibres. It mediates responses to pungent chemicals such as those found in mustard oil and wasabi (), garlic () and (cinnamaldehyde) 1-3. It is also a receptor for products of oxidative stress and some pro-inflammatory mediators 2,4, and has been proposed to have roles in mechanotransduction and cold-sensing. Recent studies have suggested that TRPA1 and other sensory-nerve expressed TRP channels are novel therapeutic targets in nociceptive conditions due to their ability to modulate spinal synaptic transmission 5-7.Neurotransmitters released from primary afferent fibres relay nociceptive information in the spinal cord and play an important role in sensory signalling. Transmitter release can be used to profile functional TRP channels expressed at central terminals. In vivo, microdialysis has commonly been used to measure transmitter release however accurate estimations of drug concentrations reaching receptors cannot be made. Another approach is the measurement of transmitter release from tissue preparations in vitro using a high throughput (96-well) format. In the current study we measured CGRP release from a synaptosomal preparation of lumbar spinal cord in response to various stimuli. CGRP release was measured from a crude synaptosomal sample prepared by dissection of the lumbar dorsal spinal cord from adult male Sprague-Dawley rats (~250- 500g). The spinal cord was homogenised allowing the formation of ‘synaptosomes’. Using an enzyme immunometric assay the amount of CGRP released from the synaptosomes was measured and compared. The effects of various TRPA1 agonists, including the pungent exogenous activators, Allyl isothiocyanate (AITC) and Cinnamaldehyde, on CGRP release were studied. Both compounds evoked CGRP release in a concentration dependent manner (AITC, EC 50 ± SEM = 58.41 ± 13.01µM, n=11: Cinnamaldehyde, 57.08 ± 9.54µM, n=14) as did the electrophilic, endogenous agonists, 4-HNE and 4-ONE (4-HNE, 19.58 ± 2.23µM n=5: 4- ONE, 64.71 ± 4.36µM, n=3). The effect of a broad spectrum TRP antagonist, Ruthenium Red (RR) on these responses was assessed. 10µM RR reduced CGRP release induced by AITC from 123.75 ± 8.34% (n=6) to 84.50 ± 8.51% of Basal Release (n=3) and Cinnamaldehyde-induced release from 125.63 ± 6.72% (n=6) to 89.92 ± 7.58% of Basal Release (n=3). In vitro neurotransmitter release assays can be used to obtain detailed information about native signalling. Moreover, synaptosomal preparations allow an exclusive look at the nerve-endings of primary afferent fibres and the ion channels which function at these central terminals. Information from these assays may be useful for understanding modulation of nociceptive signalling at the level of the spinal cord.

References: 1. Jordt, S. E.; Bautista, D. M.; Chuang, H. H.; McKemy, D. D.; Zygmunt, P. M.; Hogestatt, E. D.; Meng, I. D.; Julius, D. Nature 2004, 427 , 260-5. 2. Bandell, M.; Story, G. M.; Hwang, S. W.; Viswanath, V.; Eid, S. R.; Petrus, M. J.; Earley, T. J.; Patapoutian, A. Neuron 2004, 41 , 849-57.

112 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters 3. Bautista, D. M.; Movahed, P.; Hinman, A.; Axelsson, H. E.; Sterner, O.; Hogestatt, E. D.; Julius, D.; Jordt, S. E.; Zygmunt, P. M. Proc Natl Acad Sci U S A 2005, 102 , 12248-52. 4. Andersson, D. A.; Gentry, C.; Moss, S.; Bevan, S. The Journal of neuroscience : the official journal of the Society for Neuroscience 2008, 28 , 2485-2494. 5. Wrigley, P. J.; Jeong, H. J.; Vaughan, C. W. Br J Pharmacol 2009, 157 , 371-80. 6. Jeffry, J. A.; Yu, S. Q.; Sikand, P.; Parihar, A.; Evans, M. S.; Premkumar, L. S. PLoS One 2009, 4, e7021. 7. Andersson, D. A.; Gentry, C.; Alenmyr, L.; Killander, D.; Lewis, S. E.; Andersson, A.; Bucher, B.; Galzi, J. L.; Sterner, O.; Bevan, S.; Hogestatt, E. D.; Zygmunt, P. M. Nat Commun 2011, 2, 551.

International Workshop on Transient Receptor Potential (TRP) Channels 113 Posters Nº 67 Regulation of TRPC channels by immunophilins in human platelets

Lopez E., Berna-Erro A., Salido G.M., Rosado J.A. and Redondo P.C. Department of Physiology (Phycell group), University of Extremadura, Cáceres, Spain

Physiological platelet agonists, such as thrombin, activate TRPC channels, which are involved in Ca 2+ entry mainly through via two pathways: capacitative Ca 2+ entry, which depends on intracellular store empting upon inositol, 1,4,5-trisphosphate (IP 3) generation, and non-capacitative Ca 2+ influx operated by second messengers like diacylglycerol (DAG), both generated after stimulation of PLC β. During the last decade, many groups have focused their effort in the identification of proteins that regulate TPRC function in order to characterize the nature and sequence of their activation during Ca 2+ entry. Immunophilins have been described to control intracellular Ca 2+ channels and Ca 2+ ATPases in different cell types. Here, we present evidence supporting immunophilins as new TRPC proteins regulators in human platelets. The immunophilins FKBP12 and FKBP52 co-immunoprecipitated with several isoforms of TRPC channels in human platelets. Thapsigargin (TG)-evoked Ca 2+ and Mn 2+ entry was significantly reduced in platelets treated with immunophilin inhibitors, such as FK506 and rapamycin. Similarly, OAG-evoked Ca 2+ entry was reduced in the presence of FK506. Finally, TRPC1 and FKBP52 silencing by using sh/siRNA-based technology in the megakaryoblastic cell line, MEG01, significantly reduced TG-evoked Ca 2+ entry. Summarizing, we suggest that immunophilins represent a key target to design new drugs for controlling TRPC channel activity in patients with disorders associated to TRPC channelopaties.

Acknowledge: This work have been supported by MEC (BFU2010-21043-C02-01), Junta de Extremadura-FEDER (GR10010 & PRIBS10020). Redondo PC was supported by MEC “Ramón y Cajal Program” (RYC-20070-00349) and Lopez E is supported by NHI Carlos III Health Program (FI10/00573). Berna-Erro A was supported by University of Extremadura Posdoc- Research Contract (D-01).

114 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 68 TRPC6 confers pH sensitivity to OAG-mediated aggregation in mouse platelets

Albarran L., Berna-Erro A., Dionisio N., Redondo P.C. , Salido G.M. and Rosado J.A. Phycell Group, Department of Physiology, University of Extremadura, Cáceres, 10003, Extremadura, Spain

Introduction: Calcium (Ca 2+ ) entry into the cell is a crucial step in blood platelet function. Thus, altered inward Ca 2+ currents directly impact on platelet aggregation and therefore, in thrombosis and hemostasis. Two mechanisms of Ca 2+ entry, operated by previous depletion of intracellular Ca 2+ stores (SOCE) and by receptor-agonist interactions (ROCE), have been described as essential regulators of Ca 2+ homeostasis in these cells. Marked reduction in arterial pH as consequence of metabolic acidosis is a common event during cases of prolonged haemorrhage and hypovolemia. Decreased pH significantly impairs platelet aggregation, and studies in the mechanism by which pH affects platelet function points to alterations in SOCE. Ca 2+ channels of the TRPC family of such as TRPC6 have been described in platelets. However, their function in Ca 2+ homeostasis is still unclear in these cells. Therefore, we studied the possible impact of TRPC6 removal in murine platelet function during acidosis. Materials and Methods: Washed blood platelets were isolated in absence of extracellular Ca 2+ from TRPC6-deficient and wild-type mice, and incubated in Tyrode’s buffer in the presence 50 µM Ca 2+ . This last step helps platelets to equilibrate possible loss of intracellular Ca 2+ during the isolation process. Platelet function at physiological (pH=7.13) and acidic (pH=5.5) conditions was evaluated by aggregometry. Statistical significance assessed by Student t test. Results: TRPC6-deficient platelets aggregated normally (80% approx) under physiologic pH conditions upon ROCE stimulation with the dyacylglicerol analog 1- oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of pH 5.5, wild-type platelets aggregated significantly less (25% approx), indicating the presence of pH sensitive ROCE in murine platelets. In contrast, TRPC6-deficient platelets exhibited higher aggregation percentages (55% approx) closer to those shown in physiological pH conditions as compared to wild-type controls, suggesting a protective effect of platelet function in the absence of TRPC6 function. Conclusions: Our results suggest that TRPC6 confers pH sensitivity to mechanisms of ROCE in murine platelets. The presence of pH-insensitive mechanisms of compensation in the absence of TRPC6 function are not discarded, but deserved further investigation.

International Workshop on Transient Receptor Potential (TRP) Channels 115 Posters Nº 69 Regulation of lysosomal exocytosis by a TRP channel in the lysosome

Mohammad A. Samie , Xiang Wang, and Haoxing Xu The Department of Molecular, Cellular, and Developmental Biology, the University of Michigan, 3089 Natural Science Building (Kraus), 830 North University, Ann Arbor, MI 48109, USA

Lysosomes, the cell’s recycling centers, are required for cells to eliminate waste materials and cellular debris. Recent evidence, however, suggest that lysosomes are also involved in other cellular processes such as membrane trafficking and signal transduction. Fusion of lysosomal compartments with the plasma membrane, i.e, lysosomal exocytosis, has been shown to play critical roles in several physiological processes, such as plasma membrane repair, particle uptake, cytokine secretion, and transmitter release. Although it has been known that lysosomal exocytosis can be induced by the rise of intracellular Ca 2+ concentrations, the source of Ca 2+ and the underlying Ca 2+ channel(s) remain elusive. Mucolipin TRP channels (TRPMLs) are members of the TRP ion channel super-family that are localized exclusively on the lysosome membranes. Using a lysosome-targeted Genetically-Encoded Ca 2+ - Indicators and directly patch clamping lysosomal membranes, we found that TRPML1 is the principle Ca 2+ -permeable channel in the lysosome. By measuring Lamp1 surface staining and lysosomal enzyme release, we found that activation of TRPMLs by a potent TRPML-specific membrane-permeable small-molecule agonist is sufficient to induce lysosomal calcium release and lysosomal exocytosis. We are currently investigating the physiological stimuli that can activate TRPMLs to induce lysosomal exocytosis.

116 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 70 In vitro characterisation of TRPV1 in the inflammatory cardiovascular system

Claire Sand 1, Andrew Grant, 2 Manasi Nandi 3 1Cardiovascular Division, 2Wolfson Centre for Age Related Diseases, 3Institute of Pharmaceutical Sciences, King’s College London, UK

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective, highly Ca 2+ - permeable cation channel expressed predominantly in sensory nerve terminals. Recent evidence suggests that TRPV1 may also be expressed in vascular tissues and may play a role in the regulation of vascular function under pathological conditions. Given that TRPV1 is sensitized or directly activated by several components of the inflammatory milieu, and that TRPV1 knockout mice exhibit enhanced hypotension and increased mortality in murine models of sepsis, we hypothesized that TRPV1 may play an important vasoregulatory role in the setting of sepsis. In order to characterize vascular expression of TRPV1, we isolated mouse aortae from TRPV1 wildtype and knockout mice, and cultured bovine aortic endothelial cells (bAECs) and bovine aortic smooth muscle cells (bASMC) with and without pro-inflammatory lipopolysaccharide (LPS) treatment, as an in vitro model of sepsis. Using RT-PCR we demonstrated TRPV1 mRNA expression in mouse aorta. Western immunoblotting revealed weak protein expression in bAECs that was unaffected by LPS treatment, but strong expression in bASMC, where LPS produced an incremental effect on protein levels. We also investigated TRPV1 activity using the ratiometric fluorescent Ca 2+ dye Fura- 2/AM and the selective TRPV1 agonist capsaicin. Despite evidence of vascular TRPV1 protein expression, using this technique we found no evidence of functional TRPV1 activity bAECs, bASMCs, or isolated murine pulmonary ECs and ASMCs either with or without LPS treatment. The functional expression of TRPV1 in other vascular beds, more closely related to blood pressure control, for example, remains to be investigated, and an extensive in vivo characterization of the cardiovascular consequences of TRPV1 deletion or antagonism in the setting of sepsis may shed more light on the role of this channel in inflammatory vasoregulation.

International Workshop on Transient Receptor Potential (TRP) Channels 117 Posters Nº 71 Agonist- and Ca 2+ -dependent desensitization of TRPV1 protein targets the receptor to lysosomes for degradation

Lucía Sanz-Salvador ‡, Amparo Andrés-Borderia ‡, Antonio Ferrer-Montiel §, and Rosa Planells-Cases ‡ From the ‡Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain and the §Instituto de Biología Molecular y Celular, Universidad Miguel Hernández de Elche, 03202 Elche, Spain.

TRPV1 receptor agonists such as the vanilloid capsaicin and the potent analog resiniferatoxin are well known potent analgesics. Depending on the vanilloid, dose, and administration site, nociceptor refractoriness may last from minutes up to months, suggesting the contribution of different cellular mechanisms ranging from channel receptor desensitization to Ca 2+ cytotoxicity of TRPV1-expressing neurons. The molecular mechanisms underlying agonist-induced TRPV1 desensitization and/or tachyphylaxis are still incompletely understood. Here, we report that prolonged exposure of TRPV1 to agonists induces rapid receptor endocytosis and lysosomal degradation in both sensory neurons and recombinant systems. Agonist-induced receptor internalization followed a clathrin- and dynamin-independent endocytic route, triggered by TRPV1 channel activation and Ca 2+ influx through the receptor. This process appears strongly modulated by PKA-dependent phosphorylation. Taken together, these findings indicate that TRPV1 agonists induce long-term receptor down- regulation by modulating the expression level of the channel through a mechanism that promotes receptor endocytosis and degradation and lend support to the notion that cAMP signaling sensitizes nociceptors through several mechanisms.

118 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 72 Are ion channels of the TRP family involved in oligodendrocyte progenitor migration?

Nina K. Schwering 1, Irmgard D. Dietzel 2, Patrick Happel 1 1: Central Unit for Ion Beams and Radionuclides (RUBION) and 2: Department of Molecular Neurobiochemistry Ruhr-University of Bochum, Universitätsstraße 150, D-44780 Bochum, Germany

The development and maintenance of function of many organs depends on the ability of their cells to migrate. Besides the coordinated rearrangements of the cell's cytoskeleton, evidence is accumulating that water and ion fluxes into and out of the cell are essential requirements for cell migration. Although much is known about the occurrence of various ion fluxes in migrating cells, the mediators and the regulation of these ion fluxes still remain unknown. Using scanning ion conductance microscopy (SICM) and time lapse Ca 2+ imaging we found that the frontal end of migrating oligodendrocyte progenitor cells swells prior to the acceleration of the nucleus. Furthermore, a local increase in intracellular Ca 2+ concentration could be observed. We suppose that the local volume increase activates mechano-sensitive cation channels that in turn mediate the Ca 2+ influx. To identify potential mediators we are currently studying the mRNA expression levels of various cation channels during the development of OPCs. Promising candidates are channels that are upregulated in OPCs compared with the immotile adult oligodendrocytes. Therefore, we first investigate TRPC1 channels which are likely to be activated by mechanical stretch. Furthermore, it is known that TRPC1 forms heteromultimeres with, besides others, TRPC3 and TRPC6 in embryonic rat brain. TRPC3, in turn, is known to be upregulated in the brains of rats from embryonic day 18 until postnatal day 20, a stage of development in which myelin is formed from dividing and migrating oligodendrocyte progenitors.

International Workshop on Transient Receptor Potential (TRP) Channels 119 Posters Nº 73 Block by BCTC reveals TRPV1-independent camphor responses in rat sensory neurons

Selescu T. 1, Reid G. 2, Babes A. 1 1 Faculty of Biology, University of Bucharest, Bucharest, Romania 2 Department of Physiology, University College Cork, Cork, Ireland

Studying TRP ion channels in their native environment is challenged by the lack of selectivity of some of their chemical activators. The aim of this study was to asses the contribution of TRPV1 to the responses to camphor and 2-APB in sensory neurons, by using a potent TRPV1 inhibitor against these non-specific activators. Camphor is known as an agonist of TRPV3, a partial agonist of TRPV1 and inhibitor of TRPA1, while 2-aminoethoxydiphenylborane (2-APB) is an activator of TRPV1-3, TRPM6, TRPA1 and inhibitor of TRPC1-7 and TRPM2-5, 7 and 8. Capsazepine was shown to be only a weak and partial inhibitor of the currents elicited by 2-APB or camphor in mouse TRPV1-expressing cells. Some of the TRPV1 antagonists are shared with TRPM8 (e.g. capsazepine, BCTC). Human TRPV1 and human TRPM8 were stably expressed in HEK293 cells. DRG neuron cultures were obtained from Sprague-Dawley adult male rats (150-200 g). Intracellular calcium was imaged with Calcium Green-1 AM and the temperature was set at 25°C. The TRPV1/TRPM8 antagonist BCTC completely and persistently blocked hTRPV1 responses to 2-APB and camphor. Pre-application of 1 M BCTC was found to be sufficient for total inhibition of the response to a subsequent application of either 10 mM camphor, 150 M 2-APB or 0.3 M capsaicin. Tachyphylaxis was observed on successive 10 mM camphor for 20 s at 7 minute intervals. Using BCTC we blocked either the first or the second response. While TRPM8 is not acknowledged as a camphor activated ion channel, in our experiments hTRPM8-expressing HEK293 cells were robustly activated by 5 or 10 mM camphor and tachyphylaxis was not observed. We found BCTC to be less effective and less persistent in blocking camphor induced transients mediated by hTRPM8, compared to its effect on hTRPV1. Although, when pre- and then co-applied with 5 mM camphor, 5 M BCTC blocked the response almost completely (ca. 97%), 1 M BCTC was not able to inhibit the activation of hTRPM8 by 10 mM camphor. Inferring from the experiments on recombinant human TRPV1 and TRPM8, we tested the effect of BCTC on the responses to camphor in rat DRG neuron cultures. Calcium imaging experiments revealed neurons with transients to 10 mM camphor not blocked by 1 M BCTC. A subgroup of these cells was also menthol sensitive (100-500 M). Intracellular calcium increase was not observed under prolonged applications of 1-5 M BCTC alone. In conclusion, experiments on recombinant human TRPV1 and TRPM8 show that in millimolar concentrations camphor is a common activator of the two ion channels, while BCTC is a common antagonist of activation by camphor and is more effective on hTRPV1. The experiments on neurons suggest that in rat DRGs camphor sensitivity is not exclusively mediated by TRPV1 and that TRPM8 is also probably playing a role.

120 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 74 TRPA1-activation on central afferent terminals by 5,6- Epoxyeicosatrienoic acid (5,6-EET) upon nociceptive stimulation causes mechanical hyperagesia

Marco Sisignano 1, Chul-Kyu Park 3, Carlo Angioni 1, Dong Dong Zhang 1, Andrew Grant 4, Ruirui Lu, Ru-Rong Ji 3, Clifford J. Woolf 2, Gerd Geisslinger 1, Klaus Scholich 1 and Christian Brenneis 1,2 1Institute of Clinical Pharmacology, pharmazentrum frankfurt/ZAFES, University Hospital, Goethe-University, D-60590 Frankfurt am Main, Germany. 2F. M. Kirby Neurobiology Center, Department of Neurobiology, Harvard Medical School, Children's Hospital Boston, Boston, MA 3Department of Anesthesiology, Sensory Plasticity Laboratory, Pain Research Center, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. 4Wolfson Centre for Age Related Disease, King's College, London, UK

Epoxyeicosatrienoic acids (EETs) are CYP-epoxygenase (CYP450) derived metabolites of arachidonic acid (AA) which act as endogenous signaling molecules in multiple biological systems. We investigated the specific contribution of 5,6-EET to Transient-Receptor-Potential-(TRP)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. We found that during capsaicin-induced nociception 5,6-EET-levels increased in the DRG and it is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux [10 nM] in cultured DRG-neurons which was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices 5,6-EET dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSC) in lamina II neurons that also respond to mustard oil (AITC), indicating a presynaptic mechanism. 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1 null mice, suggesting that 5,6-EET facilitates spinal cord synaptic transmission pre- synaptically via TRPA1. Finally, intrathecal injection of 5,6-EET caused mechanical hyperalgesia in wild type but not TRPA1 null mice. We conclude that 5,6-EET is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.

International Workshop on Transient Receptor Potential (TRP) Channels 121 Posters Nº 75 Sub-saturating doses of capsaicin on primary cultured sensory neurons reveals TRPV1 sensitization by inflammatory mediators

Jared M. Sprague 1,2,3 , Clifford J. Woolf 1,2 1FM Kirby Center for Neurobiology, Children’s Hospital, Boston, USA 2Department of Neurobiology, Harvard Medical School, Boston, USA 3Biological Sciences of Dental Medicine, Graduate School of Arts and Sciences, Harvard University, Cambridge, USA

Transient receptor potential vanilloid type 1 (TRPV1) is an important receptor for transducing noxious stimuli such as heat, protons, and other compounds, both endogenous (e.g. anandamide) and exogenous (e.g. capsaicin). Because of its reactivity to heat and protons and its specificity for capsaicin, its function is frequently assayed in vitro as a model of inflammatory-based peripheral sensitization. Typically used saturating doses of capsaicin (~1 µM) result in vitro in a short-term desensitization of TRPV1 responses to repeated capsaicin challenges. Many studies looking at TRPV1 “sensitization” utilize exposure to inflammatory mediators (e.g. NGF,

IL-1β, prostaglandin E 2) to actually reduce desensitization, restoring capsaicin-induced responses to the first response. This particular method could more accurately be described as TRPV1 re sensitization, or restoring capsaicin-induced responses to pre- desensitization levels. The aim of this study was to examine TRPV1 sensitization (a reduction in threshold) in sensory neurons distinct from resensitization. We did this primarily using a calcium-based imaging system (Fura-2) on freshly dissociated and cultured sensory neurons from C57Bl/6 mice. Neurons were exposed to a very low dose of capsaicin (30 nM) (over 30 times lower than the saturating concentration usually used), followed by exposure to inflammatory compounds and subsequent re- exposure to capsaicin (30 nM). Our results show that adding inflammatory mediators to DRG neurons in vitro doubles the number responding to capsaicin at 30 nM . This doubling brings the total number of responders to roughly 40% of all neurons, corresponding with response percentages at higher doses of capsaicin (~1 µM). We are attempting to tease apart the mechanistic differences between direct sensitization of TRPV1 and a reduction of desensitization (resensitization) by using this approach that reveals two groups of capsaicin-responsive cells: those that initially respond without inflammatory priming and another group that only responds after inflammatory stimuli.

Funding: NIH NS039518 and NS072040

Conflicts of interest: None

122 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 76 Heating up TRP channel drug discovery

Sonja Stoelzle 1, Alison Obergrussberger 1, Mohamed Kreir 1, Michael George 1, Andrea Brüggemann 1, Niels Fertig 1 1Nanion Technologies GmbH, Gabrielenstr. 9, 80636 München, Germany.

Transient receptor potential (TRP) channels are an important class of receptors found widely distributed throughout the mammalian central and peripheral nervous systems. They have been shown to be activated by many stimuli including temperature, mechano-stimulation, divalent cations and pH. TRP channels are receiving much attention as potential targets for the treatment of, for example, chronic pain, asthma and diabetes isipidus. Patch clamp electrophysiology remains the gold standard for studying ion channels. We have employed a planar patch clamp workstation to study TRPV channels using a variety of stimuli. High quality data could be achieved with a high success rate for obtaining giga-seals (typically 60-80%). TRPM8 was reconstituted in planar lipid bilayers and activated by menthol and PIP2. TRPV1, TRPV3 or TRPV4 stably expressed in CHO or HEK cells were activated by ligands such as capsaicin (TRPV1), 2-APB (TRPV1 & TRPV3) or 4 α-Phorbol 12,13-didecanone (TRPV4) and inhibited by ruthenium red (TRPV1 & TRPV3). Data will be presented showing activation and inhibition of TRPV channels by a variety of agonists and antagonists. TRPV channels are also activated by temperatures >42˚C (TRPV1), >34˚C (TRPV3) or >27˚C (TRPV4). Using a heated pipette, the temperature of the added solution was increased and then rapidly applied to the cell. Very rapid changes in temperature can be achieved at the cell (within ms), from room temperature up to ~65˚C, whilst continuously recording. Whole-cell currents will be presented showing heat activation of TRPV1, TRPV3 and TRPV4 using a range of temperatures. Pharmacology can also be performed using elevated temperature as the activator of TRPV1 and a direct comparison can be made between the ability of a compound to block the ligand response, e.g., by capsaicin, and/or the temperature response of TRPV1 channels. As previously reported in the literature 1, TRPV3 channels display sensitization to repetitive stimulation either using the ligand 2-APB or heat, a phenomenon we also observed. The ability to distinguish between the different modes of action of TRP channels may have important implications for drug design and may give further clues about the physiological and pathophysiological roles of these channels.

1. Chung et al (2004) J. Neurosci. 24 pp. 5177 - 5182

International Workshop on Transient Receptor Potential (TRP) Channels 123 Posters Nº 77 Identification of the C-terminus as a critical molecular determinant of calcium-sensitivity in human TRPA1 channels

Lucie Sura 1, Vlastimil Zíma 2, Lenka Marsakova 1, Anna Hynkova 1, Ivan Barvík 2, Viktorie Vlachova 1* 1Department of Cellular Neurophysiology, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic 2Division of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, 121 16 Prague 2, Czech Republic

The ankyrin transient receptor potential channel TRPA1 is a Ca2+-permeable cation channel whose activation results from a complex synergy between distinct activation sites. One of them is particularly important for determining the sensitivity of TRPA1 to chemical, voltage and cold stimuli. From the cytoplasmic side, TRPA1 is critically regulated by Ca2+ ions and this mechanism represents a self-modulating feedback loop that first augments and then inhibits the initial activation [1,2,3,4]. To date, mechanisms underlying calcium-dependent TRPA1 modulation remain controversial, with some studies suggesting direct binding of calcium to an EF hand-like domain [5,6] and others demonstrating an EF hand- and calmodulin- independent process [4,7,8]. Therefore, other domains through which Ca2+ could activate, potentiate and inactivate TRPA1 have to be considered. In an attempt to identify the putative motif involved in Ca 2+ -dependent modulation, we investigated the contribution of the cluster of acidic residues in the distal C-terminus of TRPA1 in these processes using site-directed mutagenesis and whole-cell electrophysiological recordings. We found that the neutralization of four conserved residues, namely Glu1077 and Asp1080-Asp1082 in human TRPA1, had strong effects on the Ca 2+ - and voltage-dependent potentiation and/or inactivation of cinnamon- induced responses. Using molecular dynamics simulations, we prove the capability of the highly conserved stretch of residues I 1074 ISETEDDDS 1083 in the carboxyl terminus of TRPA1 to bind Ca 2+ . In addition, we found that truncation of the C-terminus by only 20 residues selectively slowed down the Ca 2+ -induced inactivation ~3-fold without affecting other functional parameters, substantiating the role of this domain in Ca 2+ - dependent modulation. Our findings identify the conserved acidic motif in the C-terminus that is actively involved in TRPA1 modulation by Ca 2+ and may represent its long-sought Ca 2+ -sensing domain. These results contribute to understand the processes that shape TRPA1 participation in pain, chemical sensation and cytotoxicity, but could also help to understand the fundamental relationship between the structure and function of this ion channel.

References: 1. Story GM, et al. (2003) Cell 112: 819-829. 2. Jordt SE, et al. (2004) Nature 427: 260-265. 3. Nagata K, et al. (2005) J Neurosci 25: 4052-4061. 4. Wang YY, et al. (2008) J Biol Chem 283: 32691-32703. 5. Doerner JF, et al. (2007) J Biol Chem 282: 13180-13189. 6. Zurborg S, et al. (2007) Nat Neurosci 10: 277-279. 7. Nilius B, et al. (2011) Journal of Physiology 589: 1543-1549. 8. Cordero-Morales JF, et al. (2011) PNAS 108: E1184-1191. 9. Sura L, et al. (2012) Journal of Biological Chemistry, doi:10.1074/jbc.M112.341859

* This work was supported by the Czech Science Foundation [305/09/0081], and by the Ministry of Education, Youth and Sports of the Czech Republic [1M0517, MSM0021620835, SVV-2010-261 304] and GAUK 426311.

124 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 78 Mutations within the first amino acids of the TRP domain differentially affect the function of TRPM8 channel

Francisco J. Taberner , Ainara López, Gregorio Fernández and Antonio Ferrer-Montiel Instituto de Biología Molecular y Celular, Universidad Miguel Hernández. Av. de la Universidad S/N, Elche, Spain.

Sensing potential harmful stimuli is crucial for the survival of the organisms. The detection of these stimuli relies on specialized proteins that localize on the surface of certain sensory cells. The Transient Receptor Potential ion channels are involved in sensing some of those stimuli such as temperature, pH and different chemical compounds. Upon activation, they will evoke ionic currents that would trigger action potentials in nociceptor neurons. An important unsolved question regarding to TRP channels function, is how the presence of the stimuli is translated into the aperture of the channel. This issue comprises two very closed processes, the coupling (signal transduction) and the gating (opening process). Several lines of evidence have pointed to the cytosolic C-terminus of the channels, specifically a conserved domain called the TRP domain, as an important region in the gating process. To further understand the role of this domain in the channel function, we have obtained a set of chimeras with the cold menthol receptor (TRPM8) and the capsaicin receptor (TRPV1), focusing on the small region between the last transmembrane segment (S6) and the TRP Box. Our results indicate that this small region of the TRP domain is essential for the function of TRPM8 and TRPV1. Within this region, we have found a mutation that hampers TRPM8 function and a mutation that turns TRPM8 into constitutively active. Strikingly, the constitutive activity of the channel could become regulated upon the addition of another mutation within the same region. Based on the available TRPM8 structural model we have interpreted the effect of these mutations on the channel function and we have proposed a possible mechanism for the gating process of TRPM8.

International Workshop on Transient Receptor Potential (TRP) Channels 125 Posters Nº 79 Bimodal action of cinnamaldehyde and camphor on mouse TRPA1 channels

Yeranddy A. Alpizar, Maarten Gees, Alicia Sanchez, Aurelia Apetrei, Thomas Voets, Bernd Nilius and Karel Talavera Laboratory of Ion Channel Research, KU Leuven, Herestraat 49, 3000 Leuven, Belgium.

TRPA1 is a calcium-permeable nonselective cation channel that functions as an excitatory ionotropic receptor in nociceptive neurons. TRPA1 is activated by cooling and by a myriad of chemical irritants, and serves as a broadly tuned chemonociceptor triggering pain and local inflammatory responses in visceral and peripheral tissues. One of the most intriguing properties of TRPA1 is the bimodal effect of several of its chemical modulators. Originally described either as antagonists or agonists of mouse TRPA1, several compounds were later shown to induce activation at low (µM) concentrations and inhibition at higher concentrations. Such is the case of menthol and related compounds, citral, nicotine and mustard oil. Here we report the bimodal action of two other well-known modulators of TRPA1, namely cinnamaldehyde and camphor, which are thus far known to be agonist and antagonist, respectively. Whole-cell patch-clamp experiments in TRPA1-expressing CHO cells revealed that, as previously reported, extracellular application of 100 µM CA induced a powerful stimulation of TRPA1 currents. However, subsequent application of 3 mM CA induced a fast and reversible current inhibition. Application of 3 mM CA had more complex effects on basal currents, inducing a rather small current increase, followed by current inhibition and a dramatic overshoot of current amplitude upon washout. These observations are indeed reminiscent of the effects of TRPA1 modulators having bimodal effects on this channel. The bimodal effects of CA could be also documented using fotometric measurements of intracellular Ca 2+ in intact TRPA1-expressing CHO cells and in primary cultures of mouse dorsal root ganglion (DRG) neurons. The agonist action of camphor on TRPA1 was readily observed in patch-clamp experiments performed in CHO cells over-expressing this channel. As previously reported extracellular application of 1 mM camphor induced a decrease of basal currents, but the current amplitude showed a significant overshoot upon washout. On the other hand, application of 100 µM camphor induced a 3-fold increase of the basal current amplitude measured at -75 mV. Intracellular Ca 2+ -imaging experiments in TRPA1-expressing CHO cells yielded that application of camphor induces only marginal increase in the intracellular Ca 2+ concentration, probably due to the low stimulatory potency of this compound at micromolar concentrations and its inhibitory effect at higher concentrations. However, washout of camphor triggered robust Ca 2+ rebound signals, demonstrating therefore its agonist action. Similar results were obtained in cultured mouse DRG neurons. These results highlight once more the complexity of TRPA1 pharmacology and the need for careful characterization of the effects of TRPA1 channel modulators over wide concentration ranges.

126 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 80 A positive feedback loop affecting diffusion and cell-surface expression of TRPM8-containing vesicles

Carlos A. Toro , Luis Veliz and Sebastián Brauchi Instituto de Fisiología, Facultad de Medicina, Universidad Austral de Chile, Valdivia.

Introduction: The spatial and temporal distribution of receptors constitutes an important mechanism for controlling the magnitude of cellular response. Several members of the transient receptor potential ion channel family (TRPs) can regulate its function by modulating their distribution and availability at the plasma membrane (PM) through rapid vesicular translocation and fusion. Regarding the cold receptor TRPM8, we hypothesize that the distribution of TRPM8-containing vesicles is a selfregulated process, where different agonists may stimulate the recruitment of the channel- containing vesicles to the PM by affecting their dynamics and localization. Methods: In this study DRG-derived F-11 and HEK-293 cells were transiently transfected with TRPM8-GFP. Through-the-objective TIRF microscopy was used to perform single particle tracking and to record changes in mobility. Automatized custom- made software for image analysis was used. Channel density quantification was addressed by variance analysis and biochemistry. Results: We first demonstrate constitutive expression of cold receptors in F11 cells. Detailed diffusion analysis based on single particle tracking measurements clearly show that TRPM8 agonists stimulate an increase in receptor density at the PM and affects the channel diffusion properties, allowing TRPM8-containing vesicles remain longer times at the PM. These changes are calcium-dependent. Furthermore, by the use of the selective blocker BCTC we show that this process is dependent on TRPM8 channel activity. In addition, the cell-surface increase is affected by both endocytic and exocytic mechanisms. Conclusion: We suggest that changes in the ratio of exocytic and endocytic processes after stimulation may help increase cell-surface expression of the channels in a positive feedback loop through TRPM8-dependent calcium influx. This amplification power may represent a novel regulatory process for TRPM8 channels.

Funding: FONDECYT 11070190-1110906; C.T. Is a CONICYT fellow.

International Workshop on Transient Receptor Potential (TRP) Channels 127 Posters Nº 81 Adenosine-5`-triphosphate (ATP) and phosphatidylinositol 4,5- bisphosphate (PIP 2) are regulators of transient receptor potential melastatin 3 (TRPM3) channel activity

B.I. Toth , J. Vriens and T. Voets Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven

The TRPM3, a member of the melastatin subfamily of transient receptor potential (TRP) ion channels, is a Ca 2+ permeable non-selective cation channel activated by the neurosteroid pregnenolone sulphate (PS) and by heat. Recent studies reported the expression and functional role of TRPM3 in various tissues including pancreatic β-cells and sensory neurons, but the molecular regulation of the channel remains poorly understood. Intracellular ATP and PIP 2 were shown to modulate several members of TRP family, but we lack any data regarding TRPM3. Therefore, we aimed to investigate the influence of intracellular factors on the activity of TRPM3. We carried out voltage clamp measurements using the cell-attached and inside-out configuration of the patch clamp technique on HEK293T cells overexpressing mouse TRPM3. The channels of the clamped membrane patches were stimulated by 100 M PS applied to the extracellular side of the membrane via the pipette solution. In cell attached configuration, we measured moderate channel activity, which was dramatically increased in inside-out configuration just after the excision of the membrane patch. Current potentiation after excision was followed by rapid current decay. Application of 2 mM ATP to the cytosolic side of the inside-out membrane patch restored the TRPM3 activity. Kinetic analysis of the effect of cytosolic ATP indicated a dual effect on TRPM3: direct channel inhibition, which may be due to direct binding to the channel, and slow restoration of channel activity, which may represent the action of an ATP- dependent enzyme. Our additional finding that PIP 2 also caused a partial recovery of TRPM3 current in inside-out patches, suggests that ATP may act, at least partly, by fuelling the restoration of PIP 2 levels in the plasma membrane. Supporting the above hypothesis, the non-hydrolyzable ATP analog adenosine-5’-[( β,γ)- methyleno]triphosphate (APPCP) applied to the cytosolic side of the membrane patch, in contrast to ATP, was failed to restore the channel activity. In our running experiments we are further exploring the molecular basis of the ATP-dependent TRPM3 regulation.

128 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 82 Hydrogen peroxide mediated TRPA1 receptor activation on monosodium urate crystals-induced nociceptive and edematogenic responses in rats

Gabriela Trevisan a, Carin Hoffmeister a, Mateus Fortes Rossato a, Sara Marchesan Oliveira a, Romina Nassini b, Serena Materazzi b, Camilla Fusi b, Pierangelo Geppetti b, Juliano Ferreira a. aPost-graduated Program in Biological Sciences: Toxicological Biochemistry, Department of Chemistry, Federal University of Santa Maria, Santa Maria, RS, Brazil; bDepartment of Preclinical and clinical Pharmacology and Headache Centre, University of Florence, Florence, Italy.

Gout is a common cause of painful inflammatory arthritis provoked by the accumulation of monosodium urate (MSU) crystals in the joints. However, until now the molecular mechanisms involved in the gout acute nociception have not been well explored. The transient receptor potential ankyrin 1 (TRPA1) is a non selective cation channel highly expressed in a subset of sensory fibers jointly to the vanilloid TRP vanilloid 1 (TRPV1) receptor and it is involved in diverse painful conditions. Knowing that hydrogen peroxide (H 2O2) stimulates TRPA1 and it might be produced by MSU challenge in some cells. The goal of this study was to evaluate the role and the relationship of H 2O2 and TRPA1 activation in the MSU-induced nociception, cold allodynia and edema in rodents. Initially, we showed that MSU (0.25 mg/paw) subcutaneously (s.c.) injected into the right hind paw of rats caused spontaneous nociception response, cold allodynia and edema, which are reduced by the s.c. administration of selective (HC-030031; 300 nmol/paw; 84±5, 100, and 80±7% of inhibition, respectively) and non-selective (camphor; 150 nmol/paw; 88±7, 100 and 86±7%, respectively) TRPA1 antagonists. Besides, the systemic administration of HC- 030031 (300 µmol/kg, oral route) effectively prevented the nociceptive (93±7% of inhibition), cold allodynic (100% of inhibition) and edematogenic (75±7% of inhibition) responses to MSU. The desensitization of capsaicin-sensitive afferent fibers also significantly prevented MSU-induced nociception (89±3% of inhibition), cold allodynia (100% of inhibition), and edema (73±9% of inhibition), and decrease the immunoreactivity for both TRPA1 and TRPV1 observed on sciatic nerve (42±11 and 51±16% of reduction). Moreover, in C57BL/6 mice TRPA1-deficient MSU-elicited nociception (79±4% of inhibition), cold allodynia (100% of inhibition), and edema (95±10% of inhibition) were extensively reduced. On the other hand, the uric acid or MSU crystals were not capable of inducing calcium influx in primary dorsal root ganglia (DRG) neurons culture. Once MSU- induced nociceptive and edematogenic responses seem to involve the activation of TRPA1, we further explore the role of hydrogen peroxide (H2O2), an endogenous TRPA1 activator, on these mechanisms. The H 2O2 (3 µmol/paw) when s.c. injected provoked nociceptive and edematogenic responses similar to that observed for MSU, however in an early manner. Also, the H 2O2 induced-nociceptive, cold allodynic and edematogenic responses were reduced by the TRPA1 antagonists (HC-030031 and camphor; 71±7, 100 and 96±4% of inhibition and 75±5, 100 and 94±6% respectively). The level of H 2O2 was also increased in the hind paw skin after the s.c. injection of MSU either at 5 (15-fold increase), 15 (7-fold increase), or 30 (8-fold increase) minutes after injection, and the administration of the enzyme catalase (300 UI/paw) reduced the MSU-induced nociceptive (100% of inhibition), cold allodynic (100% of inhibition), and edematogenic (95±3% of inhibition) responses. Moreover, the reducing agent dithiothreitol (DTT, 20 nmol/paw) was capable to decrease the MSU and H 2O2-elicited nociceptive (71±12 and 100% of inhibition), cold allodynic (100 and 100% of inhibition), and edematogenic (90±5 and 100% of inhibition) responses. In this study, we suggest that TRPA1 plays a relevant role in spontaneous nociceptive response, cold allodynia and edema elicited by MSU, and this is possibly mediated by the endogenous production of H 2O2.

International Workshop on Transient Receptor Potential (TRP) Channels 129 Posters Nº 83 Behavioral and electrophysiological study of the effects of thermosensitive TRP channel agonists on touch, temperature and pain sensations

Tsagareli M.G ., 1 Iodi Carstens M., 2 Tsiklauri N., 1 Carstens E. 2 1 Dept of Neurophysiology, Ivane Beritashvili Experimental Biomedicine Center, Tbilisi, Georgia 2 Section of Neurobiology, Physiology and Behavior, University of California, Davis, CA, USA

Transient receptor potential channels (TRP) have been extensively investigated over the past few years. Recent findings in the field of pain have established a subset of TRP channels that are activated by temperature (the so-called thermoTRP channels) and are capable of initiating sensory nerve impulses following the detection of thermal, as well as mechanical and chemical stimuli. A family of six thermoTRP channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) exhibits sensitivity to increases or decreases in temperature as well as to chemical substances that elicit similar hot or cold sensations. Such irritants include menthol from mint, cinnamaldehyde, , capsaicin from chili peppers, mustard oil, camphor, from , and others. In this study, we have used behavioral and electrophysiological methods to investigate if cinnamaldehyde (CA), mustard oil (allyl isothiocyanate=AITC) and menthol affect sensitivity to thermal (hot and cold), innocuous cold, and mechanical sensitivity in mail rats. Unilateral intraplantar injection of CA and AITC induced a significant, concentration- dependent reduction in latency for ipsilateral paw withdrawal from a noxious heat stimulus, i.e., heat hyperalgesia. They also significantly reduced mechanical withdrawal thresholds of the injected paw, i.e., mechanical allodynia. Bilateral intraplantar injections of CA resulted in a significant cold hyperalgesia (cold plate test) and a weak enhancement of innocuous cold avoidance (thermal preference test). These results indicate that TRPA1 channel is clearly involved in pain and TRPA1 agonists (CA and AITC) enhance heat pain sensitivity, possibly by indirectly modulating TRPV1 channel, which is co-expressed with TRPA1 in nociceptors. In contrast to CA and AITC, menthol dose-dependently increased the latency for noxious heat-evoked withdrawal, i.e. has an antinociceptive effect. Menthol did not affect mechano-sensation except for a weak allodynic effect at the highest concentration (40%), indicating that it did not exert a local anesthetic effect. Menthol had a biphasic effect on cold avoidance. High concentrations of menthol reduced cold avoidance, (i.e. cold hypoalgesia), while low menthol concentrations significantly increased cold avoidance. The highest menthol concentration resulted in cold hypoalgesia (cold plate test), while lower concentrations had no effect. These findings could be consistent with a role for TRPM8 ion channel in innocuous cold sensation but not pain. In electrophysiological experiments neuronal responses to electrical and graded mechanical and noxious thermal stimulation were tested before and after cutaneous application of AITC or CA. Repetitive application of AITC initially increased the firing rate of spinal wide-dynamic range (WDR) neurons followed by rapid desensitization that persisted when AITC was reapplied 30 min later. The responses to noxious thermal, but not mechanical, stimuli were significantly enhanced irrespective of whether the neuron was directly activated by AITC. These findings indicate that AITC produced central inhibition and peripheral sensitization of heat nociceptors. CA did not directly excite WDR neurons, and significantly enhanced responses to noxious heat while not affecting responses to skin cooling or mechanical stimulation, indicating a peripheral sensitization of heat nociceptors. Some mismatch between our behavioral and

130 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters electrophysiological data concerning CA-induced increase in mechanosensitivity and lack of CA effect on neuronal mechanosensitivity may explain by involve the route of administration of this substance. Overall, our data support a role thermosensitive TRPA1 and TRPM8 channels in pain modulation, and that these thermoTRP channels represent promising targets for the development of a new group of analgesic drugs.

Acknowledgements: This study was supported by grants from the US Civilian Research and Development Foundation (GEB1-2883-TB07), the National Institutes of Health (DE-13685 and DE013685-09S1), and from Georgian National Science Foundation (1/6-27).

International Workshop on Transient Receptor Potential (TRP) Channels 131 Posters Nº 84 Neuronal networks mediating thermotaxis in the marine annelid Platynereis dumerilii

Csaba Verasztó 1 and Gáspár Jékely 1 1Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany

Temperature is an influential factor in the migration of marine plankton, the largest diurnal migration on Earth, yet the underlying neuronal mechanisms of thermotaxis are not fully understood. The thermotactic swimming behaviors of planktonic larvae provide a simple system with which we can explore how marine animals sense and respond to temperature. We aim to elucidate the molecular and neuronal basis of thermotaxis in the larval stages of the marine annelid Platynereis dumerilii , a newly emerged model for evolutionary- and neurobiology. Its simple nervous system makes this animal ideal to investigate ancient brain functions. The TRP (transient receptor potential) superfamily of ion channels play crucial roles in animal thermosensation. We have identified more than 10 TRP channels in Platynereis dumerilii from all major families of TRP channels. Studying TRP channels in Platynereis will also shed light on how TRP channels function in marine environment. We have developed an assay to describe thermotactic response of the freely swimming larvae. We will use in situ hybridization in combination with in vivo calcium imaging to identify thermosensitive TRP channel expressing neurons in the Platynereis larva. We aim to characterize TRP channel thermosensitivity with in vitro whole-cell recording. In addition we will reconstruct the entire circuitry responsible for thermotactic behavior in an ultrastructural map of the larval brain.

132 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 85 Importance of a conserved sequence motif in transmembrane segment S3 for the gating of human TRPM8 and TRPM2

Mathis Winking , Cornelia Kühn, Andreas Lückhoff and Frank Kühn Institute of Physiology, Medical Faculty, RWTH Aachen, D52057 Aachen, Germany

For mammalian TRPM8 channels the amino acid residue G805 within the transmembrane segment S3 is essential for the sensitivity to the synthetic agonist icilin (Chuang et al., 2004). In the same study two further amino acid residues, N799 and D802, were identified which are also critical for the agonistic effect of icilin. However, the importance of these residues for the sensitivity of TRPM8 to menthol or cold was not systematically analyzed. The residues N-799 and D802 belong to a short sequence motif within S3 which is highly conserved in the entire superfamily of voltage- dependent cation channels, among them several TRP channels (Kumanovics et al., 2002). A critical interaction between this sequence motif in S3 and the S4 voltage sensor was already demonstrated for Shaker K + channels (Papazian et al., 1995). Following the hypothesis that one of the conserved residues in S3 might represent a potential interaction partner of the S4 voltage sensor of TRPM8, we performed reciprocal substitutions of N799 and D802 within the S3 sequence motif. The corresponding TRPM8 channel variants N799+D802 (wild-type), D799+D802 (mutant N799D), N799+N802 (mutant D802N) and D799+N802 (mutant N799D+D802N) were heterologeously expressed in HEK-293 cells and functionally characterized by whole- cell patch-clamp analysis as well as by Ca 2+ -imaging. The data demonstrate that the sensitivity of TRPM8 to menthol or cold was quite differently affected by these mutations. Whereas the mutation N799D almost abolished channel function, the variants D802N and N799D+D802N significantly increased the sensitivity to menthol but decreased the sensitivity to cold temperatures. Furthermore, we performed the corresponding analysis with TRPM2, the closest relative of TRPM8 within the TRP- family, which, however, is not voltage gated. TRPM2 contains the same amino acid residues within S2, S3 and S4 identified to be important for the gating of TRPM8. Surprisingly, we found that also for TRPM2 the S3 sequence motif N869+D872 (wild- type) is important for channel gating. From all the mutants only the channel variant D869+D872 formed functional channels displaying currents indistinguishable from wild- type TRPM2. In contrast, both of the variants N869+N872 and D869+N872 were functionally inactive, even if high intracellular concentrations of ADP-ribose and Ca 2+ were used. Thus, the very same mutation (the introduction of a second negatively charged aspartate) is tolerated by TRPM2 but is detrimental to the function of TRPM8. We conclude that the S3 sequence motif plays a specific role during the gating pathway of both TRPM8 and TRPM2, reflecting the different gating mechanisms and gating-related intramolecular interactions of the two channels.

This study was supported by the Deutsche Forschungsgemeinschaft (DFG KU-2271/1-1).

International Workshop on Transient Receptor Potential (TRP) Channels 133 Posters Nº 86 Knocking down of substance-P and α-CGRP on modulating inflammatory sensitization of TRPV1

Christoph Jakob Wolf Farré , Clotilde Ferrandiz-Huertas, Sakthikumar Mathivanan, Isabel Devesa, Antonio Ferrer-Montiel Instituto de Biología Molecular y Celular, Universidad Miguel Hernández Elche, Edificio Torregaita, Av. de la universidad s/n, 03202 Elche (Spain)

Transient receptor potential vanilloid subtype 1 (TRPV1), a polymodal ion channel which is predominantly expressed in dorsal root ganglion (DRG) neurons, senses distinct nociceptive stimuli acting as a major integrator of inflammatory pain. Activation of TRPV1 by several vanilloids, including capsaicin, protons and heat, promotes the entry of calcium ions which results in desensitization of the channel whereas a variety of pro-inflammatory mediators, such as ATP and bradykinin, cause sensitization to noxious stimuli both by enhancing the membrane current carried by TRPV1 and by recruiting new channels to the plasma membrane. Here, we propose that the increase of TRPV1 in the plasma membrane is due to SNARE-dependent exocytosis of LDCVs containing α-CGRP and substance P (SP), and carrying also TRPV1. For this reason, we have investigated the contribution of SP and α-CGRP on modulating TRPV1 sensitization in mice dorsal root ganglion neurons. The involvement of SP and α-CGRP was assessed using Tachykinin 1 -/- (SP -/-) and α-CGRP -/- double knockout mice. Using confocal and electron microscopy, we found that TRPV1 is colocalized with CGRP in LDCVs, and its expression is increased in the plasma membrane in presence of pro- inflammatory mediators. To study the effect of knocking down SP and α-CGRP on TRPV1 sensitization calcium-influxes were measured, showing that there is a lessened inflammatory sensitization of the channel in the absence of both neuropeptides. When SNARE-dependent exocytosis was inhibited by peptide DD04107, inflammatory sensitization was decreased stronger in wild type culture than in the knockout model. These results support the hypothesis that SP and α-CGRP are crucial modulating TRPV1 sensitization via LDCV exocytosis. Therefore, inhibition of regulated exocytosis could be a valuable therapeutic target in painful inflammatory patologies.

134 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 87 Functional characterisation of TRPM8 ionchannel of Mus musculus and Gallus gallus

Sven Zielke , Jonas Petersen, Christian Wetzel Ruhr-University Bochum, Faculty of Biology and Biotechnology, Universitätsstr. 150, D-44780 Bochum, Department of Cell Physiology, Building ND 4

Transient receptor potential (TRP) channels can be activated by a huge variety of endogenous and exogenous chemical compounds and by physical stimuli like touch and temperature. In the present study, we focused on the TRPM8 receptor of Gallus gallus (cTRPM8) and Mus musculus (mTRPM8) and compare the functional characteristics of these two ortholog receptors. The mammalian TRPM8 channels are well characterized. It is known, that the mammalian TRPM8 receptor can be activated by temperatures below 25°C, and by natural as well as synthetic chemical compounds such as menthol, eucalyptol or icilin. Furthermore, it is known that the TRPM8 receptor can be positively modulated by second messengers such as PI(4,5)P2. In addition, several intracellular signal cascades were involved in the receptor activation. Recent studies have shown, that the mice TRPM8 receptor interacts with a Gq protein. We measured temperature and menthol responsiveness of chicken sensory neurons from dissociated dorsal root ganglia and of transfected HEK-293 cells expressing mouse or chicken TRPM8. It is known, that avians and mammals have different basal body temperatures (39°C and 37°C, respectively). Fu rthermore, variations in the amino acid sequence of both species, e.g. in the coiled-coil domain which is necessary for the activation of the receptor, could be responsible for functional differences. We aimed to investigate functional properties of the ortholog receptors and compared the differences in detection of cool temperatures or after menthol stimulion between the two species. Therefore, we used the patch-clamp and Ca2+-imaging technique to characterize the activation temperature and the response behavior after stimulation with menthol. We found that the cTRPM8 has an approximately 3°C higher activation Temperature than the mouse ortholog. Furthermore the cTRPM8 leads to a lower intracellular Ca2+ increase by stimulation with menthol and cooling than the mTRPM8.

International Workshop on Transient Receptor Potential (TRP) Channels 135 Posters Nº 88 Waixenicin A inhibits cell proliferation through magnesium- dependent block of TRPM7 channels

Susanna Zierler 1,3 , Guangmin Yao 2, Zheng Zhang 1, W. Cedric Kuo 2, Peter Pörzgen 2, Reinhold Penner 1, F. David Horgen 2 and Andrea Fleig 1 1Center for Biomedical Research, The Queen’s Medical Center and John A. Burns School of Medicine, University of Hawaii, Honolulu, HI-96813, U.S.A. 2Laboratory of Marine Biological Chemistry, Department of Natural Sciences, Hawaii Pacific University, Kaneohe, HI-96744, U.S.A. 3 Present address: Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig- Maximilians-University Munich, 80336 Munich, Germany

Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. These characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. We screened a chemical library of 1,100 marine organism-derived extracts in a high- throughput assay system and identified the organic extract of the Hawaiian soft coral Sarcothelia edmondsoni as a strong inhibitor of TRPM7-mediated Mn 2+ influx. We used bioassay-linked fractionation to identify the major active component and further characterized the relevant fraction by HPLC coupled mass spectrometry (LC-MS), leading to the isolation and identification of waixenicin A. We were able to identify waixenicin A, a known metabolite from S. edmondsoni , as a strong inhibitor of overexpressed and native TRPM7 channels. Waixenicin A activity was cytosolic and potentiated by intracellular free magnesium (Mg 2+ ) concentration. Our electrophysiological recordings demonstrated, that the blocking potency of waixenicin A was regulated by intracellular free Mg 2+ ions resulting in an almost thousand-fold shift 2+ in the IC 50 to 16 nM, when a phsiologic intracellular Mg concentration of 700 µM was present. Mutating a Mg 2+ -sensitive site at the TRPM7 kinase domain (K1648R) reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg 2+ . Waixenicin A failed to inhibit the closely homologous TRPM6 channel and did not significantly affect TRPM2, TRPM4, and CRAC channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat basophilic leukemia cells. Based on the compound’s ability to inhibit cell proliferation through Mg 2+ -dependent block of TRPM7, waixenicin A or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic Mg 2+ .

136 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation Posters Nº 89 The interactome of the capsaicin receptor TRPV1

Jan Siemens Group Christina Hanack 1, Henning Kuich 1, Jana Rossius 1 and Jan Siemens 1 1 Max Delbrück Center for Molecular Medicine, Berlin, Germany

Transient receptor potential (TRP) ion channels are important components of the somatosensory system, where they are involved in the detection of pain and temperature stimuli (Fig.1A). Particularly, the capsaicin TRPV1 plays a crucial role in the perception, transduction and modulation of thermal and inflammatory stimuli (Fig.1B). By analogy to the founding member of the TRP receptor family in the Drosophila eye (Fig.1C), we hypothesize that TRP ion channels are components of supramolecular membrane-bound protein complexes that enable them to function specifically and efficiently in a context-dependent manner. We are using a genetic biochemical strategy to isolate and identify TRP channel protein complexes from native sensory ganglia of transgenic mice. Furthermore, we will analyze if the composition of protein complexes is altered under chronic pain conditions. Subsequent functional characterization of the identified molecules will provide novel insights into TRP channel function and regulation and may additionally reveal potential targets for anti- inflammatory and pain therapy.

International Workshop on Transient Receptor Potential (TRP) Channels 137 Posters Nº 90 Novel gating properties of TRPM3

Joris Vriens 1 & Thomas Voets 2 1 Laboratory of Obstetrics & Experimental Gynaecology, KU Leuven, Belgium 2 Laboratory of Ion Channel Research, KU Leuven, Belgium

Transient receptor potential melastatin-3 (TRPM3) is a broadly expressed Ca 2+ - permeable non-selective cation channel. Previous work has demonstrated robust activation of TRPM3 by the neuroactive steroid pregnenolone sulphate (PS) and nifedipine. Recently, we provided evidence for TRPM3 as a chemo- and thermosensor in the somatosensory system. TRPM3 is molecularly and functionally expressed in a large subset of small-diameter sensory neurons from dorsal root and trigeminal ganglia, and mediates the aversive and nocifensive behavioral responses to PS. In a pharmacological screen for TRPM3 modulators, we identified (Clt), as a strong potentiator of nocifensive behavioral to PS in vivo . Surprisingly, TRPM3 null mice showed no prolonged nocifensive behavioral after coinjection of PS and Clt in the hindpaw, suggesting for specific interaction with TRPM3. Our results provide insight into the structural determinants for TRPM3 gating, and provide novel tools for studying the role of this channel in vivo .

138 International Symposium on Clinical and Basic Investigation in Glioblastoma II Symposium Seve Ballesteros Foundation

Alphabetical list of authors

Abraham, William M. 68 Busserolles, Jerome 80 Abstiens, K. 62 Bütfering, Christina 110 Agarwal, Nitin 46 Aguirre-Rueda, D. 47 Caceres, Ana Isabel 60 Albarran, L. 115 Camprubí-Robles, M. 63 Alenmyr, Lisa 37 Cao, Erhu 8 Almaraz, L. 48 Cardinali, C. 50 Alpizar, Yeranddy A. 19, 49, 56, 126 Carnini, Chiara 77 Amantini, C. 50, 75, 94, 104 Carstens, E. 130 Ambudkar, Indu S. 15 Caterina, Michael J. 13 Andersson, David A. 51 Cecarini, V. 75 Andrés-Borderia, Amparo 63, 118 Chance, Mark R. 18 Angioni, Carlo 121 Chapuy, Eric 80, 85 Apetrei, Aurelia 126 Charrua, A. 61 Appendino, G. 100 Chen, X.D. 84 Aranda, M. Teresa 67 Cheong, Jessica 59 Aubdool, Aisah A. 52, 59 Ching, Y.M. 81 Chong, Jayhong A. 68 Babes, A. 120 Chubanov, Vladimir 29, 62, 84 Bang, Sangsu 54 Ciardo, M.Grazia 63 Barrière, David A. 85 Clapham, David 17 Barvík, Ivan 124 Coleman, Scott 65 Bauche, Andreas 76, 90 Colsoul, B. 109 Bautista, Diana 14 Compieta, E. 94 Bavencoffe, Alexis 80 Conte, Anna Lucía 64, 86 Belghiti, Majedeline 55 Cordero-Morales, Julio 8 Belmonte, Carlos 19, 56, 69, 96, 98, 108 Cruz, C.D. 61 Belvisi, Maria G. 25, 81 Cruz, F. 61 Benemei, S. 100 Cuesta-Garrote, N. 63 Berna-Erro, A. 114, 115 Curtis, Rory 65, 68 Bevan, Stuart 12, 51, 52, 112 Cvetkov, Teresa L. 18 Bierhaus, Angelika 51 Bindels, René J.M. 28 Daulhac, Laurence 85 Birrell, M.A. 81 Davletova, Ch. 23 Blomgren, Anders 85 de la Roche, Jeanne 66 Bodkin, Jennifer V. 20, 52, 59, 93 de la Torre Martínez, Roberto 67 Bokesch, Paula 65 De Ridder, Dirk 49, 61 Bokhobza, Alexandre 79 De Siena, G. 100 Bonfili, L. 75 Del Camino, Donato 65, 68 Bonvini, S. 81 Denlinger, Bristol 19, 56, 69 Boonen, Brett 49, 56 Desai, Bimal N. 35 Boudes, M. 61 Devesa, Isabel 101, 134 Boukalova, S. 57 Dietz, A.S. 84 Bradshaw, Heather B. 58 Dietzel, Irmgard D. 119 Brain, Susan D. 20, 52, 59, 93 Dionisio, N. 115 Brauchi, Sebastián 127 Discepoli, G. 104 Brenneis, Christian 121 Doñate Macian, P. 70 Broad, L.M. 112 Doran, Ciara 71 Brüggemann, Andrea 123 Dubuis, E. 81 Bumba, Ladislav 102

International Workshop on Transient Receptor Potential (TRP) Channels 139 Alphabetical list of authors

Earley, Scott 16, 72 Greiff, Lennart 37 Eberhardt, Mirjam 66, 73 Groschner, Klaus 95 Eleuteri, A.M. 75 Gudermann, Thomas 29, 62 Enoch, Luis 74 Guimaraes, M.Z.P. 83 Eschalier, Alain 80, 85 Guse, Andreas H. 76, 90 Estévez-Herrera, Judith 55, 106 Etienne, Monique 80, 85 Ha, Michael 88 Everaerts, Wouter 49 Hanack, Christina 137 Happel, Patrick 119 Facchinetti, Fabrizio 77 Hardie, Roger 33 Fajardo, Otto 19, 96 Harneit, Angelika 76, 90 Farcito, S. 97 Harrison, Patrick 71 Farfariello, V. 50, 75, 94, 104 Hartwig, Kerstin 36 Felipo, Vicente 55 Hayward, Neil J. 65, 68 Ferioli, Silvia 29 Heads, Richard 59 Fernandes, Elizabeth S. 20, 52, 59, 93 Heilmaier, Renate 29 Fernández Acuña, Carlos 19 Hellings, Peter 49 Fernández, Gregorio 125 Henriques, M.S.T. 83 Fernandez-Ballester, Gregorio 18, 82 Hoffmann, Daniel 92 Fernández-Carvajal, Asia 67 Hoffmann, Tal 89 Fernández-Peña, C. 23, 74 Hoffmeister, Carin 129 Ferrandiz-Huertas, Clotilde 101, 134 Hofmann, Thomas 29, 62, 84 Ferreira, Juliano 129 Högestätt, Edward D. 17, 37, 85, 103 Ferrer-Montiel, Antonio 18, 55, 63, 67, 82, Horgen, F. David 136 101, 106, 118, 125, 134 Huynh, Kevin W. 18 Fertig, Niels 123 Hwang, Sun Wook 54 Filosa, A. 50 Hynkova, Anna 124 Fiorio Pla, Alesandra 79 Fleig, Andrea 136 Iodi Carstens, M. 130 Fleming, Thomas 51 Fliegert, Ralf 76, 90 Jékely, Gáspár 132 Florez, Danny 64 Jemal, Imane 86 Fortes Rossato, Mateus 129 Jendryke, Thomas 87 Fusi, Camilla 77, 100, 105, 129 Ji, Ru-Rong 121 Jirku, Michaela 102 Gaillard, Stéphane 99 Johanson, Urban 103 Gallego, R. 23 Jordt, Sven-Eric 60, 88 Gangadharan, Vijayan 46 Julius, David 8 Garcia-Elias, Anna 78 García-López, M. Teresa 67 Kaschig, Katrin 71 Gaudet, Rachelle 9 Katz, Ben 34 Gees, Maarten 126 Kichko, Tatjana I. 19, 89 Geisslinger, Gerd 121 King, Ross 52 Genova, Tullio 79 Kirchberger, Tanja 76, 90 Gentry, Clive 51, 52 Kjellbom, Per 103 George, Michael 123 Kodji, Xenia 52 Geppetti, Pierangelo 77, 100, 129 Koivisto, Ari 27 Ghosh, Debapriya 21 Kondratskyi, Artem 80 Gilardino, A. 97 Konieczny, Vera 110 Gil-Bisquert, A. 47 Kozyreva, T.V. 91 Giménez-Garzó, Carla 55 Kreir, Mohamed 103, 123 Gkika, Dimitra 79, 80 Kühn, Cornelia 92, 133 Gomis, Ana 64, 86 Kühn, Frank 92, 133 Gonzales, Albert L. 16, 72 Kuich, Henning, 137 Gonzalez, O. 98 Kuner, Rohini 46 González-Muñiz, Rosario 67 Kuo, W. Cedric 136 González-Usano, Alba 55 Grace, M.S. 81 Leffler, Andreas 66 Gracheva, Elena 8 Leist, Anita 36 Grandl, Jorg 11 Leoni, P. 104 Grant, Andrew 117, 121 Lev, Shaya 34 Gregorio-Teruel, Lucia 82 Lewin, Gary 46

140 International Symposium on Foundation Alphabetical list of authors

Liang, Lihuan 93 Liberati, S. 50, 75, 94, 104 Obergrussberger, Alison 123 Liberta, Frédéric 85 Oberwinkler, Johannes 36 Lichtenegger, Michaela 95 Offidani, M. 104 Lim, Ji Yeon 54 Ontoria-Oviedo, I. 106 Liu, Boyi 88 Oranges, T. 105 López López, José Ramón 19 Owsianik, Grzegorz 21 López, Ainara 125 Lopez, E. 114 Pardo, Carlos 78 López-González, M.J. 96 Paredes-Brunet, P. 47 Lovisolo, D. 97 Park, Chul-Kyu 121 Lu, Ruirui 121 Parra, A. 98 Lückhoff, Andreas 92, 133 Patacchini, Riccardo 77 Luis, Enoch 19 Pedretti, Pamela 77 Lujan, Rafael 101 Penner, Reinhold 136 Perálvarez-Marín, Alex 70, 107 Madrid, R. 108 Pérez-García, María Teresa 19 Maio, V. 105 Pertovaar, Antti 27 Malapert, Pascale 99 Pertusa, M. 96, 108 Mallet, Christophe 85 Petersen, Jonas 135 Manenschjin, Jan-Albert 19, 48, 98 Philippaert, K. 109 Marchesan Oliveira, Sara 129 Pieramici, T. 50 Marics, Irène 99 Piergentili, L. 50 Marsakova, Lenka 124 Planells-Cases, Rosa 55, 63, 106, 118 Marshall, Nichola 93 Plant, Tim 110 Massi, D. 105 Pörzgen, Peter 136 Materazzi, Serena 77, 100, 105, 129 Poteser, Michael 95, 111 Mathivanan, Sakthikumar 101, 134 Potter, Barry V.L. 76, 90 Mauroy, Brigitte 80 Prevarskaya, Natalia 79, 80 Mederos y Schnitzler, M. 62 Meißner, M. 62 Qin, Feng, 82 Melo, P.A. 83 Quaglia, W. 50 Meseguer, Victor M. 19, 56, 96 Quallo, T.E. 112 Minke, Baruch 34 Quek, Samuel 59 Minor, Daniel 10 Mogg, A.J. 112 Raboune, Siham 58 Mohr, Florian 36 Ranaldi, R. 50 Moiseenkova-Bell, Vera Y. 18 Ranzuglia, V. 50 Monsen, Jennifer 68 Redondo, P.C. 114, 115 Montell, Craig 32 Reeh, Peter W. 19, 66, 89 Montoliu, Carmina 55 Reid, Gordon 71, 120 Moparthi, Lavanya 103 Reimúndez, A. 23 Moqrich, Aziz 99 Relova, J.L. 23 Moran, Magdalene M. 65, 68 Ricci-Vitiani, L. 94 Moreau, Christelle 76, 90 Romanin, Christoph 95 Morelli, M.B. 50, 75, 94, 104 Rosado, J.A. 114, 115 Morenilla, C. 98 Rossi, E. 100 Morenilla-Palao, Cruz 48, 74, 108 Rossius, Jana 137 Moretto, Nadia 77 Rubio-Moscardó, Fanny 78 Morris, John B. 88 Ruffinatti, F.A. 97 Mortin, Lawrence I. 68 Mrkonjic, Sanela 78 Salamon, Robin 20 Munaron, Luca 79 Salido, G.M. 114, 115 Munaro-Vieira, D. 83 Samie, Mohammad A. 116 Murphy, Christopher 68 Sanchez, Alicia 126 Sand, Claire 20, 117 Nabissi, M. 50, 75, 94, 104 Sanders, Lindsey 72 Nandi, Manasi 117 Santoni, G. 50, 75, 94, 104 Nassini, Romina 77, 100, 105, 129 Santoni, M. 50, 94, 104 Navia, B. 23 Sanz-Salvador, Lucía 118 Nawroth, Peter P. 51 Schäfer, S. 62 Nilius, Bernd 126 Schleifer, Hannes 95

International Workshop on Transient Receptor Potential (TRP) Channels 141 Alphabetical list of authors

Schöbel, Anja 76 Valverde, Miguel Angel 24, 78 Scholich, Klaus 121 van Gerven, Laura 49 Schwering, Nina K. 119 Vanden Berghe, Pieter 21 Segal, Andrei 21 Vázquez-Ibar, Jose-Luis 70 Selescu, T. 120 Veliz, Luis 127 Semtner, Marcus 110 Vennekens, R. 109 Señarís, R. 23 Verasztó, Csaba 132 Serini, Guido 79 Vermeulen, François 49 Shuba, Yaroslav 80 Viana, Félix 19, 23, 48, 69, 74, 96, 98, 108 Siemens, Jan 137 Vicente, Rubén 78 Simmons, David 29 Vlachova, Viktorie 57, 124 Sisignano, Marco 121 Voets, Thomas 19, 21, 49, 56, 109, 126, Skryma, Roman 80 128, 138 Sorge, J.L. 112 Vong, Chi-Teng 59 Soriano, Sergio 86 Vriens, Joris 21, 128, 138 Sorice, M. 75 Sprague, Jared M. 122 Wang, Liwen 18 Stanslowsky, Nancy 66 Wang, Rui 46 Stockner, Thomas 95 Wang, Xiang 116 Stoelzle, Sonja 123 Wegner, Florian 66 Stuart, Jordyn M. 58 Wetzel, Christian H. 87, 135 Sullivan, Michelle N. 72 Willis, Daniel N. 88 Sura, Lucie 124 Winking, Mathis 92, 133 Survery, Sabeen 103 Wisnowsky, Annika 29 Sytik, Ludmila 29 Wolf Farré, Christoph Jakob 101, 134 Woolf, Clifford J. 121, 122 Taberner, Francisco J. 125 Tajada, Sendoa 19 Xu, Haoxing 30, 116 Talavera, Arturo 19 Xu, X.Z. Shawn 31 Talavera, Karel 19, 49, 56, 126 Teisinger, Jan 57, 102 Yang, Tae-Jin 54 Thomas, Mark 76 Yang, Ying 72 Toro, Carlos A. 127 Yao, Guangmin 136 Toth, B.I. 128 Yoo, Sungjae 54 Trevisan dos Santos, G. 100 Trevisan, Gabriela 129 Zamburlin, P. 97 Tsagareli, M.G. 130 Zhang, Dong Dong 121 Tsiklauri, N. 130 Zhang, Xuming 22 Zhang, Zheng 136 Uller, Lena 37 Zholos, Alexander 80 Ulzhofer, Bettina 46 Zielke, Sven 135 Zierler, Susanna 29, 136 Valente, Pierluigi 82 Zíma, Vlastimil 124 Valero, M. 96 Zygmunt, Peter M. 17, 37, 85, 103 Vallés, S.L. 47

142 International Symposium on Foundation

Alphabetical list of participants

Agarwal , Nitin Aubdool , Aisah Pharmacology Institute Cardiovascular division University of Heidelberg Centre for Integrative Biomedicine Heidelberg, BW, Germany London, UK [email protected] [email protected]

Aguiar Alpizar , Yeranddy Bang , Sangsu Molecular and Cellular Medicine School of Medicine KU Leuven Korea University Leuven, Vlaams Brabant, Belgium Ansan-Shi, Gyeonggi-Do, Republic of Korea [email protected] [email protected]

Akin , Elizabeth Bautista , Diana Biomedical Sciences Department of Molecular & Cell Biology Colorado State University University of California Fort Collins, CO, USA Berkeley, CA, USA [email protected] [email protected]

Alenmyr , Lisa Belghiti , Majedeline Department of Clinical Chemistry and Facultad de Farmacia Pharmacology Universidad de Valencia Lund University Valencia, Spain Lund, Skåne, Sweden [email protected] [email protected] Belmonte , Carlos Almaraz , Laura Instituto de Neurociencias Physiology Universidad Miguel Hernández-CSIC Universidad Miguel Hernández San Juan de Alicante, Alicante, Spain San Juan de Alicante, Alicante, Spain [email protected] [email protected] Belvisi , Maria G. Amantini , Consuelo Pharmacology and Toxicology Section Scuola di Scienze del Farmaco National Heart & Lung Institute University of Camerino Faculty of Medicine Camerino, Macerata, Italy London, UK [email protected] [email protected]

Ambudkar , Indu Bevan , Stuart Molecular Physiology and therapeutics School of Biomedical Sciences Branch King's College London NIDCR, NIH London, UK Bethesda, Maryland, USA [email protected] [email protected] Bindels , René J.M. Andersson , David Radboud University Nijmegen Medical Wolfson CARD Centre King's College London Nijmegen, The Netherlands London, UK [email protected]; [email protected] [email protected]

International Workshop on Transient Receptor Potential (TRP) Channels 143 Alphabetical list of participants

Biró , Tamás Chubanov , Vladimir Department of Physiology Walther-Straub-Institute of Pharmacology University of Debrecen and Toxicology Debrecen, Hungary Ludwig-Maximilians University of Munich [email protected] Munich, Germany [email protected] Boonen , Brett KU Leuven Ciardo , Maria Grazia Leuven, Vlaams Brabant, Belgium Instituto de Biologia Molecular y Celular [email protected] Universidad Miguel Hernández Elche, Alicante, Spain Boukalova , Stepana [email protected] Cellular neurophysiology Institute of Physiology AS CR Clapham , David Prague, Czech Republic Children's Hospital Boston [a Harvard [email protected] Medical School affiliate] Boston, MA, USA Bradshaw , Heather [email protected] Psychological and Brain Sciences Indiana University Conte , Anna Lucia Nicoletta Bloomington, IN, USA Instituto de Neurociencias [email protected] Universidad Miguel Hernández-CSIC San Juan de Alicante, Alicante, Spain Brain , Susan [email protected] Pharmacology King's College London Curtis , Rory London, , UK Pain [email protected] Cubist Pharmaceuticals Lexington, MA, USA Brauchi , Sebastian [email protected] Physiology Universidad Austral de Chile de Cáceres Bustos , Anabel Valdivia, Chile Pharmacology [email protected] Yale University New Haven, CT, USA Caires , Rebeca [email protected] Instituto de Neurociencias Universidad Miguel Hernández-CSIC de la Peña , Elvira San Juan de Alicante, Alicante, Spain Universidad Miguel Hernández-CSIC [email protected] San Juan de Alicante, Alicante, Spain [email protected] Caprini , Marco Human and General Physiology de la Roche , Jeanne Univeristy of Bologna Anaesthesiology Bologna, Italy Medical School Hannover [email protected] Hannover, Lower Saxony, Germany [email protected] Caterina , Michael J. Dept. of Biological Chemistry and Dept. of de la Torre Martinez , Roberto Neuroscience Instituto de Biologia Molecular y Celular Center for Sensory Biology. Johns Hopkins Universidad Miguel Hernández School of Medicine Elche, Alicante, Spain Baltimore, MD, USA [email protected] [email protected] Del Camino , Donato Charrua , Ana Biology Department of Experimental Biology Hydra Biosciences Faculty of Medicine of University of Porto Cambridge, MA, USA Porto, Portugal [email protected] [email protected]

144 International Symposium on Foundation Alphabetical list of participants

Denlinger , Bristol Fernandes , Maria Instituto de Neurociencias Institute of Pharmaceutical Science Universidad Miguel Hernández-CSIC King's College London San Juan de Alicante, Alicante, Spain London, UK [email protected] [email protected]

Desai , Bimal Fernández Ballester , Gregorio Pharmacology Instituto de Biologia Molecular y Celular University of Virginia Universidad Miguel Hernández Charlottesville, Virginia, USA Elche, Alicante, Spain [email protected] [email protected]

Devesa Giner , Isabel Fernández-Carvajal , Asia Instituto de Neurociencias Instituto de Biologia Molecular y Celular Universidad Miguel Hernández-CSIC Universidad Miguel Hernández Elche, Alicante, Spain Elche, Alicante, Spain [email protected] [email protected]

Doñate Macian , Pablo Fernández-Peña , Carlos Bioquímica y Biología Molecular Instituto de Neurociencias Universidad Autónoma de Barcelona Universidad Miguel Hernández-CSIC Valencia, Spain San Juan, Alicante, Spain [email protected] [email protected]

Doran , Ciara Ferrándiz Huertas , Clotilde Physiology Instituto de Biologia Molecular y Celular University College Cork Universidad Miguel Hernández Cork, Ireland Elche, Alicante, Spain [email protected] [email protected]

Dupont , Lisa Ferrer-Montiel , Antonio Ghent University Hospital Instituto de Biologia Molecular y Celular Ghent, Oost-Vlaanderen, Belgium Universidad Miguel Hernández [email protected] Elche, Alicante, Spain [email protected] Earley , Scott Biomedical Science Fliegert , Ralf Colorado State University Center of Experimental Medicine, Institute of Fort Collins, CO, USA Biochemistry and Signal Transduction [email protected] University Medical Center Hamburg- Eppendorf Eberhardt , Mirjam Hamburg, Germany Department of Anesthesiology and Intensive [email protected] Care Medical School Hannover Florez Paz , Danny Mauricio Hannover, Lower Saxony, Germany Instituto de Neurociencias [email protected]; Universidad Miguel Hernández-CSIC [email protected] San Juan, Alicante, Spain [email protected] Enoch , Luis Baltazar Instituto de Neurociencias Fox , Philip Universidad Miguel Hernández-CSIC Biomedical Sciences San Juan de Alicante, Alicante, Spain Colorado State Univeristy [email protected] Fort Collins, Colorado, USA [email protected] Farfariello , Valerio Scuola di Scienze del Farmaco Frank , Robert University of Camerino GI-P-D Medicinal Chemistry Camerino, Macerata, Italy Grünenthal GmbH [email protected] Aachen, NRW, Germany [email protected]

International Workshop on Transient Receptor Potential (TRP) Channels 145 Alphabetical list of participants

Fusi , Camilla Gregorio Teruel , Lucía Department of Preclinical and Clinical Instituto de Biologia Molecular y Celular Pharmacology Universidad Miguel Hernández University of Florence Elche, Alicante, Spain Florence, Italy [email protected] [email protected] Gudermann , Thomas Garcia-Elias Heras , Anna Walther-Straub-Institut für Pharmakologie Universitat Pompeu Fabra und Toxikologie Barcelona, Barcelona, Spain Ludwig-Maximilians-Universitaet Muenchen [email protected] Muenchen, Germany [email protected] Gaudet , Rachelle Dept. of Molecular and Cellular Biology Guimaraes , Marilia Harvard University Pharmacology Cambridge, MA, USA Universidade Federal do Rio de Janeiro [email protected] Rio de Janeiro, RJ, Brazil [email protected] Genova , Tullio Department of Life Sciences and Systems Gupta , Rupali Biology Cell Signalling Università degli Studi di Torino Sokendai Turin, Italy Okazaki, Aichi, Japan [email protected] [email protected]

Ghosh , Debapriya Habermann , Christopher Department of Cellular and Molecular Grünenthal GmbH Medicine Aachen, NRW, Germany KU Leuven [email protected] Leuven, Flemish Brabant, Belgium [email protected] Hanack , Christina Max-Delbrück Center for Molecular Medicine Gkika , Dimitra Berlin, Berlin, Germany Cell Physiology [email protected] University of Lille Villeneuve D'Ascq, Nord, France Hardie , Roger [email protected] Dept. Physiology Development & Neuroscience Gomis , Ana Cambridge University Universidad Miguel Hernández-CSIC Cambridge, UK San Juan de Alicante, Alicante, Spain [email protected] [email protected] Hofmann , Thomas Gonzales , Albert Pharmakologisches Institut Biomedical Science Philipps-Universität Marburg Colorado State University Marburg, Hessen, Germany Fort Collins, CO, USA [email protected] [email protected] Högestätt , Edward Grace , Megan Lund University NHLI Lund, Skåne, Sweden Imperial College [email protected] London, UK [email protected] Hwang , Sun Wook School of Medicine Grandl , Jorg Korea University Department of Neurobiology Ansan, Gyeinggi-Do, Korea Duke University Medical Center, Room [email protected] 2017/MSRB 2 Durham, NC, USA [email protected]

146 International Symposium on Foundation Alphabetical list of participants

Hynkova , Anna Kühn , Frank Cellular Neurophysiology Institute of Physiology Institute of Physiology AS CR University of Aachen Prague, Czech Republic Aachen, NRW, Germany [email protected] [email protected]

Jemal , Imane Liberati , Sonia Instituto de Neurociencia Scuola di Scienze del Farmaco Universidad Miguel Hernández-CSIC University of Camerino Sant Joan d´Alacant, Alicante, Spain Camerino, Macerata, Italy [email protected] [email protected]

Jendryke , Thomas Lichtenegger , Michaela Cell Physiology Institute of Pharmaceutical Sciences - Ruhr-Universität Bochum Pharmacology and Toxicology Bochum, NRW, Germany Karl Franzens University Graz [email protected] Graz, Styria, Austria [email protected] Jirku , Michaela Institute of physiology López Córdoba , Ainara Academy of sciences CR Instituto de Biologia Molecular y Celular Prague, Czech Republic Universidad Miguel Hernández [email protected] Elche, Alicante, Spain [email protected] Johanson , Urban Lund University López González , María José Lund, Skåne, Sweden Instituto de Neurociencias [email protected] Universidad Miguel Hernández-CSIC San Juan de Alicante, Alicante, Spain Jordt , Sven-Eric [email protected] Department of Pharmacology Yale University School of Medicine Lovisolo , Davide New Haven, CT, USA Dept. of Life Sciences and Systems Biology [email protected] University of Torino Torino, Italy Julius , David [email protected] Department of Physiology University of California, San Francisco- Maes , Tania Minor Lab Dept of Respiratory Medicine (Prof. Dr. G. San Francisco, CA, USA Joos) [email protected] Ghent University Hospital Gent, East-Flanders, Belgium Khairatkar Joshi , Neelima [email protected] Biological & Chemical Research Glenmark Pharmaceuticals Limited Malapert , Pascale Navi Mumbai, Maharashtra, India IBDML [email protected] Marseille, , France [email protected] Kichko , Tatjana I. Physiology and Pathophysiology Manenschijn , Jan-Albert University Erlangen-Nuremberg Instituto de Neurosciencias Erlangen, Bavaria, Germany Universidad Miguel Hernández-CSIC [email protected] Alicante, Spain [email protected] Kirchberger , Tanja Center of Experimental Medicine, Marics , Irene Department of Biochemistry and Signal IBDML Transduction Marseille, France University Medical Center Hamburg- [email protected] Eppendorf Hamburg, Germany [email protected]

International Workshop on Transient Receptor Potential (TRP) Channels 147 Alphabetical list of participants

Materazzi , Serena Nabissi , Massimo Dept. of Preclinical and Clinical Scuola di Scienze del Farmaco Pharmacology University of Camerino Univ. of Florence Camerino, Macerata, Italy Florence, Italy [email protected] [email protected] Nassini , Romina Mathis , Winking Preclinical and clinical Pharmacology Institute of Physiology University of Florence Medical Faculty, RWTH-Aachen, University Florence, Italy Aachen, NRW, Germany [email protected] [email protected] Oberwinkler , Johannes Mathivanan , Sakthikumar Dept. of Physiology Instituto de Biologia Molecular y Celular Universität Marburg Universidad Miguel Hernández Marburg, Hessen, Germay Elche, Alicante, Spain [email protected] [email protected] Ontoria Oviedo , Imelda Meseguer Vigueras , Victor Manuel Centro de Investigación Príncipe Felipe Instituto de Neurociencias Valencia, Spain Universidad Miguel Hernández-CSIC [email protected] San Juan de Alicante, Alicante, Spain [email protected] Ordás , Purificación Instituto de Neurociencias Minke , Baruch Universidad Miguel Hernández-CSIC Medical Neurobiology San Juan de Alicante, Alicante, Spain Hebrew University [email protected] Jerusalem, Israel [email protected] Pardo Pastor, Carlos Universitat Pompeu Fabra Minor , Daniel Barcelona, Spain Cardiovascular Research Institute [email protected] Box 3122 University of California, San Francisco- Peralvarez-Marin , Alex Minor Lab Center for Biophysical Studies San Francisco, CA, USA Universidad Autónoma de Barcelona [email protected] Cerdanyola del Vallés, Barcelona, Spain [email protected] Monteiro , César Bruno Departamento de Biologia Experimental Pertovaara , Antti Guimarães, Braga, Portugal Physiology [email protected]; Institute of Biomedicine [email protected] Helsinki, Finland [email protected]; Montell , Craig [email protected] Dept. of Biological Chemistry The Johns Hopkins School of Medicine Pertusa , María Baltimore, MD, USA Biology [email protected] Universidad Santiago de Chile Santiago, Región Metropolitana, Chile Moparthi , Lavanya [email protected] Lund University Lund, Skåne, Sweden Philippaert , Koenraad [email protected] Mol and Cell Medicine - Laboratory of Ion Channel Research Moqrich , Aziz KU Leuven Institut of Developmental Biology Marseille. Leuven, Vl. Braband, Belgie Neurobiology Department [email protected] CNRS Marseille, Bouche Du Rhone, France [email protected]

148 International Symposium on Foundation Alphabetical list of participants

Planells-Cases , Rosa Sand , Claire Leibniz-Institut für Molekulare Cardiovascular Pharmakologie (FMP) and Max-Delbrück- King's College London Centrum für Molekulare Medizin (MDC) London, Greater London, UK Berlin, Germany [email protected] [email protected] Santoni , Giorgio Plant , Tim Scuola di Scienze del Farmaco Pharmakologisches Institut University of Camerino Philipps-Universität Marburg, FB-Medizin Camerino, Macerata, Italy Marburg, Hessen, Germany [email protected] [email protected] Sanz Salvador , Lucía Poteser , Michael Facultad Medicina Dept. of Biophysics Universidad de Valencia Medical University of Graz Valencia, Spain Graz, Austria [email protected] [email protected] Schmidt , Manuela Priel , Avi Somatosensory Signaling Pharmacology MPI for Experimental Medicine Hebrew University Goettingen, Lower Saxony, Germany Jerusalem, Israel [email protected] [email protected] Schwering , Nina Quallo , Talisia Rubion Wolfson Centre for Age-Related Diseases Ruhr-Universität Bochum London, UK Waltrop, NRW, Germany [email protected] [email protected]

Redondo Liberal , Pedro Cosme Selescu , Tudor Fisiologia Department of Anatomy, Physiology and Universidad de Extremadura Biophysics Cáceres, Spain University of Bucharest, Faculty of Biology [email protected] Bucharest, Bucharest, Romania [email protected] Reimúndez Dubra , Alfonso Physiology Señarís Rodríguez , Rosa CIMUS Departamento de Fisiología (CIMUS) Santiago, A Coruña, Spain Universidad de Santiago de Compostela [email protected] Santiago de Compostela, A Coruña, Spain [email protected] Remory , Isabel Anesthesiology Silke , Hobbie University hospital Brussel Respiratory research Brussel, Belgium Boehringer-Ingelheim Pharma GmbH Co KG [email protected] Biberach, BW, Germany [email protected] Sadofsky , Laura Cardiovascular and Respiratory Science Simonsen , Charlotte University of Hull Lund University Hull, East Yorkshire, UK Lund, Skåne, Sweden [email protected] [email protected]

Samie , Mohammad Sisignano , Marco Molecular Cellular and Developmental Department of Clinical Pharmacology Biology Pharmazentrum Frankfurt/ZAFES, University University of Michigan Hospital Frankfurt Ann Arbor, Michigan, USA Frankfurt, Hessen, Germany [email protected] [email protected]

International Workshop on Transient Receptor Potential (TRP) Channels 149 Alphabetical list of participants

Sprague , Jared Trevisan dos Santos , Gabriela Biological Sciences of Dental Medicine Pharmacology Harvard University Florence, Toscana, Italy Watertown, MA, USA [email protected] [email protected] Tsagareli , Merab G. Sreekrishna , Koti Nuerophysiology Biotechnology Ivane Beritashvili Experimental Biomedicine Procter and Gamble Company Center Mason, OH, USA Tbilisi, Kartli, Georgia [email protected] [email protected]

Stoelzle , Sonja Valente , Pierluigi Nanion Technologies Department of Neuroscience and Brain Munich, Bavaria, Germany Technologies, Italian Institute of Technology [email protected] Italian Institute of Technology Genova, Italy Taberner Sanchis , Francisco José [email protected] Instituto de Biologia Molecular y Celular Universidad Miguel Hernández Valverde , Miguel Ángel Elche, Alicante, Spain Dept. Ciències Experimentals i de la Salut [email protected] Universitat Pompeu Fabra Barcelona, Spain Talavera , Karel [email protected] KU Leuven Leuven, Vlaams Brabant, Belgium Van Gerven , Laura [email protected] Otorhinolaryngology, Head & Neck Surgery Catholic University Leuven Tamkun , Michael Leuven, Belgium Biomedical Sciences [email protected] Colorado State University Fort Collins, CO, USA Veraszto , Csaba [email protected] Max Planck Institute for Developmental Biology Teisinger , Jan Tübingen, Baden-Württemberg, Germany Institute of physiology [email protected] Academy of sciences CR Prague, Czech Republic Viana , Félix [email protected] Instituto de Neurociencias Universidad Miguel Hernández-CSIC Tierens , Simon Sant Joan d'Alacant, Alicante, Spain Medicine [email protected] Vrije Universiteit Brussel Londerzeel, Vlaams-Brabant, Belgium Vicente García , Rubén [email protected] Universitat Pompeu Fabra Barcelona, Spain Toro , Carlos Alejandro [email protected] Instituto de Fisiologia Universidad Austral de Chile Vlachova , Viktorie Valdivia, Region del Bio-Bio, Chile Cellular Neurophysiology [email protected] Institute of Physiology AS CR Prague, Czech Republic Toth , Balazs Istvan [email protected] Department of Cellular and Molecular Medicine Voets , Thomas KU Leuven Laboratory of Ion Channel Research Leuven, Flemish Brabant, Belgium KU Leuven, Gasthuisberg O&N1 [email protected] Leuven, Belgium [email protected]

150 International Symposium on Foundation Alphabetical list of participants

Vriens , Joris Development & Regeneration KU Leuven Leuven, Vlaams Brabant, Belgium [email protected]

Wolf , Christoph Instituto de Biologia Molecular y Celular Universidad Miguel Hernández Elche, Alicante, Spain [email protected]

Xu , Haoxing Department of MCDB University of Michigan Ann Arbor, MI, USA [email protected]

Xu , X. Z. Shawn Life Sciences Institute, Room 6115A. University of Michigan Ann Arbor, MI, USA [email protected]

Yoo , Sungjae Graduate school of medicine NeurosAnsan-Shi, Gyeonggi-Do, Korea [email protected]

Zhang , Xuming Pharmacology University of Cambridge Cambridge, Cambridgeshire, UK [email protected]

Zielke , Sven Department of Cellphysiology Ruhr-Universität Bochum Bochum, Nordrhein Westfalen, Germany [email protected]

Zierler , Suzanna Walther-Straub-Institute of Pharmacology and Toxicology Ludwig-Maximilians University of Munich Munich, Bavaria, Germany [email protected]

Zygmunt , Peter Michael Lund University Lund, Skåne, Sweden [email protected]

International Workshop on Transient Receptor Potential (TRP) Channels 151

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With the organization, collaboration and sponsorship of:

1982 - 2012 3

© FUNDACIÓN CAC • 09.2012