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 David Julius. 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. Paracetamol 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 capsaicin receptor (TRPV1) and the wasabi 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 cannabinoid 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 methylglyoxal (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 capsazepine 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-tetrahydrocannabiorcol, 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 calcium imaging, 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 menthol and icilin. 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 bradykinin 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 mustard oil, garlic and cannabis [16-18] and by environmental irritants (eg. acrolein) [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 resiniferatoxin 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 cannabinoids 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 (cinnamaldehyde) and immunohistochemistry to assess substance P-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+ pregnenolone sulfate leads to an increased Ca influx in – and subsequently to an enhanced glucose-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 ruthenium red.
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