Pharmacology of Drosophila Melanogaster TRPA1 Isoforms

Tuesday, February 14, 2017 of SmTRPA-expressing mammalian cells to capsaicin or AITC elicits an influx of calcium, as measured by Fura-2 ratiometric imaging. These results suggest that some TRPV1-mediated sensory functions may be fulfilled by schistosome TRP channels from other subfamilies, and that at least one schistosome TRP channel has atypical pharmacological sensitivities that could be selectively targeted for development of new antischistosomal therapeutics. Other schistosome TRP channels might also be exploitable for targeting. 2005-Pos Board B325 Pharmacology of Drosophila Melanogaster TRPA1 Isoforms Brett Boonen. KU Leuven, Leuven, Belgium. The TRPA1 cation channel functions as a broadly-tuned chemonociceptor in many species. Recent studies identified several splice variants of Drosophila TRPA1 (from dTRPA1(A) to (D)). These were reported to display distinct chemical and thermal sensitivities and distinct expression patterns. Some differences in the chemical sensitivities between insect and mammalian orthologues have also been reported. Given the usefulness of Drosophila as animal model for studying TRPA1 chemosensation in vivo, we re-evaluated the effects of three mammalian TRPA1 modulators (nicotine, citronellal and menthol) on dTRPA1 splice variants in HEK293T cells. Using intracellular Ca2þ fluorimetry and whole-cell patch-clamp, we confirmed that the electrophilic agonist allyl isothiocyanate activates all splice variants, whereas nicotine activates all variants except dTRPA1(B). Citronellal robustly activated all splice variants. The activation by nicotine and citronellal could be inhibited by the TRPA1 antagonist HC030031. On the other hand, menthol did not activate any dTRPA1 isoform. Our results demonstrate that the mammalian heterologous expression system HEK293T can be used for functional studies on insect TRPA1 channels, and that the chemosensory properties of these channels may strongly differ from those of the mammalian homologues. Our findings serve as starting point for further structure-function studies for the determination of the structural bases of chemical sensitivity in TRPA1 channels. 2006-Pos Board B326 Novel Role of PUFAs as Essential Cofactors for TRPV4 Function Rebeca C. Caires, Valeria Vasquez, Julio F. Cordero-Morales. Physiology, University of Tennessee, Memphis, TN, USA. Blood pressure regulation relies on the ability of membrane proteins to transduce variations in physical and chemical stimuli into electrical signals. The transient receptor potential vanilloid 4 (TRPV4) is a cation channel that has been implicated in endothelium-dependent vasorelaxation and modulation of systemic blood pressure. TRPV4 is widely expressed in the vasculature, being found in smooth muscles, vascular endothelial cells, and perivascular sensory neurons. Activation of TRPV4 downstream of cell swelling via polyunsaturated fatty acids-derived mediators promotes arterial vasodilation. The precise mechanism by which polyunsaturated fatty acids and eicosanoids regulate TRPV4 function remains elusive. Here, we use C. elegans to precisely alter fatty acid content in vivo; worms allow for easy and accurate manipulation of lipid content, have the ability to incorporate lipids back via diet supplementation, and a collection of lipid-deficient mutants. We show that chemical activation of a transgenic worm that expresses rat (r) TRPV4 in the sensory amphid neurons (ASH) by GSK1016790A (TRPV4 selective agonist), but not wild type, elicits withdrawal responses; therefore, providing a chemical strategy to study the activation mechanism of TRPV4 using C. elegans. Using this assay, we determine that TRPV4 is not functional in the background of lipid-deficient mutant’s worms (fat-3, lacking long polyunsaturated fatty acids; PUFAs). These results suggest that PUFAs are obligatory cofactors of TRPV4 function. Using confocal microscopy, we established that channel distribution and morphology of ASH neurons are indistinguishable between TRPV4::GFP and TRPV4::GFP; fat-3 worms. Moreover, diet supplementation experiments suggest that EET rescues 20 % less the withdrawal responses of TRPV4; fat3 mutants when compared to arachidonic acid (AA), implicating AA as a key fatty acid for TRPV4 function. By knocking down lysophospholipid acyltransferase enzyme (MBO-6), we found that membranes phospholipids lacking AA negatively regulate TRPV4 function. Our results support a model whereby membrane composition and lipid metabolism modulates TRPV4 function. 2007-Pos Board B327 The Vglut3Icre;Rosa26Ai14 Mouse Model as a Tool for Studying TRPM8D Sensory Neurons Theanne N. Griffith1,2, Javier E. Marquina-Solis2,3, Adrian C. Thompson2,4, Blair A. Jenkins1,2, Ellen A. Lumpkin1,2. 1 Physiology, Columbia University, New York, NY, USA, 2Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA, USA, 3David Rockefeller Graduate Program, The Rockefeller University, New York, NY, USA, 4School of Medicine, University of Tasmania, Hobart, Australia.

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Despite their importance to survival, cold-sensing TRPM8þ neurons represent an understudied sensory neuron population, comprising only 8-10% of all trigeminal (TG) and dorsal root ganglion (DRG) neurons. However, available transgenic mouse lines that label TRPM8þ sensory neurons have important caveats that hinder their use in functional studies. Thus, a mouse model that eases identification of these neurons would facilitate investigation of their unique physiological properties. To this end, we have found a novel use for the Vglut3iCre;Rosa26Ai14 mouse line in rapidly identifying menthol-sensitive sensory neurons in electrophysiological studies. In this mouse line, Vglut3 lineage neurons express cytoplasmic tdTomato and are easily identifiable without immunolabeling. Menthol is a potent activator of TRPM8 channels and thus a good proxy for identifying cold-sending neurons in vitro. Calcium microfluorimetry experiments on cultured Vglut3iCre;Rosa26Ai14 DRG neurons from adult mice (<24 h in culture) revealed 100% of neurons responding to menthol (100 mM) were small-diameter and of the Vglut3 lineage (tdTomatoþ.) These neurons did not respond to capsaicin (1 mM), a TRPV1 channel agonist, or chloroquine (1 mM), which activates TRPA1 signaling, consistent with the TRPM8þ sensory neuron phenotype. Furthermore, in current-clamp recordings, menthol evoked a train of action potentials in 100% of labeled neurons with a capacitance of < 15 pF (n = 5). Action potentials were tetrodotoxin-sensitive, confirming these neurons were not nociceptors. Thus, by targeting small (<15 pF), tdTomatoþ DRG neurons in our electrophysiological recordings, we were able to increase the probability of identifying and recording from TRPM8þ neurons by 10-fold. In conclusion, the Vglut3iCre;Rosa26Ai14 is a useful model for rapidly identifying TRPM8þ sensory neurons and could facilitate electrophysiological studies of coldresponding sensory neurons. 2008-Pos Board B328 Analog-To-Digital Conversion of Differential Metabotropic Inputs to Lateral Septal Neurons by the Combined Action of TRPC4 and GIRK Michael X. Zhu, Jin-bin Tian. Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX, USA. In a neural network, neurons frequently receive multiple coincidental transmitter inputs representing varying activities of different brain regions. These inputs not only vary in the signal pathways they activate but also in the strengths for each of the pathways. However, it remains unclear how neurons decipher co-incident inputs of varying strengths and encode discernible output messages. The vast majority of neurotransmitters signal through G proteincoupled receptors, which activate heterotrimeric G proteins to alter neuronal function via a limited set of effectors, including G protein-activated and/or receptor-operated channels that are capable of modulating neuronal excitability. For receptors that couple to Gq/11 and Gi/o, whereas the activation of Gq/11-phospholipase C pathway, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, suppresses Gi/o-mediated activation of G protein-activated inwardly rectifying Kþ (GIRK) channels, the phospholipase C signaling is required for Gi/o stimulation of the nonselective cation channel, Transient Receptor Potential Canonical 4 (TRPC4). Thus, coincident activation of Gq/11 and Gi/o pathways differentially affects GIRK and TRPC4 channel activities, shaping excitability in a manner that is very sensitive on the relative strengths of Gq/11 and Gi/o signaling. Using whole-cell slice recordings and agonist or electrical stimulation of lateral septal neurons, we demonstrate that varying stimulation intensities at Gq/11 and Gi/o-coupled receptors give rise to distinctive membrane potential waveforms that include burst firing, plateau depolarization and hyperpolarization of varying durations resulting from combined actions of TRPC4 and GIRK channels. These differential responses alter neuronal firing in distinguishable patterns. We propose that the conversion of concurrent Gq/11 and Gi/o signaling of differential strengths into discernible electrical responses by joint actions of TRPC4 and GIRK channels is vital for lateral septum to serve as the information processing center of cerebrum for higher-order brain functions. 2009-Pos Board B329 Crosstalk of Pain and Pleasure: TRPV1 is a Novel Oxytocin Receptor Yelena Nersesyan1, Lusine Demirkhanyan1, Tyler Dawson1, Swapna Asuthkar1, Deny Cabezas-Bratesco2, Sebastian Brauchi2, Eleonora Zakharian1. 1 Cancer Biology & Pharmacology, University of Illinois, Peoria, IL, USA, 2 Department of Medicine, Universidad Austral de Chile, Valdivia, Chile. The Transient Receptor Potential Vanilloid 1 (TRPV1) channel is a receptor for a wide range of noxious stimuli. In the peripheral nervous system, TRPV1 can be sensitized by heat, capsaicin, and acidic pH. However, the physiological regulation of TRPV1 by endogenous agonists is not well understood. In the present work, we discovered a new endogenous agonist of the TRPV1 channel,