Structure of a Double-Knot Tarantula Toxin Bound to the TRPV1 Channel at the Protein-Lipid Interface

Monday, February 29, 2016 Thus, we explored the mechanism of inhibition of TRPV1 by this molecule. We found that it acts from both the intra and extracellular sides by promoting the stabilization of a closed state of the channel by directly binding to the channel. The physiological response to this lipid is to inhibit the painful responses elicited by the injection of LPA into the paws of mice. These findings provide new insights into the molecular mechanisms involved in the inhibition of an important receptor of noxious stimuli. This work was supported by grants from ‘‘Programa de Apoyo a Proyectos de Investigacio´n e Innovacio´n Tecnologica (PAPIIT) IN200314 and Consejo Nacional de Ciencia y Tecnologia CB-2014-01-238399 (to T. R.), Programa de Apoyo a Proyectos de Investigacio´n e Innovacio´n Tecnologica (PAPIIT) IA202815 (to S.L.M.L). 1404-Pos Board B381 Light-Controlled PI3K Activation Mimics TRPV1 Potentiation by NGF Anastasiia Stratiievska, Sharona E. Gordon. Physiology and Biophysics, University of Washington, Seattle, WA, USA. Membrane receptor signaling is extensively studied, yet unclear. For example, activation of TrkA by nerve growth factor (NGF) triggers many downstream signaling pathways, including: Phosphoinositide 3-kinase (PI3K), PLC and MAPK. One target of TrkA signaling is the pain-transducing ion channel TRPV1, an essential player in inflammatory hyperalgesia. Although regulation of TRPV1 by NGF has been studied for over a decade, the relative contributions of PI3K, PLC, and MAPK are still controversial. Innovative approaches have recently been developed to bypass cell-surface receptors and activate specific signaling pathways using light-dependent dimerization. One new system (Levskaya et al, Nature, 2009) involves dimerization of phytochrome B (PhyB) and phytochrome interaction factor (PIF) by 650 nm light and their dissociation by 750 nm. By expressing plasma membrane anchored PhyB and PIF fused to a lipid-active enzyme, 650 nm light recruits the PIF-enzyme to the plasma membrane, ‘‘activating’’ the enzyme by colocalizing it with its substrate. This interaction is fully reversible by 750 nm light over tens of seconds. We applied this light-controlled dimerization system to dissect the signaling cascade leading to TRPV1 potentiation by NGF. Our goal was to bypass TrkA, activating only the PI3K branch, to determine if activation of PI3K with light mimics potentiation of capsaicin-induced calcium responses to the TRPV1 agonist capsaicin. The PhyB/PIF system was successful in reversibly activating PI3K as measured by levels of its product, PI(3,4,5)P3. Importantly, potentiation of capsaicin responses by light and NGF were similar. Our interpretation is that PI3K activation is sufficient to mimic potentiation of TRPV1 by NGF, at least on the acute time scale of our experiments. Although inflammatory hyperalgesia is a complex process likely involving numerous components, control of PI3K with light allowed us to identify a major player definitively. 1405-Pos Board B382 Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya Alata Ningning Wei1, Haining Lv2, Yang Wu3, Shilong Yang4. 1 neurobiology, Peking University, beijing, China, 2State Key Laboratory of Natural and Biomimetic Drugs, Peking University, beijing, China, 3 Department of Molecular and Cellular Pharmacology, Peking University, beijing, China, 4Key Laboratory of Animal Models and Human Disease Mechanisms, Chinese Academy of Sciences, Kunming Institute of Zoology, kunming, China. Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pains such as headache, rheumatic pain, and toothache. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor TRPV1 channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from active extract of M. alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1 expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. We docked the muralatin L molecule into the TRPV1 structure using Maestro Suite software. The docking results reveal that muralatin L is sequestered in a pocket that is formed by the residues Tyr511 from S3, Met547 and Thr550 from S4, and Glu570 in S4-S5 linker of TRPV1. Site-

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directed mutagenesis further confirms that indicating that the residue Y511 that forms the hydrogen bond with muralatin L is critical for muralatin L binding. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice, but not in TRPV1 knockout mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy. 1406-Pos Board B383 Structure of a Double-Knot Tarantula Toxin Bound to the TRPV1 Channel at the Protein-Lipid Interface Chanhyung Bae1, Claudio Anselmi1, Jeet Kalia1, Andres Jara-Oseguera1, Charles D. Schwieters1, Dmitriy Krepkiy1, Chul Won Lee2, Jae Il Kim3, Jose´ D. Faraldo-Go´mez1, Kenton J. Swartz1. 1 NIH, Bethesda, MD, USA, 2Chonnam National University, Gwangju, Korea, Republic of, 3GIST, Gwangju, Korea, Republic of. Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channel proteins. Although representative structures of toxin-channel complexes have been solved for toxins that bind from solution, none are available for toxins that bind within the lipid membrane. Here we solve the structure of the bivalent double-knot toxin (DkTx) from tarantula venom, develop improved atomic models of the TRPV1 channel with and without DkTx bound, and investigate the interactions of the toxin with the channel and membrane. Our results demonstrate that DkTx binds to the periphery of the external pore of TRPV1 in a counterclockwise configuration, using a limited toxinchannel interface, while inserting hydrophobic residues into the surrounding membrane to stabilize the complex. In addition, we find that DkTx maximizes the utility of bivalency by optimizing one knot for membrane partitioning and another for channel activation. 1407-Pos Board B384 Cell Unroofing Enhances TRPV1 Mobility in the Plasma Membrane Eric N. Senning, Sharona E. Gordon. Department of Physiology and Biophysics, University of Washington, Seattle, WA, USA. Mobility of an integral membrane protein within a cell membrane is determined both by the properties of the lipid bilayer and the cytoskeletal structures that attach to both lipid and protein elements therein. Our previous research on TRPV1 ion channels expressed in HEK293T/17 cells demonstrated that the channel moves laterally in the plasma membrane, exhibiting a broad distribution of mobilities, with a mean within one cell of 0.029 mm2/s. We hypothesized that interactions with the cytoskeleton limit the mobility of TRPV1, and skew the distribution of mobilities toward slower values. To test this hypothesis, we unroofed HEK293T/17 cells transiently transfected with TRPV1-tdTomato to retain the plasma membrane adhered to the glass coverslip and disrupt the cytoskeletal network. Upon unroofing, we observed a shift in the mobility of TRPV1-tdTomato to a mean of 0.16 mm2/s, more than 5-fold greater than that of TRPV1 mobility in intact cells. We estimated the height of the plasma membrane sheet using atomic force microscopy, yielding a value of 5.2 5 2.1 nm above the coverslip with no evidence of buckling or membrane undulations. We have previously shown that Ca2þ influx through TRPV1 regulates its mobility in intact dorsal root ganglion neurons and cultured mammalian cells. Together with the increased mobility of TRPV1 in unroofed cells, our data raise the question of whether regulation of mobility by Ca2þ occurs via a cytoskeleton-mediated mechanism. 1408-Pos Board B385 TRPV1 Expressed in HEK293t/17 Cells is not Regulated by Plasma Membrane Cholesterol Content Sharona E. Gordon, Marcus D. Collins, Moshe T. Gordon. University of Washington, Seattle, WA, USA. The EC50 of TRPV1 is highly variable depending on the cell type in which it is expressed. For example, the EC50 of TRPV1 is HEK293T/17 cells is ~1 mM whereas the EC50 of TRPV1 in isolated DRG neurons is ~ 100 nM. Numerous explanations have been proposed for this phenomenon, with one possible explanation being the varying lipid contents of the plasma membrane regulating the EC50. Lipids are known to regulate ion channels, for example PIP2 regulates TRPV1. The cholesterol content of the plasma membrane is a candidate for regulating TRPV1, as it is known to vary among types of cells. We measured TRPV1 function with patch-clamp electrophysiology to determine the effect of removing cholesterol from the PM on channel function. Insideout excise patches remain stable for tens of minutes, allowing for cholesterol removal and replacement by flowing a Methylated-b-Cyclodextrin, a ring of sugar molecules that bind cholesterol, across the patch. In intact HEK293T/ 17 cells, we estimated the cholesterol content to be about 30%. Thus, the