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The transient receptor potential vanilloid 1 (TRPV1) ion channel is mainly found in primary nociceptive afferents whose activity has been linked to pathophysiological conditions including pain, itch and inflammation. Consequently, it is important to identify naturally occurring antagonists of this channel. Here we show that a naturally occurring monounsaturated fatty acid, oleic acid, inhibits TRPV1 activity, and also pain and itch responses in mice by interacting with the vanilloid (capsaicin)-binding pocket and promoting the stabilization of a closed state conformation. Moreover, we report an itch-inducing molecule, cyclic phosphatidic acid, that activates TRPV1 and whose pruritic activity, as well as that of histamine, occurs through the activation of this ion channel. These findings provide insights into the molecular basis of oleic acid inhibition of TRPV1 and also into a way of reducing the pathophysiological effects resulting from its activation.

Ion channels display conformational changes in response to binding of their agonists and antagonists. The study of the relationships between the structure and the function of these proteins has witnessed considerable advances in the last two decades using a combination of techniques, which include electrophysiology, optical approaches (i.e. patch clamp fluorometry, incorporation of non-canonic amino acids, etc.), molecular biology (mutations in different regions of ion channels to determine their role in function) and those that have permitted the resolution of their structures in detail (X-ray crystallography and cryo-electron microscopy). The possibility of making correlations among structural components and functional traits in ion channels has allowed for more refined conclusions on how these proteins work at the molecular level. With the cloning and description of the family of Transient Receptor Potential (TRP) channels, our understanding of several sensory-related processes has also greatly moved forward. The response of these proteins to several agonists, their regulation by signaling pathways as well as by protein-protein and lipid-protein interactions and, in some cases, their biophysical characteristics have been studied thoroughly and, recently, with the resolution of their structures, the field has experienced a new boom. This review article focuses on the conformational changes in the pores, concentrating on some members of the TRP family of ion channels (TRPV and TRPA subfamilies) that result in changes in their single-channel conductances, a phenomenon that may lead to fine-tuning the electrical response to a given agonist in a cell.

The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Each pore is independently gated at hyperpolarized voltages by a conserved pore glutamate. Presumably, exiting chloride ions push glutamate outwardly while external protonation stabilizes it. To understand the mechanism of mouse CLC-2 opening we used homology modelling-guided structure–function analysis. Structural modelling suggests that glutamate E213 interacts with tyrosine Y561 to close a pore. Accordingly, Y561A and E213D mutants are activated at less hyperpolarized voltages, re-opened at depolarized voltages, and fast and common gating components are reduced. The double mutant cycle analysis showed that E213 and Y561 are energetically coupled to alter CLC-2 gating. In agreement, the anomalous mole fraction behaviour of the voltage dependence, measured by the voltage to induce half-open probability, was strongly altered in these mutants. Finally, cytosolic acidification or high extracellular chloride concentration, conditions that have little or no effect on WT CLC-2, induced reopening of Y561 mutants at positive voltages presumably by the inward opening of E213. We concluded that the CLC-2 gate is formed by Y561-E213 and that outward permeant anions open the gate by electrostatic and steric interactions.

Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure–function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate–gating relationship in TMEM16A remain less explored. Here, we study the gating–permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN− mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN−. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

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of noxious and non-noxious anesthetics inhibit agonist-evoked activation of mammalian but not drosophila TRPA1. Analysis of chimeric drosophila and mouse TRPA1 channels reveal that the 5th transmembrane domain (S5) is critical for anesthetic sensitivity. We identify two amino acids at the N-terminal S5 domain required for the inhibitory effects of GAs and introduction of these residues into dTRPA1 confers GA-induced block in these channels. Further, we show that activation by noxious GAs requires several amino acids in the S5, S6 and the first pore helix previously implicated in binding of the high-affinity antagonist, A-967079. Notably, isoflurane relieves A-967079mediated inhibition consistent with a competitive interaction and in silico modeling predicts that pungent GAs occupy the A-967079 binding pocket. Thus inhibition and activation by GAs are governed by distinct amino acids in the pore domain region. These data reveal the mechanism by which TRPA1 discriminates between pungent and non-pungent GAs, and provide structural requirements for designing antagonists to counter the noxious properties of GAs. channels are polymodal receptors whose open probability is under command of several stimuli such as temperature, pH and ligands. Several studies have involved TRPV1 channels in pain sensation related to inflammatory responses and tissue damage, leading the pharmaceutical industry to the search of novel compounds able to modulate their function in order to develop novel pain relief therapies. Capsazepine is the best known TRPV1 antagonist and the residues accounting for their binding to TRPV1 channels have been identified by mutagenesis and specie-specific effects. Nevertheless, the molecular mechanism underlying capsazepine-mediated channel inhibition has not been described at molecular level to date. We performed molecular dynamics simulations of the TRPV1 channel in the open state in the absence and the presence of Capsazepine and Capsaicin in order to identify the interactions accounting of TRPV1 channel. Even when Capsazepine shares in part their binding site with the well-known TRPV1 channel agonist Capsaicin, their ligand orientation resulted to be different. We also observed a dynamic hydrogen bond network at the neighbors of the vanilloid binding pocket that differs with those observed in both the absence of ligand and in the presence of capsaicin. Our results suggest that the subtle differences at the interaction network at the capsaicin binding pocket leads to completely opposite effects at the channel activation gate. 1400-Pos Board B377Extracellular Sodium is Required for Temperature-Dependent Gating in TRPV1 Channels Andres Jara-Oseguera, Chanhyung Bae, Kenton J. Swartz. Molecular Physiol/Biophys, NINDS, NIH, Bethesda, MD, USA. The TRPV1 channel in sensory neurons integrates pain-producing stimuli, including noxious temperature, acidosis, pungent vanilloid compounds and pro-inflammatory lipids. Measurements of TRPV1 channel activity at extreme positive and negative voltages in the presence of othe...