Gating and anion selectivity are reciprocally regulated in TMEM16A (ANO1).

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.

Electro-steric opening of the CLC-2 chloride channel gate.

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.

Epithelia-Sensory Neuron Cross Talk Underlies Cholestatic Itch Induced by Lysophosphatidylcholine.

BACKGROUND & AIMS: Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of antipruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus. METHODS: Pruritogenicity of lysophosphatidylcholine (LPC), LPA's precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC's pruritogenicity involving keratinocyte TRPV4 was studied using genetic and pharmacologic approaches, cultured keratinocytes, ion channel physiology, and structural computational modeling. Activation of pruriceptor sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in vitro and ex vivo Ca2+ imaging assays. Sera from patients with primary biliary cholangitis were used for measuring the levels of LPC and miR-146a. RESULTS: LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. Three-dimensional structural modeling, site-directed mutagenesis, and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4 activation by LPC induced extracellular release of miR-146a, which activated TRPV1+ sensory neurons to cause itch. LPC and miR-146a levels were both elevated in sera of patients with primary biliary cholangitis with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates. CONCLUSIONS: We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron cross talk, whereby it directly activates skin keratinocyte TRPV4, which rapidly releases miR-146a to activate skin-innervating TRPV1+ pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons, and central neural pathways supporting pruriception.

Inhibition of TRPV1 channels by a naturally occurring omega-9 fatty acid reduces pain and itch.

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.