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Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker.

ABSTRACT: BACKGROUND AND PURPOSE:Calcium-activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene-9-carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both activation and inhibition. The mechanisms underlying these effects are not fully defined. EXPERIMENTAL APPROACH:Patch-clamp electrophysiology in conjunction with concentration jump experiments was employed to define the mode of interaction of A9C with TMEM16A channels. KEY RESULTS:In the presence of high intracellular Ca(2+) , A9C inhibited TMEM16A currents in a voltage-dependent manner by entering the channel from the outside. A9C activation, revealed in the presence of submaximal intracellular Ca(2+) concentrations, was also voltage-dependent. The electric distance of A9C inhibiting and activating binding site was ~0.6 in each case. Inhibition occurred according to an open-channel block mechanism. Activation was due to a dramatic leftward shift in the steady-state activation curve and slowed deactivation kinetics. Extracellular A9C competed with extracellular Cl(-) , suggesting that A9C binds deep in the channel's pore to exert both inhibiting and activating effects. CONCLUSIONS AND IMPLICATIONS:A9C is an open TMEM16A channel blocker and gating modifier. These effects require A9C to bind to a region within the pore that is accessible from the extracellular side of the membrane. These data will aid the future drug design of compounds that selectively activate or inhibit TMEM16A channels.

SUBMITTER: Ta CM

PROVIDER: S-EPMC4728427 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker.

Ta Chau M CM Adomaviciene Aiste A Rorsman Nils J G NJ Garnett Hannah H Tammaro Paolo P

British journal of pharmacology 20160118 3

<h4>Background and purpose</h4>Calcium-activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene-9-carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both act ...[more]

PMID: 26562072

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Project description:Pressure-induced arterial depolarization and constriction (the myogenic response) is a smooth muscle cell (myocyte)-specific mechanism that controls regional organ blood flow and systemic blood pressure. Several different nonselective cation channels contribute to pressure-induced depolarization, but signaling mechanisms involved are unclear. Similarly uncertain is the contribution of anion channels to the myogenic response and physiological functions and mechanisms of regulation of recently discovered transmembrane 16A (TMEM16A), also termed Anoctamin 1, chloride (Cl(-)) channels in arterial myocytes.To investigate the hypothesis that myocyte TMEM16A channels control membrane potential and contractility and contribute to the myogenic response in cerebral arteries.Cell swelling induced by hyposmotic bath solution stimulated Cl(-) currents in arterial myocytes that were blocked by TMEM16A channel inhibitory antibodies, RNAi-mediated selective TMEM16A channel knockdown, removal of extracellular calcium (Ca(2+)), replacement of intracellular EGTA with BAPTA, a fast Ca(2+) chelator, and Gd(3+) and SKF-96365, nonselective cation channel blockers. In contrast, nimodipine, a voltage-dependent Ca(2+) channel inhibitor, or thapsigargin, which depletes intracellular Ca(2+) stores, did not alter swelling-activated TMEM16A currents. Pressure-induced (-40 mm Hg) membrane stretch activated ion channels in arterial myocyte cell-attached patches that were inhibited by TMEM16A antibodies and were of similar amplitude to recombinant TMEM16A channels. TMEM16A knockdown reduced intravascular pressure-induced depolarization and vasoconstriction but did not alter depolarization-induced (60 mmol/L K(+)) vasoconstriction.Membrane stretch activates arterial myocyte TMEM16A channels, leading to membrane depolarization and vasoconstriction. Data also provide a mechanism by which a local Ca(2+) signal generated by nonselective cation channels stimulates TMEM16A channels to induce myogenic constriction.

Dataset Information

Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker.

Publications

Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker.

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