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

Abstract

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.

Predicting Mutational Effects on Ca2+-Activated Chloride Conduction of TMEM16A Based on a Simulation Study

“…Our results also found that the free energy reduces from T 1 to the open state I 2 (Figure B), which suggests that I 2 is a stable state. When the Cl – ion reaches T 2 , the anion is surrounded by a local negatively electrostatic environment that is mainly induced by pore-lining E624 (located in the loop connecting α5 and α6 helixes) and E633 (located in the α6 helix) in the extracellular vestibule (Figure S4), despite the extracellular vestibule mainly providing a positively electrostatic environment that makes favorable contributions to chloride conduction. , The changes in electrostatic environments for chloride conduction from I 2 to T 2 account for the small barrier of I 2 –T 2 in Figure B.…”

“…Our findings on the electrostatic potential distribution also support this point. Besides, we found that the Cl – ion in the T 1 state is in a strongly negatively charged environment formed by Ca 2+ binding sites ,, (Figure A). The electrostatic repulsion of the Cl – ion and the negatively charged environment hinder the movement of anions.…”

Missense mutations in the calcium-activated chloride channel TMEM16A promote tumor growth by activating oncogenic signaling in Human Cancer

“…Complete activation of TMEM16A occurs when the concentration of intracellular calcium ([Ca 2+ ]i) surpasses 1.5 µM (6)(7)(8)(9)(10)(11). Channel´s activity is modulated by intra-and extracellular protons (12)(13)(14)(15) and the permeating anion, which seems to couple the gating and permeation processes (16)(17)(18)(19).…”