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.