Extracellular protons enable activation of the calcium‐dependent chloride channel TMEM16A

Abstract

Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca²⁺ -dependent Cl^- channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca²⁺ . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H⁺ ]_o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H⁺ ]_o from 10^-10 to 10^-5.5 m caused a progressive increase in the chloride current (I_Cl ) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca²⁺ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca²⁺ ]_i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the I_Cl was nearly insensitive to proton concentrations between 10^-5.5 and 10^-9.0 m. Additionally, I_Cl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10^-5.5 m, but 10^-9.0 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca²⁺ ]_i .