Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Ca2+-activated Cl- channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca2+ has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca2+ or by voltage in the absence of Ca2+, but Ca2+-and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca2+ and voltage sensing. Deleting (448)EAVK in the first intracellular loop dramatically decreases apparent Ca2+ affinity. In contrast, mutating the adjacent amino acids 444EEEE abolishes intrinsic voltage dependence without altering the apparent Ca2+ affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca2+ was facilitated by anions with high permeability or by an increase in [Cl-](e). Our data show that the transition between closed and open states is governed by Ca2+ in a voltage-dependent manner and suggest that anions allosterically modulate Ca2+-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic.