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

<jats:p> Ca <jats:sup>2+</jats:sup> -activated Cl <jats:sup>−</jats:sup> 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 Ca <jats:sup>2+</jats:sup> has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca <jats:sup>2+</jats:sup> or by voltage in the absence of Ca <jats:sup>2+</jats:sup> , but Ca <jats:sup>2+</jats:sup> - and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca <jats:sup>2+</jats:sup> and voltage sensing. Deleting <jats:sub>448</jats:sub> EAVK in the first intracellular loop dramatically decreases apparent Ca <jats:sup>2+</jats:sup> affinity. In contrast, mutating the adjacent amino acids <jats:sub>444</jats:sub> EEEE abolishes intrinsic voltage dependence without altering the apparent Ca <jats:sup>2+</jats:sup> affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca <jats:sup>2+</jats:sup> was facilitated by anions with high permeability or by an increase in [Cl <jats:sup>−</jats:sup> ] <jats:sub>e</jats:sub> . Our data show that the transition between closed and open states is governed by Ca <jats:sup>2+</jats:sup> in a voltage-dependent manner and suggest that anions allosterically modulate Ca <jats:sup>2+</jats:sup> -binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic. </jats:p>