Acidic Amino Acids in the First Intracellular Loop Contribute to Voltage- and Calcium- Dependent Gating of Anoctamin1/TMEM16A

Anoctamin1 (Ano1, or TMEM16A) is a Ca(2+)-activated chloride channel that is gated by both voltage and Ca(2+). We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates ((444)EEEE(447)) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca(2+) sensitivity. In the present study, we further found that deletion of (444)EEEEEAVKD(452) produced a more than 40-fold decrease in the apparent Ca(2+) sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τ(fast)) that represented voltage-dependent mode, and a slow component (τ(slow)) that reflected the Ca(2+)-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τ(fast), significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca(2+), and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca(2+) dependence of G-V curve, exhibited an increase in the τ(slow), and produced a 2–3 fold decrease in the apparent Ca(2+) sensitivity, suggesting that these acidic residues are involved in Ca(2+)-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1.