Explaining Calcium-Dependent Gating of Anoctamin-1 Chloride Channels Requires a Revised Topology

Rationale: Ca2+-activated Cl channels play pivotal roles in the cardiovascular system. They regulate vascular smooth muscle tone and participate in cardiac action potential repolarization in some species. Ca2+-activated Cl channels were recently discovered to be encoded by members of the anoctamin (Ano, also called Tmem16) superfamily, but the mechanisms of Ano1 gating by Ca2+ remain enigmatic. Objective: The objective was to identify regions of Ano1 involved in channel gating by Ca2+. Methods and Results: The Ca2+ sensitivity of Ano1 was estimated from rates of current activation, and deactivation in excised patches rapidly switched between zero and high Ca2+ on the cytoplasmic side. Mutation of glutamates E702 and E705 dramatically altered Ca2+ sensitivity. E702 and E705 are predicted to be in an extracellular loop, but antigenic epitopes introduced into this loop are not accessible to extracellular antibodies, suggesting this loop is intracellular. Cytoplasmically applied membrane-impermeant sulfhydryl reagents alter the Ca2+ sensitivity of Ano1 E702C and E705C as expected if E702 and E705 are intracellular. Substituted cysteine accessibility mutagenesis of the putative re-entrant loop suggests that E702 and E705 are located adjacent to the Cl conduction pathway. Conclusions: We propose an alternative model of Ano1 topology based on mutagenesis, epitope accessibility, and cysteine-scanning accessibility. These data contradict the popular re-entrant loop model by showing that the putative fourth extracellular loop (ECL 4) is intracellular and may contain a Ca2+ binding site. These studies provide new perspectives on regulation of Ano1 by Ca2+. (Circ Res. 2012;110:990-999.)