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

• Authors: Kuai Yu, Emory University; Charity Duran, Emory University; Zhiqiang Qu, Emory University; Yuan-Yuan Cui, Emory University; Criss Hartzell Jr., Emory University
• Journal Title: Circulation Research
• Volume: Volume 110, Number 7
• Publisher: American Heart Association | 2012-03-30, Pages 990-999
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1161/CIRCRESAHA.112.264440
• Subjects: Biology, Cell
• Keywords: chloride channel; calcium; ion channel; patch clamp; transmembrane topology

Abstract

Rationale Ca2+-activated Cl channels (CaCCs) play pivotal roles in the cardiovascular system: they regulate vascular smooth muscle tone and participate in cardiac action potential repolarization in some species. CaCCs 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 4th extracellular loop (ECL 4) is intracellular and may contain a Ca2+ binding site. These studies provide new perspectives on regulation of Ano1 by Ca2+.