Abstract
Ca-activated chloride channels play important roles in epithelial secretion, regulation of vascular tone, control of membrane excitability, olfactory transduction, and photoreception. Recently, Ano1 (TMEM16a) has been identified as a Ca2+-activated chloride channel. Activation of Ano1 exhibits both Ca2+- and voltage- dependence. However, the structures and mechanisms responsible for Ca2+- and voltage-dependent activation remain unknown. mAno1 exhibits a strong outward rectification at +100 mV). In the presence of Ca2+, the G-V relations were well fit with the Boltzmann equation. V1/2 was 73 mV at 10 μM intracellular free Ca2+. Increasing Ca2+ shifted the G-V relation to the left. We mutated the putative Ca2+ binding site by deleting the last glutamate of the cluster plus the 3 trailing amino acids (del448EAVK451), substituting the first four glutamates with alanines (444EEEE/AAAA447), and substituting of each negatively charged amino acid with alanine. These mutations altered both voltage-dependent and Ca2+-dependent gating of the channel in complex ways that are consistent with this region being an integral component of Ca2+ dependent gating of the channel.
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