Hypoxia augments the calcium-activated chloride current carried by anoctamin-1 in cardiac vascular endothelial cells of neonatal mice

BACKGROUND AND PURPOSE: The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs). EXPERIMENTAL APPROACH: Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs. KEY RESULTS: Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca(2+) and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of I(Cl)(()(C)(a)) detected in CVECs was significantly inhibited by T16A(inh)-A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of I(Cl)(()(C)(a)) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of I(Cl)(()(C)(a)) was inhibited by T16A(inh)-A01 or anti-Ano1 antibody. Hypoxia also increased the current density of I(Cl)(()(C)(a)) in Ano1 gene knockdown CVECs. CONCLUSIONS AND IMPLICATIONS: Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated I(Cl)(()(C)(a)) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca(2+). Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage.