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

Abstract

BACKGROUND AND PURPOSEThe 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 APPROACHWestern 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 RESULTSAno1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca2+ and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl(Ca) detected in CVECs was significantly inhibited by T16Ainh-A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl(Ca) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl(Ca) was inhibited by T16Ainh-A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl(Ca) in Ano1 gene knockdown CVECs.CONCLUSIONS AND IMPLICATIONSAno1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl(Ca) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca2+. 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.

Characterization of Cardiac Anoctamin1 Ca2+‐Activated Chloride Channels and Functional Role in Ischemia‐Induced Arrhythmias

“…B,b and c) and was also significantly inhibited by extracellular application of a specific pore‐targeting anti‐ANO1 antibody (1:100 dilution; Figs. C,b and c) (Thomas‐Gatewood et al, ; Wu et al, ). In contrast, this current was not inhibited by either the boiled specific pore‐targeting anti‐ANO1 antibody (1:100 dilution) or the non‐pore‐targeting anti‐ANO1 antibody (1:100 dilution; Figs.…”

The bestrophin- and TMEM16A-associated Ca2+-activated Cl–channels in vascular smooth muscles

“… 98 The recently developed inhibitor T16A inh -A01 63 effectively inhibits the TMEM16A-associated I Cl(Ca) without effect on the bestrophin-associated cGMP-dependent I Cl(Ca) in VSMCs. 17 This inhibitor is now widely used for pharmacological identification of TMEM16A-associated I Cl(Ca) and its functions, 23 , 38 , 60 , 69 , 101 including VSMC contraction. 17 Drug-screening also generated another inhibitor, CaCC inh -A01, which has been shown to inhibit TMEM16A-associated I Cl(Ca) .…”

Hydrogen peroxide suppresses TRPM4 trafficking to the apical membrane in mouse cortical collecting duct principal cells

“…For ion selectivity experiments, 145 mM NaCl in the bath was replaced by 145 mM KCl, or 145 mM NaCl in the pipettes was replaced by 145 mM NMDG-Cl, and the data were corrected for junction potentials at the ground bridge (3 M KCl in 3% agar). The free Ca 2ϩ concentration after chelation of CaCl2 with EGTA was determined using the free Web software Winamac (Stanford University, Stanford, CA), as previously described (40).…”