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


METHODS
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.


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 ICl ( C a) 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 ( C a) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl ( C a) was inhibited by T16Ainh -A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl ( C a) in Ano1 gene knockdown CVECs.


CONCLUSIONS
Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl ( 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.
Departments of Clinical Pharmacy and Cardiology, Institute of Clinical Pharmacy, The 2nd Affiliated Hospital, Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, Harbin Medical University, Harbin, China.
Ming-Ming
MM
Wu
Jie
J
Lou
Bin-Lin
BL
Song
Yuan-Feng
YF
Gong
Yan-Chao
YC
Li
Chang-Jiang
CJ
Yu
Qiu-Shi
QS
Wang
Tian-Xing
TX
Ma
Ke
K
Ma
H Criss
HC
Hartzell
Dayue Darrel
DD
Duan
Dan
D
Zhao
Zhi-Ren
ZR
Zhang
D000818:Animals; D000831:Animals, Newborn; D000075369:Anoctamin-1; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D049109:Cell Proliferation; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics; D006352:Heart Ventricles / Q000166:cytology; D000860:Hypoxia / Q000503:physiopathology*; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014162:Transfection
C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D049452:Green Fluorescent Proteins; D002118:Calcium
anoctamin; calcium-activated chloride channels; cardiac vascular endothelial cells; hypoxia