title: TMEM16A knockdown abrogates two different Ca(2+)-activated Cl (-) currents and contractility of smooth muscle in rat mesenteric small arteries.
abstract: The presence of Ca(2+)-activated Cl(-) channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca(2+)-activated Cl(-) currents (I Cl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent I Cl(Ca) depends on bestrophin expression, while the "classical" I Cl(Ca) is not. Downregulation of bestrophins did not affect arterial contraction but inhibited the rhythmic contractions, vasomotion. In this study, we have used in vivo siRNA transfection of rat mesenteric small arteries to investigate the role of a putative CaCC, TMEM16A. Isometric force, [Ca(2+)]i, and SMC membrane potential were measured in isolated arterial segments. I Cl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent I Cl(Ca) and the "classical" I Cl(Ca) in SMCs. TMEM16A downregulation also reduced expression of bestrophins. TMEM16A downregulation suppressed vasomotion both in vivo and in vitro. Downregulation of TMEM16A reduced agonist (noradrenaline and vasopressin) and K(+)-induced contractions. In accordance with the depolarizing role of CaCCs, TMEM16A downregulation suppressed agonist-induced depolarization and elevation in [Ca(2+)]i. Surprisingly, K(+)-induced depolarization was unchanged but Ca(2+) entry was reduced. We suggested that this is due to reduced expression of the L-type Ca(2+) channels, as observed at the mRNA level. Thus, the importance of TMEM16A for contraction is, at least in part, independent from membrane potential. This study demonstrates the significance of TMEM16A for two SMCs I Cl(Ca) and vascular function and suggests an interaction between TMEM16A and L-type Ca(2+) channels.
authors.affiliation: Department of Biomedicine, MEMBRANES, Aarhus University, Ole Worms Alle bygn.4, 1163, Aarhus, C 8000, Denmark.
authors.forename: Vibeke Secher
authors.initials: VS
authors.lastname: Dam
authors.forename: Donna M B
authors.initials: DM
authors.lastname: Boedtkjer
authors.forename: Jakob
authors.initials: J
authors.lastname: Nyvad
authors.forename: Christian
authors.initials: C
authors.lastname: Aalkjaer
authors.forename: Vladimir
authors.initials: V
authors.lastname: Matchkov
mesh_terms: D000200:Action Potentials; D000818:Animals; D000075369:Anoctamin-1; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D006152:Cyclic GMP / Q000494:pharmacology; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014661:Vasoconstriction
chemical_list: C552942:ANO1 protein, rat; D000075369:Anoctamin-1; D020746:Calcium Channels, L-Type; D018118:Chloride Channels; D012333:RNA, Messenger; D006152:Cyclic GMP; D002118:Calcium