title: TRPC1 and Orai1 interact with STIM1 and mediate capacitative Ca(2+) entry caused by acute hypoxia in mouse pulmonary arterial smooth muscle cells.
abstract: Previous studies in pulmonary artery smooth muscle cells (PASMCs) showed that acute hypoxia activates capacitative Ca(2+) entry (CCE) but the molecular candidate(s) mediating CCE caused by acute hypoxia remain unclear. The present study aimed to determine if transient receptor potential canonical 1 (TRPC1) and Orai1 interact with stromal interacting molecule 1 (STIM1) and mediate CCE caused by acute hypoxia in mouse PASMCs. In primary cultured PASMCs loaded with fura-2, acute hypoxia caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. Acute hypoxia also increased the rate of Mn(2+) quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni(2+), La(3+), and Gd(3+), exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive rise in [Ca(2+)](i) and the increase in Mn(2+) quench rate were both inhibited in cells treated with TRPC1 antibody or TRPC1 small interfering (si)RNA, in STIM1 siRNA-transfected cells and in Orai1 siRNA-transfected cells. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca(2+)](i) and Mn(2+) quench rate caused by acute hypoxia, and they were reduced in cells treated with TRPC1 antibody and in cells transfected with Orai1 siRNA. Furthermore, TRPC1 and Orai1 coimmunoprecipitated with STIM1 and the precipitation levels of TRPC1 and Orai1 were increased in cells exposed to acute hypoxia. Immunostaining showed colocalizations of TRPC1-STIM1 and Orai1-STIM1, and the colocalizations of these proteins were more apparent in acute hypoxia. These data provide direct evidence that TRPC1 and Orai1 channels mediate CCE through activation of STIM1 in acute hypoxic mouse PASMCs.
authors.affiliation: Dept. of Pharmacology/318, Univ. of Nevada School of Medicine, 1664 North Virginia St., Reno, NV 89557, USA. lcng@medicine.nevada.edu
authors.forename: Lih Chyuan
authors.initials: LC
authors.lastname: Ng
authors.forename: Kathryn G
authors.initials: KG
authors.lastname: O'Neill
authors.forename: Dominique
authors.initials: D
authors.lastname: French
authors.forename: Judith A
authors.initials: JA
authors.lastname: Airey
authors.forename: Cherie A
authors.initials: CA
authors.lastname: Singer
authors.forename: Honglin
authors.initials: H
authors.lastname: Tian
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Shen
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Hume
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D005682:Gadolinium / Q000494:pharmacology; D020868:Gene Silencing; D007093:Imidazoles / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000503:physiopathology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009532:Nickel / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D000071740:ORAI1 Protein; D011651:Pulmonary Artery / Q000187:drug effects / Q000503:physiopathology*; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000235:genetics / Q000276:immunology / Q000502:physiology*
chemical_list: D057134:Antibodies, Neutralizing; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D007093:Imidazoles; D008562:Membrane Glycoproteins; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D008345:Manganese; D007811:Lanthanum; D009532:Nickel; D005682:Gadolinium; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D009543:Nifedipine; D002118:Calcium