Orai1 and STIM1 Mediate Capacitative Ca2+ Entry in Mouse Pulmonary Arterial Smooth Muscle Cells

Abstract

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that TRPC1 and STIM1 mediates the sustained component of capacitative Ca2+ entry (CCE) but the molecular candidate(s) that mediate the transient component of CCE remains unknown. The aim of the present study was to further examine if Orai1 mediates the transient component of CCE through activation of STIM1 protein in mouse PASMCs.In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i).The transient but not the sustained rise in [Ca2+]i was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca2+]i and the increase in Mn2+ quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 siRNA. Moreover, over-expression of STIM1 enhanced the rise in [Ca2+]i and the increase in Mn2+ quench of fura-2 fluorescence caused by CPA and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to co-immunoprecipitate with STIM1 and immunostaining showed co-localization of Orai1 and STIM1 proteins. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel through activation of STIM1 in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]