Orai1 interacts with STIM1 and mediates capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells

Ng LC, Ramduny D, Airey JA, Singer CA, Keller PS, Shen XM, Tian H, Valencik M, Hume JR. Orai1 interacts with STIM1 and mediates capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells. Am J Physiol Cell Physiol 299: C1079-C1090, 2010. First published August 25, 2010; doi: 10.1152/ajpcell.00548.2009.-Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that cannonical transient receptor potential channel TRPC1 and stromal interaction molecule 1 (STIM1) mediate the sustained component of capacitative Ca2+ entry (CCE), but the molecular candidate(s) that mediate the transient component of CCE remain unknown. The aim of the present study was to examine whether Orai1 mediates the transient component of CCE through activation of STIM1 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 small interfering (si) RNA. Moreover, overexpression 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 coimmunoprecipitate with STIM1, and the precipitation level of Orai1 was increased in cells subjected to store-depletion. Immunostaining revealed colocalization of Orai1 and STIM1 proteins, and the colocalization of these proteins was more apparent after store-depletion. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel as a result of STIM1 activation in mouse PASMCs.