TRPC1 Mediates Capacitative Calcium Entry through Activation of STIM1 in Mouse Pulmonary Arterial Smooth Muscle Cells

Abstract

The present study aimed to determine if TRPC1 and STIM1 mediate capacitative Ca2+ entry (CCE) in mouse pulmonary arterial smooth muscle cells (PASMCs). In primary cultured 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 sustained rise in [Ca2+]i was partially inhibited by nifedipine but they were both inhibited by SKF 96365, Ni2+, La3+ and Gd3+. In addition, CPA increased the rate of Mn2+ quench of fura‐2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. The nifedipine‐insensitive rise in [Ca2+]i and the increase in Mn2+ quench rate were both inhibited in cells pretreated with antibodies raised against extracellular epitopes of TRPC1 and STIM1. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca2+]i and Mn2+ quench rate caused by CPA, and they were reduced by TRPC1 antibody. RT‐PCR and Western blot analysis revealed TRPC1 and STIM1 mRNAs and proteins. Furthermore, TRPC1 was found to co‐immunoprecipitate with STIM1. Taken together, store‐depletion causes activation of voltage‐operated Ca2+ entry and CCE. These data provide direct evidence that TRPC1 channel mediates CCE through activation of STIM1 in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]