Evidence for STIM1‐ and Orail‐dependent storeoperated calcium influx through I _CRAC in vascular smooth muscle cells: role in proliferation and migration

Abstract

The identity of store-operated calcium (Ca(2+)) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of I(CRAC), a highly Ca(2+)-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called "synthetic") display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of I(CRAC). Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced I(CRAC) in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show I(CRAC) in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.

Thapsigargin activates Ca2+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

“…Interestingly, even though the silencing efficiency was of between 65 and 80% at the protein level, an important residual Ca 2+ entry persisted upon store depletion. Such a "partial" SOCE reduction was already observed, for instance in smooth muscle cells [52][53][54] or HEK cells [20], but in most cell types, the inhibition of SOCE upon STIM1 invalidation is very pronounced [3,39,55,56]. This finding prompted us to investigate the mechanism of the residual TG-induced Ca 2+ entry.…”

Attenuated mesangial cell proliferation related to store-operated Ca2+ entry in aged rat: the role of STIM 1 and Orai 1

“…The sequence for rat STIM-1 siRNA was UAAGGGAAGACCUCAAUUA (sense strand) and UAAUUGAGGUCUUCCCUUA (antisense strand) (Potier et al 2009). The Orai-1 siRNA sequence was CAACAGCAAUCCGGAGCUU (sense strand) and AAGCUCCGGAUUGCUGUUG (antisense strand) (Potier et al 2009). siRNA delivery was achieved using Lipofectamine RNAiMAX reagent according to the manual.…”

TRPC channels as STIM1-regulated SOCs

“…This may explain a recent report claiming that TRPC3 and TRPC6 expressed in HEK cells do not function as SOCs 19 and that knockdown of TRPC6 does not affect SOC activity in proliferative vascular smooth muscle cells. 20 when expressed at high levels, both channels showed spontaneous activity that was independent of STIM1. 16 This implies that TRPC3 and TRPC6 can function in two modes.…”

“…14,[16][17][18] The expression studies generated the most controversy since the findings were not uniform, with several groups reporting that the expressed TRPC channels function as SOCs, 14,[16][17][18] while others using the same expression system concluded that the same TRPC channels do not function as SOCs. 19,20 Several of these differences could be traced to expression level of the channels 16,21 and most likely because several of the TRPC channels can function both in SOC and non-SOC modes (see below).…”