Dynamic Assembly of TRPC1-STIM1-Orai1 Ternary Complex Is Involved in Store-operated Calcium Influx

Abstract

Store-operated calcium entry (SOCE) is a ubiquitous mechanism that is mediated by distinct SOC channels, ranging from the highly selective calcium release-activated Ca 2؉ (CRAC) channel in rat basophilic leukemia and other hematopoietic cells to relatively Ca 2؉ -selective or non-selective SOC channels in other cells. Although the exact composition of these channels is not yet established, TRPC1 contributes to SOC channels and regulation of physiological function of a variety of cell types. Recently, Orai1 and STIM1 have been suggested to be sufficient for generating CRAC channels. Here we show that Orai1 and STIM1 are also required for TRPC1-SOC channels. Knockdown of TRPC1, Orai1, or STIM1 attenuated, whereas overexpression of TRPC1, but not Orai1 or STIM1, induced an increase in SOC entry and I SOC in human salivary gland cells. All three proteins were co-localized in the plasma membrane region of cells, and thapsigargin increased co-immunoprecipitation of TRPC1 with STIM1, and Orai1 in human salivary gland cells as well as dispersed mouse submandibular gland cells. In aggregate, the data presented here reveal that all three proteins are essential for generation of I SOC in these cells and that dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in activation of SOC channel in response to internal Ca 2؉ store depletion. Thus, these data suggest a common molecular basis for SOC and CRAC channels.

Regulation of store-operated Ca2+entry by CD38 in human airway smooth muscle

“…There is now considerable evidence for store-operated or capacitative Ca 2ϩ entry in different smooth muscle types. The presence of store-operated Ca 2ϩ influx pathways has been demonstrated previously in guinea pig, rat, human, and porcine airway smooth muscle (6,34,35,48,58). Previous studies from our group have established that SOCE exists in human ASM (6,58).…”

“…However, this does not rule out CD38 regulation of TRPC3 channels per se. This may occur within caveolae expressing CD38 and TRPC3, via direct complexing of CD38 and TRPC3, or through modulation of the recently recognized trigger for SOCE, stromal interaction molecule (STIM1) (34,36,40). Our additional studies showing partial blockade of SOCE (or of TNF␣ and CD38 overexpression effects) by 8-Br-cADPR suggest that at least some of the CD38 effects may be mediated via cADPR (which is produced by CD38 activation following agonist exposure).…”

TRPC3 Regulates Agonist-stimulated Ca2+ Mobilization by Mediating the Interaction between Type I Inositol 1,4,5-Trisphosphate Receptor, RACK1, and Orai1

“…For the shOrai1, the sense sequence was 5Ј-CACCTCACTGGT-TAGCCATAAGACGAATCTTATGGCTAACCAGTGA-3Ј, and the antisense sequence was 5Ј-AAAACCTTTACACGCTAGATGGTttcgTCTTATGGCTAACCAGTGA-3Ј. These sequences were synthesized and hybridized as previously described (33,34). Plasmids were used for silencing experiments at 1 g/l.…”

TRPC channels as STIM1-regulated store-operated channels

“…It is now clear that Orai1 [4][5][6][7]9,[33][34][35], and perhaps Orai2 and Orai3 [34,36] are the proteins that mediate I crac . However, recently it was shown that Orai1 forms a complex with STIM1 and TRPC1 [37]. Of particular significance is the finding that KD of TRPC1 partially inhibited SOCs-mediated Ca 2+ influx and I soc , whereas KD of Orai1 completely inhibited Ca 2+ influx and I soc [37].…”