Orai proteins interact with TRPC channels and confer responsiveness to store depletion

Abstract

The TRPC (C-type transient receptor potential) class of ion channels has been hypothesized to participate in store-operated Ca 2؉ entry (SOCE). Recently, however, STIM1 and Orai1 proteins have been proposed to form SOCE channels. Whether TRPCs participate in SOCE that is dependent on or regulated by Orai has not been explored. Here we show that Orai1 physically interacts with the N and C termini of TRPC3 and TRPC6, and that in cells overexpressing either TRPC3 or TRPC6 in a store-depletion insensitive manner, these TRPCs become sensitive to store depletion upon expression of an exogenous Orai. Thus, Orai-1, -2, and -3 enhanced thapsigargin-induced calcium entry by 50 -150% in cells stably overexpressing either TRPC3 or TRPC6. Orai1 expression had no significant effect on endogenous, thapsigargin-induced calcium entry in wildtype cells (HEK-293, COS1), in HEK cells expressing a thapsigarginsensitive variant of TRPC3 (TRPC3a), or in HEK cells overexpressing another membrane protein, V1aR. Single-channel cation currents present in membrane patches of TRPC3-overexpressing cells were suppressed by expression of Orai1. We propose that Orai proteins by interacting with TRPCs act as regulatory subunits that confer STIM1-mediated store depletion sensitivity to these channels.capacitative calcium entry ͉ ion channels ͉ thapsigargin ͉ transmembrane signaling

Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels

“…If this were the case, we reasoned, expression of Orai1 in cells expressing excess TRPCs might lead to increases in SOCE, as seen in Fura2 imaging experiments. ¶ We indeed found that Orai1 increased SOCE in cells stably expressing TRPC3 or TRPC6 but not in untransformed HEK293 cells or HEK cells that were expressing unrelated molecules, such as the V2 or the V1a vasopressin receptors (8). In these studies we also showed that expression of SOCE-enhancing levels of Orai1 tends to ''silence'' ʈ spontaneous activity of TRPC3 in cells that stably overexpress these channels.…”

“…Expression of low levels of Orai (typically between 50 and 90 ng of Orai1-carrying pcDNA3 plasmid to transfect the cells grown to near confluence in a 60-mm dish) leads to an enhancement of SOCE between 50% and 150% for cells stably expressing either TRPC3 or TRPC6 (8). Orai1 also enhances SOCE in TRPC1-and TRPC7-expressing cells (data not shown).…”

“…Based on the foregoing data, and recognizing that Orai/ CRACMs are part of SOCE/Icrac channels, we proposed that SOCE/Icrac channels may be heteromeric complexes of TRPC and Orai proteins (8). If this were the case, we reasoned, expression of Orai1 in cells expressing excess TRPCs might lead to increases in SOCE, as seen in Fura2 imaging experiments.…”

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

“…Orai1 was also shown to be associated with the complex formed by STIM1 and TRPC. Hence, the idea emerged that the function of TRPC channels might well depend on their association or not with the STIM/Orai molecules [30][31][32][33][34][35]. Our experimental evidence suggested that TRPC3 function independently from STIM/Orai and that its activity is not linked to the Ca 2+ content of the store.…”

“…Antisense From siRNA STIM1 GGCUCUGGAUACAGUGCUCtt GAGCACUGUAUCCAGAGCCtt Ambion Orai1 CGUGCACAAUCUCAACUCGtt CGAGUUGAGAUUGUGCACGtt Ambion TRPC1 CGGACUUCUAAAUAUGCUAtt UAGCAUAUUUAGAAGUCCGAAtt Oiagen TRPC3 GCAGCAGCUCUUGACGAUCUGGUAU AUACCAGAUCGUCAAGAGCUGCUGC Invitrogen TRPC4 UGUCUAUGUUGGAGAUGCUCUAUUA UAAUAGAGCAUCUCCAACAUAGACA Invitrogen TRPC5 CCCAAGUCAUUUCUAUACCUUGGUA UACCAAGGUAUAGAAAUGACUUGGG Invitrogen TRPC6 CCCAAGGAAUAUUUGUUUGAGUUGU ACAACUCAAACAAAUAUUCCUUGGG Invitrogen PKC CCAGAUUUGUGGCUUAUAAtt UUAUAAGCCACAAAUCUGGtt Qiagen PKC␦ GCCGGGACACUAUAUUCCAtt UGGAAUAUAGUGUCCCGGCUtt Ambion PCR primers STIM1 TGACAGGGACTGTGCTGAAG AAGAGAGGAGGCCCAAAGAG Invitrogen Orai1 GCTCATGATCAGCACCTGCAC GGGACTCCTTGACCGAGTTG Invitrogen TRPC1 GAGGTGATGGCGCTGAAGG GCACGCCAGCAAGAAAAGC Invitrogen TRPC3 CTGGCTCTGCTTGTGTTCAATG AGTCAGTAACTGTGATATTGGATATTG Invitrogen TRPC4 TCAGCACATCGACAGGTCAGAC CCACGGTAATATCATCCACTCGAC Invitrogen TRPC5 CTCTGATTGCGGAAGCCTCT GCAACGAACTTAAAATGTTGGATATTG Invitrogen TRPC6 AAGTATAAGGAGCTCAGAAATTTCCAT TCTGATATTGTCTTGGAGGATTATTGA Invitrogen GusB CCACCAGGGACCATCCAAT AGTCAAAATATGTGTTCTGGACAAAGTAA Invitrogen TBP GCCCGAAACGCCGAATATA CGTGGCTCTCTTATCCTCATGA Invitrogen GAPDH GCACAAGAGGAAGAGAGAGACC AGGGGAGATTCAGTGTGGTG Invitrogen EE-EF1 AGCAAAAATGACCCACCAATG GGCCTGGATGGTTCAGGATA Invitrogen reported that Orai1 and TRPC proteins form complexes together with STIM1 that participate to Ca 2+ entry [30][31][32][33][34][35].…”