Native Store-operated Ca2+ Influx Requires the Channel Function of Orai1 and TRPC1.
With the discovery of STIM1 and Orai1 and gating of both TRPC and Orai1 channels by STIM1, a central question is the role of each of the channels in the native store-operated Ca(2+) influx (SOCs). Here, we used a strategy of knockdown of Orai1 and of TRPC1 alone and in combination and rescue by small interfering RNA-protected mutants (sm) of smOrai1 and smTRPC1 to demonstrate that in human embryonic kidney (HEK) cells, rescue of SOCs required co-transfection of low levels of both smOrai1 and smTRPC1. The pore mutant Orai1(E106Q) failed to rescue the SOCs in the presence or absence of TRPC1 and, surprisingly, the pore mutant TRPC1(F562A) failed to rescue the SOCs in the presence or absence of Orai1. TRPC1 is gated by electrostatic interaction between TRPC1(D639D,D640D) with STIM1(K684K, K685K). Strikingly, the channel-dead TRPC1(D639K,D640K) that can be rescued only by the STIM1(K684E,K685E) mutant could restore SOCs only when expressed with Orai1 and STIM1(K684E,K685E). Accordingly, we found a mutual requirement of Orai1 and TRPC1 for their interaction with the native STIM1 in HEK cells. By contrast, SOC and the CRAC current in Jurkat cells were inhibited by knockdown of Orai1 but were not influenced by knockdown on TRPC1 or TRPC3. These findings define the molecular makeup of the native SOCs in HEK cells and the role of a STIM1-Orai1-TRPC1 complex in SOC activity.
Kim
Min Seuk
MS
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
Zeng
Weizhong
W
Yuan
Joseph P
JP
Shin
Dong Min
DM
Worley
Paul F
PF
Muallem
Shmuel
S
D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D000071740:ORAI1 Protein; D012313:RNA / Q000737:chemistry; D015398:Signal Transduction; D050052:TRPC Cation Channels / Q000378:metabolism*; D014404:Tubulin / Q000737:chemistry
D015220:Calcium Channels; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D014404:Tubulin; C096351:transient receptor potential cation channel, subfamily C, member 1; D012313:RNA; D002118:Calcium