Endogenous TRPC1, TRPC3, and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells.
Endogenously expressed canonical transient receptor potential (TRPC) homologs were investigated for their role in forming store-operated, 1-oleoyl-2-acetyl-sn-glycerol-stimulated, or carbachol (CCh)-stimulated calcium entry pathways in HEK-293 cells. Measurement of thapsigargin-stimulated Ba(2+) entry indicated that the individual suppression of TRPC1, TRPC3, or TRPC7 protein levels, by small interfering RNA (siRNA) techniques, dramatically inhibited (52-68%) store-operated calcium entry (SOCE), whereas suppression of TRPC4 or TRPC6 had no effect. Combined suppression of TRPC1-TRPC3, TRPC1-TRPC7, TRPC3-TRPC7, or TRPC1-TRPC3-TRPC7 gave only slightly more inhibition of SOCE (74-78%) than seen with suppression of TRPC1 alone (68%), suggesting that these three TRPC homologs work in tandem to mediate a large component of SOCE. Evidence from co-immunoprecipitation experiments indicates that a TRPC1-TRPC3-TRPC7 complex, predicted from siRNA results, does exist. The suppression of either TRPC3 or TRPC7, but not TRPC1, induced a high Ba(2+) leak flux that was inhibited by 2-APB and SKF96365, suggesting that the influx is via leaky store-operated channels. The high Ba(2+) leak flux is eliminated by co-suppression of TRPC1-TRPC3 or TRPC1-TRPC7. For 1-oleoyl-2-acetyl-sn-glycerol-stimulated cells, siRNA data indicate that TRPC1 plays no role in mediating Ba(2+) entry, which appears to be mediated by the participation of TRPC3, TRPC4, TRPC6, and TRPC7. CCh-stimulated Ba(2+) entry, on the other hand, could be inhibited by suppression of any of the five endogenously expressed TRPC homologs, with the degree of inhibition being consistent with CCh stimulation of both store-operated and receptor-operated channels. In summary, endogenous TRPC1, TRPC3, and TRPC7 participate in forming heteromeric store-operated channels, whereas TRPC3 and TRPC7 can also participate in forming heteromeric receptor-operated channels.
Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Illinois 60637, USA.
Tatiana K
TK
Zagranichnaya
Xiaoyan
X
Wu
Mitchel L
ML
Villereal
D001464:Barium / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000494:pharmacology; D050052:TRPC Cation Channels; D050053:TRPM Cation Channels
D015220:Calcium Channels; D004075:Diglycerides; D007473:Ion Channels; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; C498501:TRPC7 protein, human; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; C096351:transient receptor potential cation channel, subfamily C, member 1; D001464:Barium; C038237:1-oleoyl-2-acetylglycerol; D002217:Carbachol; D002118:Calcium