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Papers In Press, published online ahead of print June 22, 2005
Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, IL 60637
Corresponding Author: mitch{at}drugs.bsd.uchicago.edu, mitch@bsd.uchicago.edu
Endogenously expressed TRPC homologs were investigated for their role in forming store-operated, OAG-stimulated, or carbachol (CCh)-stimulated calcium entry pathways in HEK-293 cells. Measurement of thapsigargin-stimulated Ba2+ entry indicated that the individual suppression of TRPC1, TRPC3 or TRPC7 protein levels, by siRNA techniques, dramatically inhibited (52-68%) store-operated calcium entry (SOCE), while 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 indicate 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 Ba2+ leak flux, that was inhibited by 2-APB and SKF96365, suggesting the influx is via leaky store-operated channels. The high Ba2+ leak flux is eliminated by co-suppression of TRPC1/TRPC3 or TRPC1/TRPC7. For OAG-stimulated cells, siRNA data indicate that TRPC1 plays no role in mediating Ba2+ entry, which appears to be mediated by the participation of TRPC3, TRPC4, TRPC6 and TRPC7. CCh-stimulated Ba2+ 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, while TRPC3 and TRPC7 can also participate in forming heteromeric receptor-operated channels.
J. Biol. Chem, 10.1074/jbc.M505842200
Submitted on May 31, 2005
Accepted on June 22, 2005
Endogenous TRPC1, TRPC3 and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells
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