RECEPTOR-ACTIVATED CA2- EVIDENCE FOR A NON-CAPACITATIVE CA2+ ENTRY( INFLUX VIA HUMAN TRP3 STABLY EXPRESSED IN HUMAN EMBRYONIC KIDNEY (HEK)293 CELLS )

Ca2+ release from its internal stores as a result of activation of pho spholipase C is accompanied by Ca2+ influx from the extracellular spac e. Ca2+ influx channels may be formed of proteins homologous to Drosop hila Trp. At least six non-allelic Trp genes are present in the mouse genome. Full-length human, bovine, mouse, and rat cDNAs for Trp1, 3, 4 , 6 have been cloned. Expression of these genes in various mammalian c ells has provided evidence that Trp proteins form plasma membrane Ca2-permeant channels that can be activated by an agonist that activates phospholipase C, by inositol 1,4,5-trisphosphate, and/or store depleti on. We have stably expressed human Trp3 (hTrp3) in human embryonic kid ney (HEK)293 cells. Measurement of intracellular Ca2+ concentrations i n Fura2-loaded cells showed that cell lines expressing hTrp3 have sign ificantly higher basal and agonist-stimulated influxes of Ca2+, Mn2+ B a2+, and Sr2+ than control cells. The increase in Ca2+ entry attributa ble to the expression of hTrp3 obtained upon store depletion by thapsi gargin was much lower than that obtained by stimulation with agonists acting via a G(q)-coupled receptor. Addition of agonists to thapsigarg in-treated Trp3 cells resulted in a further increase in the entry of d ivalent cations. The increased cation entry in Trp3 cells was blocked by high concentrations of SKF 96365, verapamil, La3+, Ni2+, and Gd3+. The Trp3-mediated Ca2+ influx activated by agonists was inhibited by a phospholipase C inhibitor, U73122. We propose that expression of hTrp 3 in these cells forms a nonselective cation channel that opens after the activation of phospholipase C but not after store depletion. In ad dition, a subpopulation of the expressed hTrp3 may form heteromultimer ic channels with endogenous proteins that are sensitive to store deple tion.