Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry.
Ca2+ release from its internal stores as a result of activation of phospholipase C is accompanied by Ca2+ influx from the extracellular space. Ca2+ influx channels may be formed of proteins homologous to Drosophila 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 cells 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 depletion. We have stably expressed human Trp3 (hTrp3) in human embryonic kidney (HEK)293 cells. Measurement of intracellular Ca2+ concentrations in Fura2-loaded cells showed that cell lines expressing hTrp3 have significantly higher basal and agonist-stimulated influxes of Ca2+, Mn2+, Ba2+, and Sr2+ than control cells. The increase in Ca2+ entry attributable to the expression of hTrp3 obtained upon store depletion by thapsigargin was much lower than that obtained by stimulation with agonists acting via a Gq-coupled receptor. Addition of agonists to thapsigargin-treated Trp3 cells resulted in a further increase in the entry of divalent 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 hTrp3 in these cells forms a non-selective cation channel that opens after the activation of phospholipase C but not after store depletion. In addition, a subpopulation of the expressed hTrp3 may form heteromultimeric channels with endogenous proteins that are sensitive to store depletion.
Zhu
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Department of Pharmacology and Neurobiotechnology Center, Ohio State University, Columbus, Ohio 43210, USA.
Jiang
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Birnbaumer
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D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002413:Cations, Divalent; D002460:Cell Line; D004789:Enzyme Activation; D000939:Epitopes; D006031:Glycosylation; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D017136:Ion Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D015220:Calcium Channels; D002413:Cations, Divalent; D000939:Epitopes; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium