We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP 3 ) in capacitative calcium entry and calcium releaseactivated calcium current (I crac ) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with U73122 blocked calcium entry and I crac activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I crac . The membrane-permeant IP 3 receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I crac . However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP 3 receptor because the drug was equally effective in wildtype DT40 B-cells and in DT40 B-cells whose genes for all three IP 3 receptors had been disrupted. Intracellular application of another potent IP 3 receptor antagonist, heparin, failed to inhibit activation of I crac . Finally, the inhibition of I crac activation by U73122 or wortmannin was not reversed or prevented by direct intracellular application of IP 3 . These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP 3 and IP 3 receptor in this mechanism, at least in these particular cell types.

Mammalian Trp proteins are candidates for plasma membrane calcium channels regulated by receptor activation or by intracellular calcium store depletion [capacitative calcium entry (CCE)]. One extensively investigated member of the Trp family, the human Trp3 (hTrp3), behaves as a receptor-activated, calcium-permeable, nonselective cation channel when expressed in cell lines and does not appear to be activated by store depletion. Nonetheless, there is good evidence that Trp3 can be regulated by interacting with inositol trisphosphate receptors (IP3Rs), reminiscent of the conformational coupling mode of CCE. To investigate the role of Trp3 in CCE, and its regulation by IP3R, we transiently expressed hTrp3 in the wild-type DT40 chicken B lymphocyte cell line and its variant lacking IP3R. Expression of hTrp3 in either wild-type or IP3R-knockout cells did not increase basal membrane permeability, but resulted in a substantially greater divalent cation entry after thapsigargin-induced store depletion. This hTrp3-dependent divalent cation entry was significantly greater in the wild type than in IP3R-knockout cells. Thus, it appears that in this cell line, hTrp3 forms channels that are store-operated by both IP3R-dependent and IP 3R-independent mechanisms. Trp3, or one of its structural relatives, is a candidate for the store-operated, nonselective cation channels observed in smooth muscle cells and other cell types.

. Canonical transient receptor potential TRPC7 can function as both receptor-and store-operated channel in HEK-293 cells.