The role of calmodulin-binding sites in the regulation of the Drosophila TRPL cation channel expressed in Xenopus laevis oocytes by Ca2+, inositol 1,4,5-trisphosphate and GTP-binding proteins

摘要

pThe roles of calmodulin-binding sites in the regulation by Casup2+/sup, inositol 1,4,5-trisphosphate (InsPsub3/sub) and GTP-binding regulatory proteins (G-proteins) of the iDrosophila melanogaster/i TRPL (transient-receptor-potential-like) non-specific Casup2+/sup channel were investigated. Wild-type TRPL protein and two mutant forms, TRPL (W713G) and TRPL (W814G), in which a key tryptophan residue in each of the two putative calmodulin-binding sites (Sites 1 and 2, respectively) was replaced by glycine, were expressed heterologously in iXenopus/iilaeiv/iis/i oocytes. Immunofluorescence studies indicated that the expressed TRPL, TRPL (W713G) and TRPL (W814G) proteins are located at the plasma membrane. TRPL oocytes (oocytes injected with itrpl/i cRNA) and TRPL (W814G) oocytes [oocytes injected with itrpl/i (W814G) cRNA] exhibited substantially greater rates of basal (constitutive) Casup2+/sup inflow (measured using fluo-3 and the Casup2+/sup add-back protocol) than mock-injected oocytes (mock oocytes). In TRPL (W713G) oocytes, this difference was abolished. In TRPL and TRPL (W814G) [oocytes injected with itrpl/i (W713G) cRNA], but not in TRPL (W713G) oocytes, basal Casup2+/sup inflow was inhibited by W13, an inhibitor of calmodulin action. Calmodulin (3 iμ/iM intracellular) inhibited basal Casup2+/sup inflow in TRPL but not in TRPL (W713G) or TRPL (W814G) oocytes. Staurosporin, an inhibitor of protein kinase C (PKC), inhibited, while PMA (an activator of PKC) stimulated, basal Casup2+/sup inflow in TRPL oocytes. In oocytes incubated in the presence of PMA (to suppress Casup2+/sup inflow through endogenous receptor-activated Casup2+/sup channels), the InsPsub3/sub-induced stimulation of Casup2+/sup inflow through TRPL channels was more clearly evident than in oocytes incubated in the absence of PMA. InsPsub3/sub caused a significant stimulation of Mnsup2+/sup inflow in TRPL but not in mock oocytes. Rates of InsPsub3/sub-stimulated Casup2+/sup inflow through the TRPL, TRPL (W713G) and TRPL (W814G) channels were similar. The ability of GTPγS to stimulate Casup2+/sup inflow through TRPL channels was inhibited by 50% in TRPL (W713G) oocytes but was unaffected in TRPL (W814G) oocytes. It is concluded that, in the environment of the iXenopus/i oocyte, the iDrosophila/i TRPL channel is activated by (a) interaction with Casup2+/sup/calmodulin at calmodulin-binding Site 1; (b) PKC; (c) InsPsub3/sub in a process that does not involve Casup2+/sup and calmodulin; and (d) a trimeric G-protein(s) through both a Casup2+/sup/calmodulin-dependent and a Casup2+/sup/calmodulin-independent mechanism./p