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: 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 (Biochemical Journal (1998) 330 (1149-1158))

The roles of calmodulin-binding sites in the regulation by Ca²⁺, inositol 1,4,5-trisphosphate (InsP₃) and GTP-binding regulatory proteins (G-proteins) of the Drosophila melanogaster TRPL (transient-receptor-potential-like) non-specific Ca²⁺ 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 Xenopus laevis 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 trpl cRNA) and TRPL (W814G) oocytes [oocytes injected with trpl (W814G) cRNA] exhibited substantially greater rates of basal (constitutive) Ca²⁺ inflow (measured using fluo-3 and the Ca²⁺ 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 trpl (W713G) cRNA], but not in TRPL (W713G) oocytes, basal Ca²⁺ inflow was inhibited by W13, an inhibitor of calmodulin action. Calmodulin (3 μM intracellular) inhibited basal Ca²⁺ 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 Ca²⁺ inflow in TRPL oocytes. In oocytes incubated in the presence of PMA (to suppress Ca²⁺ inflow through endogenous receptor-activated Ca²⁺ channels), the InsP₃-induced stimulation of Ca²⁺ inflow through TRPL channels was more clearly evident than in oocytes incubated in the absence of PMA. InsP₃ caused a significant stimulation of Mn²⁺ inflow in TRPL but not in mock oocytes. Rates of InsP₃-stimulated Ca²⁺ inflow through the TRPL, TRPL (W713G) and TRPL (W814G) channels were similar. The ability of GTPyS to stimulate Ca²⁺ 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 Xenopus oocyte, the Drosophila TRPL channel is activated by (a) interaction with Ca²⁺/calmodulin at calmodulin-binding Site 1; (b) PKC; (c) InsP₃ in a process that does not involve Ca²⁺ and calmodulin; and (d) a trimeric G-protein(s) through both a Ca²⁺/calmodulin-dependent and a Ca²⁺/calmodulin-independent mechanism.