THE ROLE OF CALMODULIN-BINDING SITES IN THE REGULATION OF THE DROSOPHILA TRPL CATION CHANNEL EXPRESSED IN XENOPUS-LAEVIS OOCYTES BY CA2-TRISPHOSPHATE AND GTP-BINDING PROTEINS(, INOSITOL 1,4,5)

The roles of calmodulin-binding sites in the regulation by Ca2+ inosit ol 1,4,5-trisphosphate (InsP(3)) and GTP-binding regulatory proteins ( G-proteins) of the Drosophila melanogaster TRPL (transient-receptor-po tential-like) non-specific Ca2+ channel were investigated. Wild-type T RPL protein and two mutant forms, TRPL (W713G) and TRPL (W814G), in wh ich a key tryptophan residue in each of the two putative calmodulin-bi nding sites (Sites 1 and 2, respectively) was replaced by glycine, wer e expressed heterologously in Xenopus laevis oocytes. Immunofluorescen ce studies indicated that the expressed TRPL, TRPL (W713G) and TRPL (W 814G) proteins are located at the plasma membrane. TRPL oocytes (oocyt es injected with trpl cRNA) and TRPL (W814G) oocytes [oocytes injected with trpl (W814G) cRNA] exhibited substantially greater rates of basa l (constitutive) Ca2+ inflow (measured using fluo-3 and the Ca2+ add-b ack protocol) than mock-injected oocytes (mock oocytes), In TRPL (W713 G) oocytes, this difference was abolished, In TRPL and TRPL (W814G) [o ocytes injected with trpl(W713G) cRNA], but not in TRPL (W713G) oocyte s, basal Ca2+ inflow was inhibited by W13, an inhibitor of calmodulin action. Calmodulin (3 mu M 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 acti vator of PKC) stimulated: basal Ca2+ inflow in TRPL oocytes. In oocyte s incubated in the presence of PMA (to suppress Ca2+ inflow through en dogenous receptor-activated Ca2+ channels), the InsP(3)-induced stimul ation of Ca2(+) inflow through TRPL channels was more clearly evident than in oocytes incubated in the absence of PMA. InsP(3) caused a sign ificant stimulation of Mn2+ inflow in TRPL but not in mock oocytes. Ra tes of InsP(3)-stimulated Ca2+ inflow through the TRPL, TRPL (W713G) a nd TRPL (W814G) channels were similar. The ability of GTP gamma S to s timulate Ca2+ inflow through TRPL channels was inhibited by 50% in TRP L (W713G) oocytes but was unaffected in TRPL (W814G) oocytes. It is co ncluded 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) InsP(3) in a process that does not involve Ca2+ and calmodulin; and (d) a trimeric G-protein(s) thro ugh both a Ca2+/calmodulin-dependent and a Ca2+/calmodulin-independent mechanism.