Lan L, Brereton H, Barritt G J
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, G.P.O. Box 2100, Adelaide, South Australia, 5001, Australia.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1149-58. doi: 10.1042/bj3301149.
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 muM 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 GTPgammaS 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.
研究了钙调蛋白结合位点在果蝇TRPL（类瞬时受体电位）非特异性Ca2+通道受Ca2+、肌醇1,4,5-三磷酸（InsP3）和GTP结合调节蛋白（G蛋白）调控中的作用。野生型TRPL蛋白以及两种突变形式TRPL（W713G）和TRPL（W814G）（其中两个假定的钙调蛋白结合位点[分别为位点1和位点2]中的每个位点的关键色氨酸残基被甘氨酸取代）在非洲爪蟾卵母细胞中进行了异源表达。免疫荧光研究表明，表达的TRPL、TRPL（W713G）和TRPL（W814G）蛋白位于质膜上。TRPL卵母细胞（注射了trpl cRNA的卵母细胞）和TRPL（W814G）卵母细胞[注射了trpl（W814G）cRNA的卵母细胞]表现出比模拟注射卵母细胞（模拟卵母细胞）更高的基础（组成性）Ca2+内流速率（使用fluo-3和Ca2+回补方案测量）。在TRPL（W713G）卵母细胞中，这种差异消失了。在TRPL和TRPL（W814G）[注射了trpl（W713G）cRNA的卵母细胞]中，但不在TRPL（W713G）卵母细胞中，基础Ca2+内流受到钙调蛋白作用抑制剂W13的抑制。钙调蛋白（细胞内3 μM）抑制TRPL中的基础Ca2+内流，但不抑制TRPL（W713G）或TRPL（W814G）卵母细胞中的基础Ca2+内流。蛋白激酶C（PKC）抑制剂星形孢菌素抑制，而PKC激活剂佛波酯（PMA）刺激TRPL卵母细胞中的基础Ca2+内流。在存在PMA（以抑制通过内源性受体激活的Ca2+通道的Ca2+内流）孵育的卵母细胞中，InsP3诱导的通过TRPL通道的Ca2+内流刺激比在不存在PMA孵育的卵母细胞中更明显。InsP3在TRPL中引起显著的Mn2+内流刺激，但在模拟卵母细胞中未引起。通过TRPL、TRPL（W713G）和TRPL（W814G）通道的InsP3刺激的Ca2+内流速率相似。在TRPL（W713G）卵母细胞中，GTPγS刺激通过TRPL通道的Ca2+内流的能力被抑制了50%，但在TRPL（W814G）卵母细胞中未受影响。得出的结论是，在非洲爪蟾卵母细胞的环境中，果蝇TRPL通道通过以下方式被激活：（a）在钙调蛋白结合位点1与Ca2+/钙调蛋白相互作用；（b）PKC；（c）在不涉及Ca2+和钙调蛋白的过程中的InsP3；以及（d）通过Ca2+/钙调蛋白依赖性和Ca2+/钙调蛋白非依赖性机制的三聚体G蛋白。
