Callamaras N, Parker I
Department of Psychobiology, University of California, Irvine.
Cell Calcium. 1994 Jan;15(1):66-78. doi: 10.1016/0143-4160(94)90105-8.
Inositol 1,4,5-trisphosphate receptors (InsP3R) in Xenopus laevis oocytes were localized and their regulation by Ca2+ was investigated. Antibodies raised against the C-terminal region of the mouse cerebellar InsP3R (cAb) cross-reacted with a 255 kD protein in Western blots of Xenopus microsomal membranes. Immunolocalization of this protein in cryosections of oocytes revealed diffuse staining of the cytoplasm, intense staining of the sub-plasma membrane region of the animal hemisphere, and punctate staining in association with the germinal vesicle. In the presence of 40 microM free Ca2+, isolated oocyte membranes exhibited a high affinity binding site for Ins 1,4,5-P3 (KD = 5nM) and a binding capacity of 450 fmol/mg protein. The specific binding capacity of oocyte membranes for [3H]-Ins 1,4,5-P3 increased as the level of free Ca2+ present in binding assays was raised from < 0.1 nM to 4.0 microM, with an apparent EC50 of 60 nM. Increasing the concentration of free Ba2+ failed to facilitate [3H]-Ins1,4,5-P3 binding. Other inositol phosphates competed for Ins1,4,5-P3 binding sites with approximate IC50 values of: Ins1,3,4,5-P4 = 79 nM, Ins2,4,5-P3 = 455 nM and L-Ins1,4,5-P3 = 20 microM. In addition, 150 micrograms/ml (approximately 12 microM) heparin displaced 50% of bound [3H]-Ins1,4,5-P3, whereas caffeine (10 mM) had little effect. Functional reconstitution of solubilized InsP3Rs into lipid bilayers revealed that Ca2+ was a necessary co-agonist for activation of the InsP3R. When InsP3 (5 microM) and Ca2+ (5 microM) were applied together, conductance steps were observed. InsP3 or Ca2+ alone had little effect. These results suggest that the subcellular organization of InsP3Rs and the facilitation of InsP3 binding and channel opening by Ca2+ contribute to the Ins1,4,5-P3-mediated Ca2+ spikes, waves, and oscillations observed in Xenopus oocytes.
研究了非洲爪蟾卵母细胞中肌醇1,4,5 -三磷酸受体（InsP3R）的定位及其受Ca2+的调节。针对小鼠小脑InsP3R C末端区域产生的抗体（cAb）在非洲爪蟾微粒体膜的蛋白质免疫印迹中与一种255 kD的蛋白质发生交叉反应。该蛋白质在卵母细胞冰冻切片中的免疫定位显示，细胞质呈弥漫性染色，动物半球的亚质膜区域染色强烈，且与生发泡相关的点状染色。在存在40 microM游离Ca2+的情况下，分离的卵母细胞膜对肌醇1,4,5 -三磷酸（Ins 1,4,5 -P3）表现出高亲和力结合位点（KD = 5nM），结合容量为450 fmol/mg蛋白质。随着结合测定中游离Ca2+水平从< 0.1 nM升高到4.0 microM，卵母细胞膜对[3H]-肌醇1,4,5 -三磷酸的特异性结合容量增加，表观EC50为60 nM。增加游离Ba2+的浓度未能促进[3H]-肌醇1,4,5 -三磷酸的结合。其他肌醇磷酸酯竞争肌醇1,4,5 -三磷酸结合位点，其近似IC50值为：肌醇1,3,4,5 -四磷酸（Ins1,3,4,5 -P4）= 79 nM，肌醇2,4,5 -三磷酸（Ins2,4,5 -P3）= 455 nM，L-肌醇1,4,5 -三磷酸（L-Ins1,4,5 -P3）= 20 microM。此外，150微克/毫升（约12 microM）肝素可置换50%结合的[3H]-肌醇1,4,5 -三磷酸，而咖啡因（10 mM）影响很小。将溶解的InsP3R功能重建到脂质双层中显示，Ca2+是激活InsP3R的必要协同激动剂。当同时施加肌醇1,4,5 -三磷酸（5 microM）和Ca2+（5 microM）时，观察到电导阶跃。单独的肌醇1,4,5 -三磷酸或Ca2+影响很小。这些结果表明，InsP3R的亚细胞组织以及Ca2+对肌醇1,4,5 -三磷酸结合和通道开放的促进作用有助于在非洲爪蟾卵母细胞中观察到的肌醇1,4,5 -三磷酸介导的Ca2+尖峰、波和振荡。
