Neylon C B, Nickashin A, Tkachuk V A, Bobik A
Baker Medical Research Institute, Prahran, Victoria, Australia.
Cell Calcium. 1998 May;23(5):281-9. doi: 10.1016/s0143-4160(98)90024-0.
In vascular smooth muscle, pertussis toxin (PT) inhibits thrombin-induced Ca2+ release by a mechanism independent of its effect on IP3 formation. Thus, the possibility of a direct role of G alpha i proteins in regulating IP3-sensitive Ca2+ release was investigated by examining whether G alpha i proteins are associated with the IP3 receptor complex. Purified microsomal membranes were prepared and separated by sucrose density gradient centrifugation. The relative density of [3H]-IP3 binding sites between the microsomal fractions was inversely related to the distribution of the plasma membrane marker. The relative distribution of G alpha i3 determined by immunoblotting was closely correlated with the density of [3H]-IP3 binding. Levels of G alpha i2 were more evenly distributed with highest levels present in plasma membrane-enriched fractions. IP3 receptor immunoprecipitated from triton-solubilized microsomal membranes contained G alpha i3 immunoreactivity. To determine whether G alpha i proteins influence IP3-induced Ca2+ release, the effect of PT on Ca2+ release from digitonin-permeabilized cell suspensions using Fluo-3 was examined. Exposure to PT (0.1 microgram/ml, 5 min) attenuated the initial rate of IP3 (1 microM)-induced Ca2+ release. Together, these findings are consistent with the hypothesis that a heterotrimeric G alpha i protein directly regulates IP3-dependent Ca2+ release.
在血管平滑肌中，百日咳毒素（PT）通过一种独立于其对肌醇三磷酸（IP3）形成影响的机制，抑制凝血酶诱导的钙离子（Ca2+）释放。因此，通过检测Gαi蛋白是否与IP3受体复合物相关联，研究了Gαi蛋白在调节IP3敏感性Ca2+释放中直接发挥作用的可能性。制备纯化的微粒体膜，并通过蔗糖密度梯度离心进行分离。微粒体组分之间[3H]-IP3结合位点的相对密度与质膜标记物的分布呈负相关。通过免疫印迹法测定的Gαi3的相对分布与[3H]-IP3结合的密度密切相关。Gαi2的水平分布更为均匀，在富含质膜的组分中含量最高。从曲拉通溶解的微粒体膜中免疫沉淀的IP3受体含有Gαi3免疫反应性。为了确定Gαi蛋白是否影响IP3诱导的Ca2+释放，使用Fluo-3检测了PT对洋地黄皂苷通透的细胞悬液中Ca2+释放的影响。暴露于PT（0.1微克/毫升，5分钟）会减弱IP3（1微摩尔）诱导的Ca2+释放的初始速率。总之，这些发现与异源三聚体Gαi蛋白直接调节IP3依赖性Ca2+释放的假说一致。
