Transient receptor potential protein subunit assembly and membrane distribution in human platelets
Transient receptor potential protein subunit assembly and membrane distribution in human platelets
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DOI：
10.1160/th05-06-0391
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发表时间：
2005-10-01
影响因子：
6.7
通讯作者：
Sage, SO
中科院分区：
文献类型：
--
作者：
Brownlow, SL;Sage, SO
我们之前曾提出，果蝇瞬时受体电位蛋白 TRPC 1 的人类同源物参与了人类血小板中钙库操纵的 Ca2+ 内流 (SOCE)，因为针对 TRPC I 成孔区的抗体可抑制 SOCE。在这里，我们研究了人血小板中 TRPC I 的质膜表达，并探讨了这些细胞中是否存在其他 TRPC 蛋白。生物素化揭示了静息血小板质膜中存在 TRPC I。 Ca2+ 储备耗尽或凝血酶刺激后，表面表达未发生明显变化。蛋白质印迹证明血小板裂解物中存在 TRPC1、TRPC3、TRPC4、TRPC5 和 TRPC6。 TRPC1、TRPC4 和 TRPC5 发生免疫共沉淀，TRPC3 和 TRPC6 也发生免疫共沉淀。 TRPC 1、TRPC4 和 TRPC5 与耐去垢剂的血小板膜相关，当使用甲基-β-环糊精去除细胞中的胆固醇时，它们会从血小板膜上部分释放。 TRPC3 和 TRPC6 在可溶部分和膜部分之间的分布不受甲基-β-环糊精处理的影响。这些结果表明TRPC 1、TRPC4和TRPC5形成与血小板脂筏结构域相关的异多聚体，而TRPC3和TRPC6独立于脂筏相关联。
We have previously suggested that the human homologue of the Drosophila transient receptor potential protein, TRPC 1, is involved in conducting store-operated Ca2+ entry (SOCE) in human platelets since an antibody raised against the pore-forming region of TRPC I inhibited SOCE. Here we have investigated plasma membrane expression of TRPC I in human platelets and have probed for the presence of other TRPC proteins in these cells. Biotinylation revealed the presence of TRPC I in the plasma membrane of resting platelets. Surface expression was not detectibly changed following Ca2+ store depletion or stimulation with thrombin. Western blotting demonstrated the presence of TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 in platelet lysates. TRPC1,TRPC4 and TRPC5 coimmunoprecipitated, as did TRPC3 and TRPC6. TRPC 1, TRPC4 and TRPC5 were associated with detergent-resistant platelet membranes, from which they were partially released when the cells were cholesterol-depleted using methyl-beta-cyclodextrin. The distributions of TRPC3 and TRPC6 between soluble and membrane fractions were not affected by methyl-beta-cyclodextrin treatment. These results suggest that TRPC 1,TRPC4 and TRPC5 form a heteromultimer associated with platelet lipid raft domains, whereas TRPC3 and TRPC6 associate independently of lipid rafts.