Jardín Isaac, López José J, Salido Ginés M, Rosado Juan A
Department of Physiology (Cellular Physiology Research Group), University of Extremadura, 10071 Cáceres, Spain.
Cell Signal. 2008 Apr;20(4):737-47. doi: 10.1016/j.cellsig.2007.12.010. Epub 2007 Dec 23.
Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is regulated by the filling state of the intracellular Ca2+ stores. We have previously reported that a de novo conformational coupling between the type II IP3 receptor (IP3RII) and hTRPC1 channel occurs after depletion of the intracellular Ca2+ stores in human platelets, which might be involved in the activation of SOCE in these cells. Here we present for the first time direct evidence for the functional relevance of the coupling between hTRPC1 and IP3RII in SOCE in human platelets. Our data suggest that at least two pathways may contribute to SOCE in these cells. An early component, insensitive to cytochalasin D (Cyt D), is followed by a late component which is sensitive to Cyt D. Introduction of a peptide corresponding to IP3RII(317-334) (IP3BD-peptide(317-334)) in the cells by electrotransjection impairs the coupling between hTRPC1 and IP3RII but not the interaction between hTRPC1 and STIM1 induced by store depletion. Coimmunoprecipitation experiments indicated that endogenously expressed hTRPC1 interacts with the IP3BD-peptide(317-334). Electrotransjection of cells with IP3BD-peptide(317-334), significantly attenuated the late stage of Ca2+ and Mn2+ entry induced by 10 nM thapsigargin (TG) or 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ), providing evidence for a functional role of the de novo coupling between hTRPC1 and IP3RII in the activation of SOCE in human platelets.
储存性钙内流（SOCE）是非兴奋性细胞中钙内流的主要机制，受细胞内钙库的充盈状态调节。我们之前报道过，在人血小板细胞内钙库耗竭后，II型肌醇三磷酸受体（IP3RII）与hTRPC1通道之间会发生全新的构象偶联，这可能参与了这些细胞中SOCE的激活。在此，我们首次提供了hTRPC1与IP3RII之间的偶联在人血小板SOCE中功能相关性的直接证据。我们的数据表明，至少有两条途径可能促成这些细胞中的SOCE。一个早期成分对细胞松弛素D（Cyt D）不敏感，随后是一个对Cyt D敏感的晚期成分。通过电转染将对应于IP3RII(317 - 334)的肽（IP3BD - 肽(317 - 334)）导入细胞，会损害hTRPC1与IP3RII之间的偶联，但不会损害由钙库耗竭诱导的hTRPC1与基质相互作用分子1（STIM1）之间的相互作用。免疫共沉淀实验表明，内源性表达的hTRPC1与IP3BD - 肽(317 - 334)相互作用。用IP3BD - 肽(317 - 334)电转染细胞，显著减弱了由10 nM毒胡萝卜素（TG）或20 μM 2,5 - 二 - （叔丁基）- 1,4 - 对苯二酚（TBHQ）诱导的钙和锰内流的晚期阶段，为hTRPC1与IP3RII之间全新偶联在人血小板SOCE激活中的功能作用提供了证据。
