Harper Alan G S, Sage Stewart O
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK.
Cell Calcium. 2007 Dec;42(6):606-17. doi: 10.1016/j.ceca.2007.02.004. Epub 2007 Mar 23.
We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP(3) receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.
我们之前已经证明，肌动蛋白细胞骨架的重组在人血小板的储存-操作性钙内流（SOCE）中发挥作用，并将此解释为SOCE激活过程中涉及II型IP(3)受体和含血小板hTRPC1的储存-操作性通道（SOC）的从头构象偶联步骤的证据。在此，我们提出了挑战该模型的证据。肌动蛋白聚合抑制剂细胞松弛素D或拉特罗毒素A显著降低了Ca2+进入经毒胡萝卜素（TG）处理的血小板，但未降低Mn2+或Na+的进入。诱导肌动蛋白聚合的茉莉酮酸甲酯也抑制了Ca2+的进入，但未抑制Mn2+或Na+的进入。然而，抗hTRPC1抗体抑制了TG诱发的所有三种阳离子的进入，表明它们都通过含hTRPC1的储存-操作性通道（SOC）渗透。这些结果表明，肌动蛋白细胞骨架的重组不参与SOC的激活。Na+/Ca2+交换体（NCX）的抑制剂KB-R7943或SN-6对Ca2+进入经TG处理的血小板产生剂量依赖性抑制，但对Mn2+或Na+的进入没有抑制作用。NCX抑制剂的作用与肌动蛋白聚合抑制剂的作用没有相加性，提示存在共同的作用点。这些结果表明，在人血小板中，Ca2+储存耗竭后激活了两条Ca2+可渗透途径：一条是Ca2+可渗透的、含hTRPC1的SOC，另一条是反向Na+/Ca2+交换，它在Na+通过SOC进入后被激活，并且需要功能性的肌动蛋白细胞骨架。
