López Esther, Berna-Erro Alejandro, Salido Ginés M, Rosado Juan A, Redondo Pedro C
Department of Physiology Cellular Physiology Research Group, University of Extremadura, 10003 Cáceres, Spain.
Biochim Biophys Acta. 2013 Mar;1833(3):652-62. doi: 10.1016/j.bbamcr.2012.11.029. Epub 2012 Dec 8.
Immunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca(2+) channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca(2+) entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes (Ca(2+)), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca(2+) mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn(2+) entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP(3)R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets.
亲免素是一类能与FK506结合的蛋白质，它们通过调节位于质膜或糙面内质网（RE）中的Ca(2+)通道参与钙稳态的调节。我们研究了亲免素是否参与人血小板和MEG 01细胞中储存式钙内流（SOCE）的调节。两种细胞类型均用fura-2负载以测定胞质钙浓度变化（[Ca(2+)]c），或进行刺激和固定，通过免疫沉淀和蛋白质印迹法评估蛋白质相互作用谱。我们发现用FK506孵育人血小板会增加Ca(2+)的动员。用亲免素拮抗剂处理血小板后，毒胡萝卜素（TG）诱发的、苏氨酸诱发的SOCE以及TG诱发的Mn(2+)内流均显著减少。我们通过免疫沉淀证实亲免素与人血小板中的瞬时受体电位通道1（TRPC1）和Orai1相互作用。FK506和雷帕霉素减少了人血小板中TRPC1和Orai1与FK506结合蛋白（52）（FKBP52）之间的关联，以及TRPC1与II型IP(3)R之间的关联，已知这种关联对维持人血小板中的SOCE至关重要。如单细胞形态成像技术所示，在MEG 01细胞中使用SiRNA FKBP52沉默FKBP52证实了FKBP52在SOCE激活中的作用。同时沉默TRPC1以及使TRPC1和FKBP52细胞耗竭会损害MEG 01细胞中TG诱发的SOCE激活。最后，在与FK506孵育的MEG 01细胞中，我们观察到TRPC1/FKBP52偶联减少，同样，在SOCE期间，沉默FKBP52会减少II型IP3R与TRPC1之间的关联。总之，这些结果表明亲免素参与人血小板中SOCE的调节。
