Lopez Esther, Berna-Erro Alejandro, Salido Gines M, Rosado Juan A, Redondo Pedro C
Department of Physiology, Cell Physiology Research Group, University of Extremadura, 10003 Cáceres, Spain.
Department of Physiology, Cell Physiology Research Group, University of Extremadura, 10003 Cáceres, Spain.
Biochim Biophys Acta. 2015 Oct;1853(10 Pt A):2684-96. doi: 10.1016/j.bbamcr.2015.07.023. Epub 2015 Aug 1.
Non-capacitative calcium entry (NCCE) contributes to cell activation in response to the occupation of G protein-coupled membrane receptors. Thrombin administration to platelets evokes the synthesis of diacylglycerol downstream of PAR receptor activation. Diacylglycerol evokes NCCE through activating TRPC3 and TRPC6 in human platelets. Although it is known that immunophilins interact with TRPCs, the role of immunophilins in the regulation of NCCE remains unknown. Platelet incubation with FK506, an immunophilin antagonist, reduced OAG-evoked NCCE in a concentration-dependent manner, an effect that was independent on the inactivation of calcineurin (CaN). FK506 was unable to reduce NCCE evoked by OAG in platelets from TRPC6-/- mice. In HEK-293 cells overexpressing TRPC6, currents through TRPC6 were altered in the presence of FK506. We have found interaction between FKBP38 and other FKBPs, like FKBP25, FKBP12, and FKBP52 that were not affected by FK506, as well as with calmodulin (CaM). FK506 modified the pattern of association between FKBP25 and TRPCs as well as impaired OAG-evoked TRPC3 and TRPC6 coupling in both human and mouse platelets. By performing biotinylation experiments we have elucidated that FKBP25 and FKBP38 might be found at different cellular location, the plasma membrane and the already described intracellular locations. Finally, FKBP25 and FKBP38 silencing significantly inhibits OAG-evoked NCCE in MEG-01 and HEK293 cells, while overexpression of FKBP38 does not modify NCCE in HEK293 cells. All together, these findings provide strong evidence for a role of immunophilins, including FKBP25 and FKBP38, in NCCE mediated by TRPC6.
非容量性钙内流（NCCE）有助于细胞在G蛋白偶联膜受体被占据时的激活。向血小板中注入凝血酶会在PAR受体激活的下游引发二酰甘油的合成。二酰甘油通过激活人血小板中的TRPC3和TRPC6来引发NCCE。尽管已知免疫亲和素与TRPC相互作用，但免疫亲和素在NCCE调节中的作用仍不清楚。用免疫亲和素拮抗剂FK506孵育血小板，以浓度依赖性方式降低了OAG引发的NCCE，该效应与钙调神经磷酸酶（CaN）的失活无关。FK506无法降低TRPC6基因敲除小鼠血小板中OAG引发的NCCE。在过表达TRPC6的HEK-293细胞中，FK506存在时通过TRPC6的电流发生了改变。我们发现FKBP38与其他FKBPs（如不受FK506影响的FKBP25、FKBP12和FKBP52）以及钙调蛋白（CaM）之间存在相互作用。FK506改变了FKBP25与TRPCs之间的结合模式，并损害了人和小鼠血小板中OAG引发的TRPC3和TRPC6偶联。通过进行生物素化实验，我们阐明了FKBP25和FKBP38可能存在于不同的细胞位置，即质膜和已描述的细胞内位置。最后，FKBP25和FKBP38基因沉默显著抑制了MEG-01和HEK293细胞中OAG引发的NCCE，而FKBP38的过表达并未改变HEK293细胞中的NCCE。总之，这些发现为包括FKBP25和FKBP38在内的免疫亲和素在由TRPC6介导的NCCE中的作用提供了有力证据。
