Interaction between ion channels and membrane skeleton

离子通道与膜骨架之间的相互作用

基本信息

批准号：
03833019
负责人：
INUI Makoto
金额：
$ 1.15万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

The membrane skeleton plays a critical role in a number of biological processes including mobilization of membrane receptors, endocytosis, exocytosis, cell polarity and morphology, cell adhesion, and membrane lipid turnover. Ca^<2+> has profound effects on the membrane skeleton in a variety of cells, and there must exist regulation system(s) of the membrane skeletal proteins by Ca^<2+>. Since Ca^<2+> comes into cells through Ca^<2+> channels, there may be functional interaction between Ca^<2+> channels and the membrane skeleton. In the present study, we focused on a Ca^<2+> - and phospholipid-binding protein, annexin VI, in brain, and examined whether annexin VI is involved in the regulation of membrane skeletal proteins by Ca^<2+>. Annexin VI bound to about 14 proteins in rat brain whole homogenate in a Ca^<2+>/phospholipid-dependent manner. Of these, 5 proteins were enriched in the cytoskeletal fraction, and one of them was identified to be calspectin (brain spectrin or fodrin). When examined with purified calspectin in the native state, the binding of annexin VI to calspectin was also Ca^<2+> - dependent. The Ca^<2+> affinity of the binding (KCa) was about 20 muM. The affinity for annexin VI (Kd) was about 270 nM. Annexin VI bound to beta subunit of calspectin but not to alpha subunit. When the effect of annexin VI on the interaction between F-actin and calspectin was examined by low-shear viscometry, annexin VI inhibited the F-actin cross-linking activity of calspectin in a Ca^<2+>/phospholipid-dependent manner. Cosedimentation assay showed that annexin VI dissociates calspectin from F-actin only in the presence of Ca^<2+> and phospholipid. These results indicate that annexin VI can dissociate and redistribute calspectin in a Ca2+/phospholipid-dependent manner under the plasma membrane, and that annexin VI may regulate the membrane skeleton of neuronal cells in response to Ca^<2+>.

膜骨架在许多生物过程中发挥着关键作用，包括膜受体的动员、胞吞作用、胞吐作用、细胞极性和形态、细胞粘附和膜脂周转。 Ca^2+对多种细胞的膜骨架具有深远的影响，必然存在Ca^2+对膜骨架蛋白的调节系统。由于Ca^<2+>通过Ca^<2+>通道进入细胞，因此Ca^<2+>通道与膜骨架之间可能存在功能性相互作用。在本研究中，我们关注大脑中的Ca^2+和磷脂结合蛋白膜联蛋白VI，并检查膜联蛋白VI是否参与Ca^2+对膜骨骼蛋白的调节。膜联蛋白VI以Ca 2+ /磷脂依赖性方式与大鼠脑全匀浆中的约14种蛋白质结合。其中，5 种蛋白质在细胞骨架部分中富集，其中一种被鉴定为钙血影蛋白（脑血影蛋白或胞质蛋白）。当用天然状态下的纯化钙观蛋白进行检查时，膜联蛋白VI与钙观蛋白的结合也是Ca 2+ 依赖性的。结合的Ca 2+ 亲和力(KCa)为约20μM。对膜联蛋白 VI (Kd) 的亲和力约为 270 nM。膜联蛋白 VI 与 calspectin 的 β 亚基结合，但不与 α 亚基结合。当通过低剪切粘度测定法检查膜联蛋白VI对F-肌动蛋白和钙观蛋白之间相互作用的影响时，膜联蛋白VI以Ca 2+ /磷脂依赖性方式抑制钙观蛋白的F-肌动蛋白交联活性。共沉淀测定表明，仅在Ca 2+ 和磷脂存在的情况下，膜联蛋白VI才将钙高蛋白与F-肌动蛋白解离。这些结果表明，膜联蛋白VI可以在质膜下以Ca 2+ /磷脂依赖性方式解离和重新分布calspectin，并且膜联蛋白VI可以响应于Ca 2+ 调节神经元细胞的膜骨架。