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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起关键作用，包括膜受体的动员、内吞作用、胞吐作用、细胞极性和形态、细胞粘附和膜脂质周转。Ca^<2+>对多种细胞的膜骨架有着深刻的影响，一定存在Ca^<2+>对膜骨架蛋白的调控系统。由于Ca^2+是通过Ca^2+通道进入细胞的，因此Ca^2+通道与细胞膜骨架之间可能存在功能性相互作用。在本研究中，我们集中在钙和磷脂结合蛋白，膜联蛋白VI，在大脑中，并检查是否膜联蛋白VI参与调节膜骨架蛋白的Ca^<2+>。膜联蛋白VI以Ca^2+/磷脂依赖性方式与大鼠全脑匀浆中的约14种蛋白质结合。其中，5种蛋白质在细胞骨架组分中富集，其中一种被鉴定为钙网蛋白（脑血影蛋白或胞衬蛋白）。当用天然状态的纯化钙光凝蛋白检测时，膜联蛋白VI与钙光凝蛋白的结合也是Ca^2+依赖性的。结合的Ca^2+亲和力（KCa）约为20 μ M。对膜联蛋白VI的亲和力（Kd）为约270 nM。膜联蛋白VI结合钙斑蛋白的β亚基，但不结合α亚基。当通过低剪切粘度法检测膜联蛋白VI对F-肌动蛋白和钙网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网蛋白的F-肌动蛋白交联活性。共沉降分析表明，膜联蛋白VI仅在Ca^2+和磷脂存在的情况下才能将钙斑蛋白从F-肌动蛋白上解离。这些结果表明，膜联蛋白VI可以在质膜下以Ca 2 +/磷脂依赖性的方式解离和重新分布钙spectin，并且膜联蛋白VI可以响应于Ca^<2+>调节神经细胞的膜骨架。