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+>.

膜骨架在许多生物学过程中起着至关重要的作用，包括动员膜受体，内吞作用，胞吞作用，细胞极性和形态，细胞粘附和膜脂质转移。 Ca^<2+>对各种细胞中的膜骨骼具有深远的影响，并且必须通过Ca^<2+>进行膜骨骼蛋白的调节系统。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相互作用。在本研究中，我们专注于脑中的Ca^<2+> - 和磷脂结合蛋白，膜联蛋白VI，并检查了通过Ca^<2+>通过Ca^<2+>调节膜联蛋白VI是否参与了膜骨骼蛋白的调节。与磷脂依赖性的方式，膜联蛋白VI以大鼠大脑全匀浆的大约14种蛋白质结合。其中，将5种蛋白质富集在细胞骨架级分中，其中一种被确定为Calspectin（脑谱蛋白或禽类蛋白）。当用纯化的calspectin在本地状态检查时，膜联蛋白VI与calspectin的结合也受到ca^<2+> - 依赖性。结合（KCA）的Ca^<2+>亲和力约为20妈妈。对膜联蛋白VI（KD）的亲和力约为270 nm。与Calspectin的Beta亚基绑定，但不与Alpha亚基相结合。当通过低剪切粘膜测定法检查膜联蛋白VI对F-肌动蛋白和Calspectin之间的相互作用的影响时，膜联蛋白VI抑制了CA^<2+>/磷脂依赖性方式中Calspectin的F-肌动交联活性。 COSESIONTAINS分析表明，仅在Ca^<2+>和磷脂的情况下，膜联蛋白VI仅在F-肌动蛋白中解离Calspeciatin。这些结果表明，膜联蛋白VI可以在质膜下以Ca2+/磷脂依赖性方式解离和重新分布Calspectin，并且膜联蛋白VI可以调节神经元细胞的膜骨架，以响应于Ca^<2+>。