Interaction between ion channels and membrane skeleton

膜骨架在许多生物学过程中起着关键作用，包括膜受体的动员、内吞作用、胞吞作用、细胞极性和形态、细胞黏附和膜脂代谢。Ca~(2+)&gt；对多种细胞的膜骨架有深刻的影响，必须存在Ca~(2+)&gt；对膜骨架蛋白的调控系统(S)。由于Ca~(2+)通过Ca~(2+)通道进入细胞，钙通道与膜骨架之间可能存在功能上的相互作用。在本研究中，我们重点研究了脑中的一种钙离子和磷脂结合蛋白--膜联蛋白VI，并研究了膜联蛋白VI是否参与了钙离子对膜骨架蛋白的调节。膜联蛋白VI以钙离子/磷脂依赖的方式与大鼠脑全匀浆中约14种蛋白质结合。在这些蛋白质中，有5个蛋白质在细胞骨架组分中被富含，其中一个被鉴定为钙调素(脑血影蛋白或Fodrin)。在天然状态下，当用纯化的Calspectin检测时，Annexin VI与Calspectin的结合也是钙依赖的。结合的Ca^-lt；2+-gt；亲和力约为20um。对膜联蛋白VI的亲和力约为270 nM。膜联蛋白VI与钙调蛋白的β亚基结合，但不与α亚基结合。当用低切变粘度法检测Annexin VI对F-肌动蛋白与Calspectin相互作用的影响时，Annexin VI以钙离子/磷脂依赖的方式抑制Calspectin的F-肌动蛋白交联活性。共降解实验表明，只有在钙离子和磷脂存在的情况下，Annexin VI才能将钙调蛋白从F-肌动蛋白中解离出来。这些结果表明，Annexin VI可以钙/磷脂依赖的方式在质膜下解离和重新分布Calspectin，并且Annexin VI可能调节神经细胞膜骨架对Ca~(2+)和gt~(2+)的响应。

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+>.