|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1992 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1991 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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+>.