The roles of calpastatin and calmodulin in the regulation of calcium channel

电压门控型L钙通道控制多种生理功能，包括基因转录、节律性放电、突触传递、激素分泌和兴奋-收缩偶联。L型钙离子通道在胞质侧灌流人工生理溶液时，其活性降低(衰弱现象)。这种衰败的机制目前还不清楚。我们以前报道过钙调蛋白(CaM)和钙调蛋白(CS)都能在钙离子通道衰竭后恢复钙通道活动。本研究探讨了钙调素和钙调蛋白在钙通道激活和钙拮抗过程中的相互关系和作用机制。主要研究结果如下。(1)CaM的逆转作用依赖于通道的磷酸化状态。CaM激酶II使通道处于一种可以被CaM重新激活的状态。(2)CS的L域激活L型钙通道。通过筛选L结构域的短肽，我们发现CS的有效区域位于L结构域的45-氨基酸残基。(3)为了研究CS和CaM的关系，用GST-融合蛋白下拉实验检测了CS和CaM分子之间的结合。结果表明，在Ca~(2+)和Gt~(2+)存在下，CS与CaM相互结合。(4)研究了CS与Ca~(2+)&gt~(2+)通道的结合部位。CS可能与已确认为CaM结合区的区域结合。

Voltage-gated L-type Ca^<2+> channels control diverse physiological functions including gene transcription, rhythmic firing, synaptic transmission, hormone secretion and excitation-contraction coupling. Activity of L-type Ca^<2+> channel decreases when the cytoplasmic side of the channel is perfused with an artificial physiological solution (run-down phenomenon). The mechanism of run-down is still unknown. We have previously reported that both calpastatin(CS) and calmodulin(CaM) can recover Ca^<2+> channel activity after run-down. In this study, we have investigated the relations and the mechanisms of calmodulin and calpastatin in the activation of Ca^<2+> channel and the reversion of run-down. The main results are as follows. (1)The run-down reversing effect of CaM depends on channel phosphorylation state. CaM kinase II keeps the channel in a state that can be reactivated by CaM. (2)Domain L of CS reactivates the L-type Ca^<2+> channel. By screening the short peptides of domain L, we found that the effective region of CS is located in the amino acid residues 45-64 of domain L. (3)To investigate the relations of CS and CaM, binding between these two molecules were examined with GST-fusion protein pull-down assay. The result shows that CS and CaM bind each other in the presence of Ca^<2+>. (4)Binding sites of CS to Ca^<2+> channel were also investigated. CS might bind to the region that has already confirmed as CaM binding region.