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

电压门控L型Ca^<2+>通道控制着多种生理功能，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。当L型Ca^<2+>通道的细胞质侧用人工生理溶液灌注时，该通道的活性降低（流下现象）。其机理尚不清楚。我们以前曾报道过钙蛋白酶抑制蛋白（CS）和钙调素（CaM）都能在钙通道失活后恢复其活性。本研究探讨了钙调素和钙蛋白酶抑制素在钙通道激活和钙衰逆转中的相互关系及其作用机制。主要结果如下。（1）钙调素的逆转作用依赖于通道磷酸化状态。CaM激酶II保持通道处于可被CaM重新激活的状态。（2）CS的L结构域激活L型Ca^<2+>通道。通过对结构域L的短肽进行筛选，发现CS的有效区域位于结构域L的45-64位氨基酸残基。(3)To探讨CS和CaM的关系，采用GST融合蛋白下拉法检测这两种分子之间的结合。结果表明，CS和CaM在Ca^<2+>存在时相互结合.（4）研究了CS与Ca^2+通道的结合位点。CS可能与已经确定为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.