Study on the new regulatory mechanisms of L-type calcium channels

l型钙通道在神经和肌肉组织中起着重要的作用。虽然通过ca介导的失活、A激酶和/或C激酶的磷酸化以及G蛋白的直接抑制/刺激作用，其调控机制已被报道，但仍有许多工作要做。我们以前发现无细胞系统中心脏钙通道的衰竭是由细胞质因子和ATP逆转的。本研究是为了验证我们的假设，即Ca通道是由未确定的细胞质因子调节的。通过凝胶过滤，细胞质似乎含有至少两种因子（P因子和H因子）。通过电泳、免疫学和电生理实验，确定P因子为内源性钙蛋白酶抑制剂calpastatin （CS）。虽然我们已经在离子交换色谱和胰蛋白酶和磷脂酶敏感性中阐明了H因子的性质，但其鉴定仍有待完成。我们还研究了负责Ca通道调节的CS区域。CS由n端L结构域（功能未知）和侧翼4个同源calpain抑制结构域（D1-D4）组成。L结构域，而非D1-D4结构域，部分逆转了Ca通道的运行，表明L结构域负责通道调节。在其他实验中，也发现细胞质因子的作用似乎不涉及A激酶介导的通道磷酸化。这些结果支持了我们的假设，即Ca通道受细胞质调节蛋白的调节。

L-type Ca channel plays important roles in nerve and muscle tissues. Althrough Ca-mediated inactivation, phosphorylation by A kinas and/or C kinase and direct inhitory/stimulatory action of G proteins are reported for its regulatory mechanisms, much work remains to be done. We have previously found that run-down of cardiac Ca channel in cell-free systems is reversed by cytoplasmic factor(s) and ATP.This study is carried out to evaluate our hypothesis that Ca channel is regulated by unindentified cytoplasmic factors. By gel filtration, cytoplasm seems to contain at least two factors (P factor and H factor). The P factor is identified as calpastatin (CS), the endogenous calpain inhibitor, based on the experiments of eletrophoresis, immunology and electro-physiology. Althrough we have clarified properties of H factor in ion-exchange chromatography and trypsin and phospholipase sensitivity, its idenfication remains to be done. We have also examined the region of CS responsible for Ca channel regulation. The CS consists of N-terminal L domain (function unknown) and flanking 4 homologous calpain inhibitory domains (D1-D4). The L domain, but not D1-D4 domain, partially reversed run-down of Ca channel, suggesting L domain to be responsible for the channel regulation. In other experiments, it is also found that the action of the cytoplasmic factors does not seem to involve channel phosphorylation mediated by A kinase.These results support our hypothesis that Ca channel is regulated by cytoplasmic regulatory proteins.