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

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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.