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

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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.