Calpastatin binds to a calmodulin-binding site of cardiac Cav1.2 Ca2+ channels

Calpastatin is an endogenous inhibitor of calpain and composed of domain L (CSL), which interacts with the Cav1.2 channels, and four repetitive calpain inhibitory domains. We have previously found that CSL reprimes activity of the Cav1.2 channels in cell-free patches of cardiac myocytes [L.Y. Hao, A. Kameyama, S. Kuroki, J. Takano, E. Takano, M. Maki, M. Kameyama, Calpastatin domain L is involved in the regulation L-type of Ca2+ channels in guinea pig cardiac myocytes, Biochem. Biophys. Res. Commun. 279 (2000) 756–761; E. Minobe, L.Y. Hao, Z.A. Saud, J.J. Xu, A. Kameyama, M. Maki, K.K. Jewell, T. Parr, R.G. Bardsley, M. Kameyama, A region of calpastatin domain L that reprimes cardiac L-type Ca2+channels, Biochem. Biophys. Res. Commun. 348 (2006) 288–294]. In this study, we explored the CSL interaction site in the Ca2+ channel by the pull-down method, using glutathione-S-transferase-fused fragment peptides of the Cav1.2 channel. CSL bound directly to a proximal region of the C-terminal tail of the channel, but not with the N-terminal tail, a distal region of the C-terminal tail or cytoplasmic loops between repeats I–II, II–III or III–IV. Furthermore IQ domain, but not EF-hand-like region or CB domain, in the C-terminal tail was found to bind with CSL in a partially Ca2+-dependent manner and in a probably competitive manner with calmodulin. These results suggest that CSL modulates Ca2+-channel activity through interacting with the calmodulin-binding site on the C-terminal tail of the Cav1.2 channel.