Structural Regions of the Cardiac Ca Channel α1C Subunit Involved in Ca-dependent Inactivation

We investigated the molecular basis for Ca-dependent inactivation of the cardiac L-type Ca channel. Transfection of HEK293 cells with the wild-type α1C or its 3′ deletion mutant (α1C−3′del) produced channels that exhibited prominent Ca-dependent inactivation. To identify structural regions of α1C involved in this process, we analyzed chimeric α1 subunits in which one of the major intracellular domains of α1C was replaced by the corresponding region from the skeletal muscle α1S subunit (which lacks Ca-dependent inactivation). Replacing the NH2 terminus or the III–IV loop of α1C with its counterpart from α1S had no appreciable effect on Ca channel inactivation. In contrast, replacing the I–II loop of α1C with the corresponding region from α1S dramatically slowed the inactivation of Ba currents while preserving Ca-dependent inactivation. A similar but less pronounced result was obtained with a II–III loop chimera. These results suggest that the I–II and II–III loops of α1C may participate in the mechanism of Ca-dependent inactivation. Replacing the final 80% of the COOH terminus of α1C with the corresponding region from α1S completely eliminated Ca-dependent inactivation without affecting inactivation of Ba currents. Significantly, Ca-dependent inactivation was restored to this chimera by deleting a nonconserved, 211–amino acid segment from the end of the COOH terminus. These results suggest that the distal COOH terminus of α1S can block Ca-dependent inactivation, possibly by interacting with other proteins or other regions of the Ca channel. Our findings suggest that structural determinants of Ca-dependent inactivation are distributed among several major cytoplasmic domains of α1C.