Structural Regions of the Cardiac Ca Channel α_1C 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 α_1C or its 3′ deletion mutant (α_1C−3′del) produced channels that exhibited prominent Ca-dependent inactivation. To identify structural regions of α_1C involved in this process, we analyzed chimeric α₁ subunits in which one of the major intracellular domains of α_1C was replaced by the corresponding region from the skeletal muscle α_1S subunit (which lacks Ca-dependent inactivation). Replacing the NH₂ terminus or the III–IV loop of α_1C with its counterpart from α_1S had no appreciable effect on Ca channel inactivation. In contrast, replacing the I–II loop of α_1C with the corresponding region from α_1S 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 α_1C may participate in the mechanism of Ca-dependent inactivation. Replacing the final 80% of the COOH terminus of α_1C with the corresponding region from α_1S 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 α_1S 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 α_1C.