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1C Subunit Involved in Ca-dependent Inactivation
Department of Cellular and Molecular Physiology, and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536
We investigated the molecular basis for Ca-dependent inactivation of the cardiac L-type Ca channel. Transfection of HEK293 cells with the wild-type
1C or its 3' deletion mutant (1C–3'del) produced channels that exhibited prominent Ca-dependent inactivation. To identify structural regions of 1C involved in this process, we analyzed chimeric 1 subunits in which one of the major intracellular domains of 1C was replaced by the corresponding region from the skeletal muscle 1S subunit (which lacks Ca-dependent inactivation). Replacing the NH2 terminus or the III–IV loop of 1C with its counterpart from 1S had no appreciable effect on Ca channel inactivation. In contrast, replacing the I–II loop of 1C with the corresponding region from 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 1C may participate in the mechanism of Ca-dependent inactivation. Replacing the final 80% of the COOH terminus of 1C with the corresponding region from 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 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 1C.
Key Words: 1S • skeletal muscle • L-type Ca channel • chimeric proteins • heart
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