Photochemical localization of the semotiadil binding region within the cardiac Ca2+ channel alpha1 subunit. Comparison with the skeletal muscle counterpart

Abstract

We have previously identified the binding region of a new Ca2+ antagonist semotiadil in the skeletal muscle Ca2+ channel. To the same semotiadil derivatives, the cardiac counterpart showed distinct and different binding characteristics: semotiadil and its photoaffinity analog D51-4700 inhibited [3H]PN200-110 binding to cardiac membrane preparations with IC50 values of 13-20 microM, which are 10 times higher than those in skeletal muscle. Hill slopes of the binding inhibition were 0.94-1.0 for the cardiac channels compared to 0.63-0.67 for the skeletal muscle channels. A possible explanation for the difference is that the semotiadil binding site is differently conferred in cardiac and skeletal muscle Ca2+ channels. To reveal this within the primary structure, photoaffinity labeling of cardiac membranes was employed. [3H]D51-4700 was photo-incorporated in several polypeptides but only the alpha1 subunit of the Ca2+ channel was photolabeled in a specific manner. Antibody mapping of the [3H]D51-4700-labeled alpha1 subunit with several anti-peptide antibodies revealed that the labeled site was located solely in a peptide fragment between Cys1461 and Lys1529. This region encompasses the labeled site of skeletal muscle, but contains several non-identical amino acid residues, which may participate in expressing different binding characteristics between the two muscle type Ca2+ channels.