Photochemical localization of the semotiadil binding region within the cardiac Ca2+ channel alpha1 subunit. Comparison with the skeletal muscle counterpart.
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.
Ii
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Faculty of Pharmaceutical Sciences, Kumamoto University, Japan.
Kuniyasu
A
A
Kawahara
K
K
Shibano
T
T
Schwartz
A
A
Nakayama
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002121:Calcium Channel Blockers / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D017275:Isradipine / Q000493:pharmacokinetics; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D019920:Photoaffinity Labels; D010777:Photochemistry; D013552:Swine; D013843:Thiazines / Q000494:pharmacology; D013844:Thiazoles / Q000378:metabolism* / Q000494:pharmacology
D001386:Azides; D002121:Calcium Channel Blockers; D015220:Calcium Channels; C110875:D 51-4700; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D013843:Thiazines; D013844:Thiazoles; C064356:sesamodil; D017275:Isradipine