Identification of the benzothiazepine-binding polypeptide of skeletal muscle calcium channels with (+)-cis-azidodiltiazem and anti-ligand antibodies.
The purified dihydropyridine-sensitive calcium channel from skeletal muscle transverse tubules consists of several subunits, termed alpha 1, alpha 2, beta, gamma and delta. From its associated drug receptors, those for 1,4-dihydropyridines and phenylalkylamines have been shown previously by photoaffinity labeling to reside on the alpha 1 subunit. In the present study the arylazide photo-affinity ligand, (+)-cis-azidodiltiazem ((+)-cis-(2S,3S)-5-[2-(4- azidobenzoyl)aminoethyl]-2,3,4,5-tetrahydro-3-hydroxy-2-(4-methoxyphenyl )-4- oxo-1,5-benzothiazepine), and the respective tritiated derivative, (+)-cis-[3H]azidodiltiazem (45 Ci/mmol), were developed to identify directly the benzothiazepine binding subunit. (+)-cis-Azidodiltiazem binds competitively to the benzothiazepine receptor in rabbit skeletal muscle transverse tubule membranes. Upon ultraviolet irradiation of the (+)-cis-[3H]azidodiltiazem-purified calcium channel complex, the ligand photoincorporates exclusively into the alpha 1 subunit. Photoincorporation is protected by 100 microM (-)-desmethoxyverapamil and 100 microM (+)-cis-diltiazem. A polyclonal antiserum directed against (+)-cis-azidodiltiazem was employed to detect (+)-cis-azidodiltiazem immunoreactivity photoincorporated into the purified calcium channel complex, confirming the exclusive labeling of the alpha 1 subunit. Our data provide direct evidence that, together with the drug receptors for 1,4-dihydropyridines and phenylalkylamines, the benzothiazepine binding domain of skeletal muscle calcium channels is located on the alpha 1 subunit. We conclude that our anti-ligand antibodies could be used successfully to affinity purify the photolabeled proteolytic fragments of the alpha 1 subunit which are expected to form part of the benzothiazepine binding domain.
Striessnig
J
J
Institut für Biochemische Pharmacologie, Universität Innsbruck, Austria.
Scheffauer
F
F
Mitterdorfer
J
J
Schirmer
M
M
Glossmann
H
H
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D015220:Calcium Channels / Q000032:analysis* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D046911:Macromolecular Substances; D009132:Muscles / Q000032:analysis*; D010777:Photochemistry; D011817:Rabbits; D011955:Receptors, Drug / Q000032:analysis* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism; D013841:Thiazepines / Q000378:metabolism; D014316:Tritium
D000345:Affinity Labels; D001386:Azides; D015220:Calcium Channels; D046911:Macromolecular Substances; D011955:Receptors, Drug; D011978:Receptors, Nicotinic; D013841:Thiazepines; C062217:benzothiazepine receptor; D014316:Tritium; C060117:azidodiltiazem