Identification of the benzothiazepine-binding polypeptide of skeletal muscle calcium channels with (+)-cis-azidodiltiazem and anti-ligand antibodies

Journal Article · Fri Jan 05 04:00:00 EST 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:7168417

Striessnig, J; Scheffauer, F; Mitterdorfer, J; Schirmer, M; Glossmann, H ^[1]

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1. Universitaet Innsbruck (Austria)

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.

• OSTI ID: 7168417
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:1; ISSN 0021-9258; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ANIMALS
BIOASSAY
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL COMPOSITION
ENZYME IMMUNOASSAY
HYDROGEN COMPOUNDS
IMMUNOASSAY
ISOTOPE APPLICATIONS
KINETICS
LIGANDS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR STRUCTURE
MUSCLES
ORGANIC COMPOUNDS
PORINS
PROTEINS
RABBITS
REACTION KINETICS
RECEPTORS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES