Characterization of the receptor for the 1,4-dihydropyridine Ca sup 2+ channel modulators in skeletal muscle

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1989

OSTI ID:5545480

Leung, A T

The 1,4-dihydropyridine receptor was purified in high yield from isolated rabbit skeletal muscle triads. The purified receptor contains four integral subunits: {alpha}{sub 1} (170 kDa), {alpha}{sub 2} (175/150 kDa), {beta} (52 kDa) and {gamma} (32 kDa) in a 1:1:1:1 stoichiometry. The {alpha}{sub 2} subunit is a glycoprotein that binds wheat germ agglutinin and its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels is increased upon reduction whereas the {alpha}{sub 1} subunit does not exhibit these properties but is recognized by specific monoclonal antibodies. These monoclonal antibodies to the {alpha}{sub 1} subunit, as well as a specific monoclonal antibody to the {beta} subunit, are capable of immunoprecipitating dihydropyridine binding activity from digitonin-solubilized skeletal muscle triads as well as co-immunoprecipitating the {alpha}{sub 1} and {beta} subunits. The {alpha}{sub 1} and {beta} subunits are highly labile to mild trypsin digestion in isolated triads but the dihydropyridine binding activity of the receptor does not depend on an intact {alpha}{sub 1} (dihydropyridine binding) subunit. The {alpha}{sub 2} and {gamma} subunits are resistant to trypsin digestion and the proteolytic fragments of the {alpha} subunit remain associated with the {alpha}{sub 2} subunit even after solubilization of the receptor with digitonin. A binding site for the dihydropyridines is proposed based on the identification of a 28 kDa peptide that is both photoaffinity labeled with ({sup 3}H)azidopine and contains the epitope of a specific monoclonal antibody. Polyclonal antibodies raised against a synthetic peptide corresponding to the deduced carboxyl-terminus of the {alpha}{sub 1} subunit of the receptor did not react with the purified dihydropyridine receptor and skeletal muscle membranes from various sources.

• Research Organization: Iowa Univ., Iowa City, IA (United States)
• OSTI ID: 5545480
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ANTIBODIES
AZINES
BIOCHEMICAL REACTION KINETICS
CHEMICAL COMPOSITION
ELECTROPHORESIS
FRACTIONATION
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
MAMMALS
MEMBRANE PROTEINS
MONOCLONAL ANTIBODIES
MUSCLES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
PYRIDINES
RABBITS
REACTION KINETICS
RECEPTORS
SEPARATION PROCESSES
STOICHIOMETRY
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES