Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit.
The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (alpha 1), 175,000 Da (alpha 2), 52,000 Da (beta), and 32,000 Da (gamma) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Subunit-specific polyclonal antibodies have now been prepared and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. Immunoblot analysis of fractions collected during purification of the dihydropyridine receptor shows that the 32,000-Da polypeptide copurified with alpha 1 and alpha 2 subunits at each step of the purification. In addition, monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex which includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. In addition, polyclonal antibodies against the rabbit skeletal muscle 32,000-Da polypeptide specifically react with similar proteins in skeletal muscle of other species including avian and amphibian species. Thus, our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.
Sharp
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AH
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242.
Campbell
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KP
D000818:Animals; D000906:Antibodies; D001921:Brain / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels; D002846:Chromatography, Affinity; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D046911:Macromolecular Substances; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D015388:Organelles / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011978:Receptors, Nicotinic / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism
D000906:Antibodies; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic