Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit

Abstract

The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (α1), 175,000 Da (α2), 52,000 Da (β), and 32,000 Da (γ) 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 (γ 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 α1 and α2 subunits at each step of the purification. In addition, monoclonal antibodies against the α1 and β subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the α2 subunit and the γ 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 α2 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 (γ subunit) is an integral and distinct component of the dihydropyridine receptor.