A COMBINED NONDENATURING AND DENATURING GEL-ELECTROPHORETIC ANALYSIS OF THE SUBUNIT COMPOSITION OF A MEMBRANE-PROTEIN - THE SKELETAL-MUSCLE L-TYPE CALCIUM-CHANNEL

Using a non-denaturing digitonin-based polyacrylamide gradient gel electrophoretic system we identified the dihydropyridine-sensitive Ca2+ channel from skeletal muscle as a high molecular weight protein of > 700 kDa. When this protein was excised from the native gels and re-electrophoresed into SDS gels, it dissociated into the α1, α2, β, γ and δ peptides previously suggested to be putative subunits of these Ca2+ channels. The stoichiometry of the α1:α2:β:γ peptides was 1:1:1:1. The presence of the α1 and α2 peptides in the high molecular weight native complex was directly demonstrated with anti-α1 and anti-α2 antibodies. The apparent specific association of the peptides was demonstrated by the finding that the previously separated α1 and α2 peptides did not co-migrate with the native complex in non-denaturing gels. The results of this previously untried analysis support the concept that the skeletal muscle Ca2+ channels are multisubunit proteins. The combined non-denaturing and denaturing gel analyses may be of general utility for the analysis of other membrane proteins. © 1990.