A combined non-denaturing 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 greater than 700 kDa. When this protein was excised from the native gels and re-electrophoresed into SDS gels, it dissociated into the alpha 1, alpha 2, beta, gamma and delta peptides previously suggested to be putative subunits of these Ca2+ channels. The stoichiometry of the alpha 1:alpha 2:beta:gamma peptides was (-)1:1:1:1. The presence of the alpha 1 and alpha 2 peptides in the high molecular weight native complex was directly demonstrated with anti-alpha 1 and anti-alpha 2 antibodies. The apparent specific association of the peptides was demonstrated by the finding that the previously separated alpha 1 and alpha 2 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.
Chang
C F
CF
Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611.
Hosey
M M
MM
D000818:Animals; D015220:Calcium Channels / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D009132:Muscles / Q000737:chemistry*; D011489:Protein Denaturation; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate
D015220:Calcium Channels; D046911:Macromolecular Substances; D008565:Membrane Proteins; D012967:Sodium Dodecyl Sulfate