Purification of the calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubules

Abstract

[3H]Nitrendipine binds with high affinity to a calcium antagonist receptor preferentially localized to the transverse tubule membrane of skeletal muscle. Digitonin was used to solubilize a [3H]nitrendipine-receptor complex from transverse tubule membranes. The digitonin-[3H]nitrendipine-receptor complex was purified 330-fold by a combination of wheat germ agglutinin chromatography, ion-exchange chromatography, and sedimentation through sucrose gradients to yield a preparation estimated to be 41% homogeneous on the basis of specific activity. Analysis by sodium dodecyl sulfate gel electrophoresis demonstrated that three polypeptides termed alpha [molecular weight (Mr) 130 000], beta (Mr 50 000), and gamma (Mr 33 000) quantitatively comigrated with the [3H]nitrendipine-receptor complex on sucrose gradients and represented 62% of the total protein staining. These three polypeptides are associated noncovalently. However, the apparent molecular weight of the alpha polypeptide is reduced from 160 000 to 130 000 upon reduction, consistent with the presence of an internal disulfide bond. Our results suggest that these three polypeptides are the subunits of the calcium antagonist receptor and are major components of the transverse tubule voltage-sensitive calcium channel.