Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle

Abstract

Digitonin and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1 -propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine-Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine-binding activity of 2500 -2800 pmol/mg protein (60 -70% purity) from digitonin-solubilized membranes by a combination of wheat-germ-agglutinin -Sepharose, anion-exchange and dihydropyridine-Sepharose chromatography steps.The individual subunits were isolated in dodecyl-sulfate-denatured form from the preparation of the receptor, enriched by a two-step large-scale procedure applied to Chapso-solubilized membranes. The 160-kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160-kDa subunit is cross-linked by intramolecular S-S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N-glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.Dihydropyridine calcium antagonists, such as nifedipine, are known to be the most potent blockers of potential-dependent calcium channels [I], which play a key role in a number of secretory and regulatory processes in living cells. In a variety of muscle cells the voltage-operated calcium channels are involved in excitation/contraction coupling [2 -51 which underlies the use of specific calcium antagonists in the treatment of a number of cardiovascular disorders [6].The identification of the dihydropyridine receptors, generally considered as putative calcium channels, is now accessible since a number of radioactively labeled dihydropyridine derivatives have become available [7, 81. Rabbit white muscle transverse tubule membranes are certainly the richest source of the dihydropyridine receptor known [9]. These membranes have been reported to be suitable for the receptor purification when solubilized in digitonin [lo] or Chaps [ll]. The subunit composition of the purified receptor preparations, however, differed significantly. Receptor, purified as a slowly dissociating complex with [3H]nitrendipine, solubilized by digitonin, co...