Solubilization and partial purification of putative calcium channels labelled with [3H]-nimodipine.

High-affinity binding sites for the potent 1,4-dihydropyridine calcium channel blocker [3H]-nimodipine were solubilized from guinea-pig skeletal muscle microsomes with digitonin and CHAPS [3-(3-cholamidopropyl)-dimethyl-ammonio-l-propanesulfonate]. Detergent-solubilized binding sites could not be sedimented by centrifugation (50,000 X g, 4 h), passed freely through 0.2 micron nitrocellulose filters and were stable at 4 degrees C with half-lives of greater than 60 h. The solubilized 1,4-dihydropyridine binding sites were precipitable with polyethyleneglycol 6000 on Whatman GF/C filters. Saturation analysis of solubilized microsomes with [3H]-nimodipine revealed a single class of binding sites (Bmax = 0.5 to 1.7 pmol per mg of protein) with a KD of 2.2-3.6 nmol/l at 37 degrees C. Specific binding of the 1,4-dihydropyridine calcium channel label was fully reversible (k-1 = 1.5 min-1, at 37 degrees C). The solubilized drug receptors discriminated between the optical enantiomers of chiral 1,4-dihydropyridine calcium channel blockers, (-)- and (+)D-600 as well as between l-cis and d-cis-diltiazem. d-cis-Diltiazem stimulated the binding of [3H]-nimodipine (ED50:3.6 mumol/l), by increasing the Bmax and slowed the dissociation rate of the labelled 1,4-dihydropyridine calcium channel blocker. The solubilized binding sites were sensitive to pronase, alpha-chymotrypsin and phospholipases A and C indicating their protein nature as well as their lipid requirement. Chelation of endogeneous divalent cations by EDTA, EGTA or CDTA inhibits high-affinity [3H]-nimodipine binding, demonstrating that divalent cations are required for high affinity [3H]-nimodipine binding. Detergent-solubilized binding sites are adsorbed by several sepharose-immobilized lectins, including concanavalin A, wheat germ agglutinin and lentil-lectin but not by helix pomatia lectin. Preparative chromatography on concanavalin A sepharose was performed and the adsorbed [3H]-nimodipine binding sites were selectively eluted by alpha-methylmannoside; NaCl (1 mol/l) being completely ineffective as elutant. The purification factors by this method were 17-40-fold. The binding sites could be also purified (up to 10-fold) by sucrose density centrifugation. The S20, w value of the drug receptors is 12.9 s. It is concluded that the 1,4-dihydropyridine binding sites of the putative calcium channel are intimately associated with carbohydrate containing structures. Since the detergent-solubilized material shows allosteric regulation of 1,4-dihydropyridine binding, interaction with chemically different classes of calcium channel blockers, metalloprotein nature and a S20, w value which is indicative of structure large enough to span the membrane, we conclude that we have solubilized and partially purified the putative calcium channel.