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

Summary

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×g, 4h), passed freely through 0.2 μm nitrocellulose filters and were stable at 4° C with half-lives of >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 (B _max=0.5 to 1.7 pmol per mg of protein( with a K _D of 2.2 – 3.6 nmol/l at 37°C. Specific binding of the 1,4-dihydropyridine calcium channel label was fully reversible (k ₋₁=1.5 min⁻¹, at 37°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 μmol/l), by increasing the B _max and slowed the dissociation rate of the labelled 1,4-dihydropyridine calcium channel blocker.

The solubilized binding sites were sensitive to pronase, alpha-cymotrypsin 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 selectivelyeluted 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 s _20,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 s _{20, 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.