Molecular approach to the calcium channel.

Tritiated 1,4-dihydropyridines (nimodipine, nitrendipine, nifedipine, PN 200-110) and [3H]D-cis-diltiazem as well as [3H]verapamil were employed to directly identify calcium channels in membranes for excitable tissues. The channels, when probed with 1,4-dihydropyridines, exhibit the following properties: 1,4-Dihydropyridine calcium channel blockers bind in a temperature-dependent, reversible manner and with high affinity (dissociation constants 0.2-2 nM at 37 degrees C) to a finite number of sites. For chiral 1,4-dihydropyridines, the binding is stereoselective. Hill slopes of approximately 1.0 are observed. In brain, heart, and solubilized skeletal-muscle membranes, an absolute requirement for certain divalent cations exists in order to bind the ligands with high affinity. Cooperativity (negative and positive) between Me2+ and 1,4-dihydropyridine binding sites is observed. 1,4-Dihydropyridine binding sites are down-regulated in a complex manner by the optically pure enantiomers of D-600 and verapamil. These channel blockers induce, to a different extent, a low-affinity state of the 1,4-dihydropyridine binding site. It is postulated that this allosteric site, at which these blockers act, is closely coupled to the 1,4-dihydropyridine binding site and that a spectrum of compounds exists that differ in their affinity as well as their intrinsic activity to induce the down-regulation. The 1,4-dihydropyridine binding sites are up-regulated by D-cis-diltiazem and KB-944. The up-regulation is temperature-dependent and induces a high-affinity state for 1,4-dihydropyridine channel blockers, accompanied by distinct alterations of the kinetics as well as the pharmacological profile of the 1,4-dihydropyridine binding sites. Complex interactions exist between the channel blockers that induce up-regulation and those that induce down-regulation of the binding. For a given radiolabeled 1,4-dihydropyridine, a tissue-specific (but not species-specific) equilibrium binding dissociation constant is observed. Thus, all heart (human, rat, guinea pig, frog, bovine) have the same KD (0.25 nM at 37 degrees C) for, [3H]nimodipine. The same is observed for brain (KD = 0.5 nM) and for skeletal muscle (KD = 1-2 nM). Three subtypes of channels can be distinguished on the basis of the KD and the tissue-specific up-regulation by D-cis-diltiazem. Subtype-selective drugs exist; e.g., AQA 39 is an inhibitor of [3H]nimodipine binding at skeletal-muscle calcium channels, but not at brain channels. Despite their different pharmacological and kinetic profiles, calcium channels in skeletal muscle and brain have the same molecular size (Mr) when probed by radiation inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)