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 from excitable tissues. The channels, when probed with 1,4-dihydropyridines, exhibit the following properties: 1. 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°C) to a finite number of sites. For chiral 1,4-dihydropyridines, the binding is stereoselective. Hill slopes of approximately 1.0 are observed. 2. 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. 3. 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. 4. 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 downregulation of the binding. 5. For a given radiolabeled 1,4-dihydropyridine, a tissue-specific (but not species-specific) equilibrium binding dissociation constant is observed. Thus, all hearts (human, rat, guinea pig, frog, bovine) have the same K D (0.25 nM at 37°C) for, [3H]nimodipine. The same is observed for brain (K D = 0.5 nM) and for skeletal muscle (KD = 1–2 nM). Three subtypes of channels can be distinguished on the basis of the K D 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. 6. 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. The apparent Mr of the brain channel (probed with [3H]nimodipine) is 185,000; the Mr of the skeletal-muscle channel is 178,000. 7. The Mr of the channel, as evaluated by radiation inactivation, is decreased by 60,000— 75,000 when channels are up-regulated by D-cis-diltiazem. The action of D-cis-diltiazem is stereospecific, since D-cis-diltiazem is inactive. In addition, neither benzodiazepine receptor Mr (in brain) nor acetylcholinesterase Mr (in skeletal muscle) is decreased by D-cis-diltiazem. 8. Different 1,4-dihydropyridines do not label the same density of binding sites, e.g., in skeletal-muscle membranes, in the absence or presence of D-cis-diltiazem. The concept of intrinsic activity for a given 1,4-dihydropyridine is introduced, based on its ability to induce or stabilize a high-affinity state. Most notable is that [3H]-PN 200–110 labels more sites in skeletal muscle than nifedipine, nimodipine, or nitrendipine. 9. [3H]-PN 200–110 binding to skeletal-muscle microsomes is stimulated by the allosteric regulator D-cis-diltiazem. However, although the kinetic constants are changed by D-cis-diltiazem, there is, in contrast to [3H]nimodipine or [3H]nitrendipine, only a small increase with respect to the density of sites labeled by [3H]-PN 200–110. 10. The Mr of the skeletal-muscle calcium channel, determined by radiation inactivation and with [3H]-PN-200–110 as ligand, is 138,000, this being 40,000 mass units smaller than that determined with [3H]nimodipine. These findings, taken together with the stereospecific effects of D-cis-diltiazem on the Mr of [3H]nimodipine-labeled channels in brain and skeletal muscle, are indicative of an oligomeric nature of the channel and support the concept of a continuum of 1,4-dihydropyridines ranging from agonists to antagonists. 11. The allosteric regulatory sites that interact with the 1,4-dihydropyridine site have been directly labeled with [3H]D-cis-diltiazem in skeletal muscle. Binding of [3H]D-cis-diltiazem is temperature-dependent, and maximal labeling of 11 pmole binding sites with a K D of 39 nM occurs at 2°C. The ratio of allosteric sites to 1,4-dihydropyridine binding sites appears to be 1 : 1 or 1 : 2, depending on the radioligand. Binding of D-cis-diltiazem is regulated in a complex manner by calcium-channel agonists and antagonists. At temperatures greater than 20°C, 1,4-dihydropyridine-channel antagonists stimulate; at 2°C, they are inhibitors. The rank order of efficacies (as well as the respective IC50 or EC50 values) differs for stimulation and inhibition and is typical for a given 1,4-dihydropyridine. On the basis of these findings, agonists and antagonists (which keep channels in unshut and shut states) are discriminated. 12. Skeletal-muscle calcium channels can be purified in t-tubular membranes. The density of channels is extremely high (≈ 60,000 fmoles/mg protein). The channel has been solubilized in good yield with detergents and is stable at 4°C with a half-life of 60 hr or more. The S20,w value is 12.9 S, and sucrose-gradient-purified channels are still up-regulated by D-cis-diltiazem. The channel is a glycoprotein, since it is selectively adsorbed by lectin-affinity columns and desorbed (17- to 40-fold purified) by corresponding sugars.