THE DEVAPAMIL-BINDING SITE OF THE PURIFIED SKELETAL-MUSCLE RECEPTOR FOR ORGANIC-CALCIUM CHANNEL BLOCKERS IS MODULATED BY MICROMOLAR AND MILLIMOLAR CONCENTRATIONS OF CA2+

The interaction of 2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan-7-aza-9-(3-methoxyphenyl) nonahydro-chloride (devapamil), a stereospecific analog of {3-[2-(3,4-dimethoxyphenyl)ethyl]-methylaminopropyl-3,4-dimethoxy-(1-methylethyl)benzeneacetonitrile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4-degrees-C and 30-degrees-C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel alpha-1 subunit, with an apparent K(d) of 13 +/- 2.6 nM at 4-degrees-C in the presence of 0.4-mu-M Ca2+. The affinity, and not the density, of the devapamil-binding site was decreased by lowering the pH from 8.5-6.5, or by increasing the Ca2+ concentration from 0.4-mu-M to 100 mM. The same results were obtained at 30-degrees-C, although the ligand . receptor complex was not stable at Ca2+ concentrations below 10-mu-M. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo-first-order and a second-order reactions were identical and yielded fourfold lower k-1/k+1 (K(D)) values than the equilibrium experiments performed using 1 nM and 0.4-mu-M Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k-1/k+1 (K(D)) by decreasing 10-fold the association rate at 4-degrees-C. The dissociation rate was increased about 10-fold by 5-mu-M devapamil or 100-mu-M D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low-affinity site. Incubation of the CaCB receptor in the absence of Ca2+ and devapamil at 30-degrees-C, but not at 4-degrees-C, resulted in an apparent loss of devapamil-binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4-mu-M. The apparent half-maximal inactivation times of the devapamil-binding site were 90 s and 12 min in the presence of 1 nM and 0.4-mu-M Ca2+, respectively. These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high-affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low-affinity state of the devapamil-binding site on a stable CaCB receptor.