The devapamil‐binding site of the purified skeletal muscle receptor for organic‐calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca 2+

The interaction of 2,7‐dimethyl‐3‐(3,4‐dimethoxyphenyl)‐3‐cyan‐7‐aza‐9‐(3‐methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of {3‐[2‐(3,4‐dimethoxyphenyl)ethyl]‐methylaminopropyl‐3,4‐dimethyoxy‐(1‐methylethyl)benzeneacetonitrile (verapamil), with the purfied skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4°C and 30°C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium‐channel α 1 subunit, with an apparent K d of 13 ± 2.6 nM at 4°C in the presence of 0.4 μM Ca 2+ . 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 Ca 2+ concentration from 0.4 μM to 100 mM. The same results were obtained at 30°C, although the ligand · receptor complex was not stable at Ca 2+ concentrations below 10 μM. 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 equilbrium experiments performed using 1 nM and 0.4 μM Ca 2+ , but the same values using 1 mM Ca 2+ · 1 mM Ca 2+ increased the k ‐1 / k +1 ( K D ) by decreasing 10‐fold the association rate at 4°C. The dissociation rate was increased about 10‐fold by 5 μM devapamil or 100 μM d ‐ cis ‐diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca 2+ concentrations and by the occupation of a second low‐affinity site. Incubation of the CaCB receptor in the absence of Ca 2+ and devapamil at 30°C, but not at 4°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 Ca 2+ with an apparent median effective concentration of 0.4 μM. The apparent half‐maximal inactivation times of the devepamil‐binding site were 90 s and 12 min in the presence of 1 nM and 0.4 μM Ca 2+ , respectively. These results show that micromolar Ca 2+ concentrations stabilize the CaCB receptor in a conformation which allows high‐affinity binding of phenylalkylamines. Millimolar Ca 2+ concentrations induce a low‐affinity state of the devapamil‐binding site on a stable CaCB receptor.