The devapamil‐binding site of the purified skeletal muscle receptor for organic‐calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca²⁺

Abstract

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-dimethoxy-(1 -methylethyl)benzeneacetonitrile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4OC and 30 "C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel c (~ subunit, with an apparent Kd of 13 & 2.6 nM at 4°C in the presence of 0.4 pM 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 pM to 100 mM. The same results were obtained at 30"C, although the ligand . receptor complex was not stable at Ca2+ concentrations below 10 pM. 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 -l / k + (KD) values than the equilibrium experiments performed using 1 nM and 0.4 pM Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k -l / k + l (KD) by decreasing 10-fold the association rate at 4°C. The dissociation rate was increased about 10-fold by 5 pM devapamil or 100 pM D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second lowaffinity site.Incubation of the CaCB receptor in the absence of CaZ+ and devapamil at 3 0 T , 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 Ca2+ with an apparent median effective concentration of 0.4 pM. 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 pM Ca2+, respectively.These results show that micromolar Ca2 111, which allow extremely high selectivity for Ca2+, while retaining a rapid rate of ion transport. These contradictory properties have been explained by the presence of high-affinity and low-affinity calcium-binding sites, either both within the conducting pore [12, 131 or one within the pore and second elsewhere [14]. Different studies [3 -91 suggested that the inactivated channel binds Ca2+ and CaCB with high affinity, whereas the conducting channel binds one Caz + with high affinity and at least a second CaZ+ and the CaCB with low affinity.It was therefore postulated that the affinity of the purified calcium-channel protein for CaCB should vary with the concentration of Ca2+ [15, 161. Previous work by several laboratories showed that high-affinity binding of dihydropyridines to the CaCB receptor required micromolar to millimolar concentrations of Ca2 + [15 -261, while the binding of phenylalkylamines was inhibited by the same concentrations of Ca2+ [15, 16, 18 -20, 26...

L-Type Calcium Channels: Binding Domains for Dihydropyridines and Benzothiazepines Are Located in Close Proximity to Each Other

“…As shown previously, the association reaction followed second-order kinetics, the initial association rate increasing linearly with the concentration of ligand (Brauns et al, 1995). As discussed in the Materials and Methods section, the association reaction was accompanied by an irreversible inactivation of binding activity due to slow denaturation of the channel in detergent solution (Schneider et al, 1991).…”

Molecular Determinants of Cardiac Ca2+ Channel Pharmacology

“…The regulatory effects of (Ϫ)-D600 and diltiazem are thought to be coupled to or mediated by Ca 2ϩ binding sites in L-type Ca 2ϩ channels (32,33). In previous studies, we demonstrated that the effect of (Ϫ)-D600 differed in skeletal muscle and in cells expressing the skeletal muscle ␣ 1 subunit (34).…”

Differential Effects of an L‐type Ca2+ Channel Antagonist on Activity‐ and Phosphorylation‐enhanced Release of Acetylcholine at the Neuromuscular Junction of the Frog In Vitro

“…This agent blocks the same cardiac Ca2+-currents as the dihydropyridine antagonists (Lee and Tsien, 1983) and an w-conotoxininsensitive current in motor nerves (Anderson and Harvey, 1987). Using the rate constant data for the binding of verapamil to purified channel protein (Schneider et al, 1991), assuming a mean duration of the action potential of 1 ms and pseudo-first order kinetics with no dissociation between impulses, then it would appear that in angel's experiments -95% of channels should have been bound with the antagonist at the end of a 200 impulse train if they were opened.…”