Thermodynamic and kinetic aspects of agonist and antagonist binding to 1,4-dihydropyridine receptors

The kinetic and equilibrium binding properties of the 1,4-dihydropyridine activator [³H](-)-S-Bay K 8644 and the antagonist [³H](+)-PN 200-110 were determined in rat heart membrane particulate preparations at temperatures between 4 and 37°C. The binding of [³H](-)-S-Bay K 8644 was temperature-dependent with a single binding site with K_D = 3.57 nM and B_max = 330 fmol/mg · protein at 25°C. The association and dissociation rate constants were 3.4 × 10⁷ min^-1 M^-1 and 0.095 min^-1 respectively at 25°C and decreased slightly at lower temperatures. In contrast, [³H](+)-PN 200-110 bound to high K_D(H) = 0.032 nM B_max(H) = 316 fmol/mg · protein) and low affinity sites K_D(L) = 27.6 nM and B_max(L) = 6432 fmol/mg · protein) at 25°C in rat heart preparation. A similar two-site binding of [³H](+)-PN 200-110 was found in rat brain preparation, but only a single binding site was detected in rat skeletal muscle. Binding of [³H](+)-PN 200-110 to the high and low affinity sites in cardiac membranes was sensitive and insensitive respectively to temperature. Association and dissociation rates of [³H](+)-PN 200-110 at the high affinity binding sites were best fitted as mono-exponential functions. Association and dissociation rates of [³H](+)-PN 200-110 were 3.94 × 10⁸ min^-1 M^-1 and 7.86 × 10^-3 min^-1 at 25°C. The association rate varied only slightly (3-fold), but the rate of dissociation decreased significantly (200-fold) with temperature from 37 to 4°C. Thermodynamic analysis of equilibrium binding showed that the binding of activator was enthalpy driven, whereas the binding of antagonist to the high affinity site was both entropy- and enthalpy-driven and to the low affinity site was totally entropy-driven.