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 degrees C. The binding of [3H](-)-S-Bay K 8644 was temperature-dependent with a single binding site with KD = 3.57 nM and Bmax = 330 fmol/mg.protein at 25 degrees C. The association and dissociation rate constants were 3.4 x 10(7) min-1 M-1 and 0.095 min-1 respectively at 25 degrees C and decreased slightly at lower temperatures. In contrast, [3H](+)-PN 200-110 bound to high (KD(H) = 0.032 nM, Bmax(H) = 316 fmol/mg.protein) and low affinity sites (KD(L) = 27.6 nM and Bmax(L) = 6432 fmol/mg.protein) at 25 degrees 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 x 10(8) min-1 M-1 and 7.86 x 10(-3) min-1 at 25 degrees C. The association rate varied only slightly (3-fold), but the rate of dissociation decreased significantly (200-fold) with temperature from 37 to 4 degrees 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.