European Journal of Pharmacolo~' - Molecular Pharmacolo,~.' Section, 208 (1991) 137-147 © 1991 ElsevierScience PublishersB,V. All rights reserved 0922-4106/91/$03.50 ADONIS 0922410691001752 137 EJPMOL 90226 Thermodynamic and kinetic aspects of agonist and antagonist binding to 1,4-dihydropyridine receptors Wei Zheng, Mark Hawthorn and David J. Triggle Department of Biochemical Pharmacology. School of Pharmacy. State UnicersiIy of New York at Buffalo, Buffalo. NY 14260. U.S.A. Received 20 Febrtmry1991,revisedMS received 12 June 1991,accepted 25 June 1991 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 o C. The binding of [3H](-)-S-Bay K 8644 was temperature-dependent with a single binding site with K o = 3.57 nM and Bma x = 330 fmol/mg.protein at 25°C. The association and dissociation rate constants were 3.4 × 10 7 min -j M -1 and 0.095 min -l respectively at 25 ° C and decreased slightly at lower temperatures. In contrast, [3H](+ )-PN 200-I 10 bound to high (KD~H) = 0.032 riM, BmaxtH)=316 fmol/mg' protein) and low affinity sites (Kotl.~= 27.6 nM and B,,~xcL)=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 rain -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. 1,4-Dihydropyridines; Ca 2+ channel activator; Thermodynamics; Kinetics; Radioligand binding; Ca2+ I. Introduction Voltage-gated calcium channels have been impli- cated in the pathophysiology of a number of cardiovas- cular diseases (Braunwald, 1982; Triggle, 1990) and nifedipine, verapamil and diltiazem, representing three major classes of calcium channel antagonists, the 1,4- dihydropyridines, phenylalkylamines and benzothia- zepines, have been clinically used to treat cardiovascu- lar diseases including angina, hypertension and certain arrhythmias. Simultaneously, these agents have proved to be valuable molecular tools with which to probe Ca2+channel structure and function. Despite many in- vestigations, the mechanisms of drug action at voltage- gated calcium channels are not completely understood (for reviews, see Janis et al., 1987; Triggle, 1990). Thus, for the 1,4-dihydropyridine series several important questions remain and, in particular, elucidation of the Correspondence to: David J. Triggle, Department of Biochemical Pharmacology,School of Pharmacy, State University of New York at Buffalo, Buffalo, NY 14260,U.S.A. Tel. 716-836-2823;Fax 716-636- 3688. molecular distinctions between activator and antago- nist deserves further consideration. In 1,4-dihydropyridines, activator and antagonist properties at voltage-gated calcium channels may re- side in the enantiomers of chiral molecules such as Bay K 8644 and 202 791 (Franckowiak et al., 1985; Hof et al., 1985; Triggle and Rampe, 1989). Additionally, a single enantiomer can demonstrate both activator and antagonist properties (Franckowiak et al., 1985; Hof et al., 1985; Kongsamut et al., 1985). Indeed, the demon- stration of activator properties may be a universal property of 1,4-dihydropyridines, although often ex- pressed in transitory fashion with potent antagonists (Thomas et al., 1984; Dube et al., 1985; Lee et al., 1987). Thermodynamic analyses of drug receptor binding data have been explored as a potentially useful method to analyze the molecular basis of interactions (for re- view, see Hitzemann, 1988; Raffa and Porreca, 1989). Ligand binding to a number of systems including the /3-adrenoceptor (Weiland et al., 1979; Contreras et al., 1986a, b), muscarinic (Barlow and Burston, 1979; Gies et al., 1986), benzodiazepine (Quast et al., 1982; Doble,