Temperature effects on [alpha]2-adrenergic receptor-Gi interactions

Abstract

The effect of temperature on the binding of [alpha]2-adrenergic agonists and antagonists to human platelet membranes was studied. Equilibrium binding of the [alpha]2 antagonist, [3H]yohimbine, was affected minimally, whereas the rate of dissociation changed 40-fold over a temperature range of 5-35[deg]. The antagonist dissociation rates were characterized by a linear Arrhenius plot and an activation energy of 20.5 kcal/mol. The equilibrium binding of the full [alpha]2 agonist, [3H]UK 14,304 [5-bromo-6-N-2-4,5-dihydroimidazolyl)quinoxaline tartrate] showed a 50% decrease in Bmax at 5[deg] as well as a 2-fold decrease in affinity. The kinetics of [3H]UK 14,304 binding were affected more significantly by decreases in temperature. The agonist exhibited fast and slow phases of binding. The fast binding was minimally sensitive to temperature in the range of 0-30[deg] with only a 6-fold change in rate. The slow binding rates changed nearly 100-fold over the same temperature range. Also, the slow rate of agonist binding was characterized by a nonlinear Arrhenius plot with a "break" at approximately 17[deg], which was found previously to be the phase transition temperature of platelet membrane lipids [Lohse et al., Molec. Pharmac. 29, 228 (1986)]. Despite the reduction of high affinity [3H]UK 14,304 binding at 5[deg], approximately half of the binding remained sensitive to guanine nucleotides. The data are interpreted in the context of a model in which the fast agonist binding represents a bimolecular interaction of ligand with two pre-existing conformations of the [alpha]2 receptor, one coupled to Gi and the other permanently uncoupled. The slow binding of agonist appears to require protein diffusion in the lipid membrane or a protein conformational change which is dependent on the lipid environment.