Kinetic evidence for isomerization of the dopamine receptor-raclopride complex.

The binding kinetics of the specific dopamine D2 antagonist [3H]raclopride to dopamine D2 receptors in rat neostriatum were studied. The pseudo-first-order rate constants of [3H]raclopride binding with these membranes revealed a hyperbolic dependence upon the antagonist concentration, indicating that the reaction had at least two consecutive and kinetically distinguishable steps. The first step was fast binding equilibrium, characterized by the dissociation constant KA = 12 +/- 3 nM. The following step corresponded to a slow isomerization of the receptor-antagonist complex, characterized by the isomerization equilibrium constant Ki = 0.11. The dissociation constant Kd = 1.3 nM, calculated from these kinetic data, was similar to Kd = 2.4 nM, determined from equilibrium binding isotherm for the radioligand. Implications of the complex reaction mechanism on dopamine D2 receptor assay by [3H]raclopride were discussed.