Interaction of [3H]flunitrazepam with the benzodiazepine receptor: evidence for a ligand-induced conformation change.

The interaction of [3H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 micro M endogenous GABA at 0 degrees at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 +/- 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K = 30 nM). The dissociation kinetics of the [3H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant Koff = (7.7 +/- 0.3 X 10(-4)/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]0 in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate r-1. In the higher concentration range [L]0 greater than or equal to 10 nM, r-1 as a function of [L]0 deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C: (see formula in text) where K1 = ([R][L]/([RL] and [RL]/[C] = k-2/k2 = k2. Nonlinear parameter estimation yielded K1 = 24.2 +/- 7.1 nM, k2 = (2.8 +/- 0.5) X 10(-2)/sec and K-2 = (9 +/- 2) X 10(-4)/sec. The isomerization step might reflect ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.