Stimulatory and protective effects of benzodiazepines on GABA receptors labeled with [3H]muscimol.

We investigated the effects of benzodiazepines on [3H]muscimol binding to rat brain membranes and on heat inactivation of GABA receptors. Scatchard analysis of [3H]muscimol binding to frozen and 0.05% Triton X-100 treated membranes revealed two components; a higher affinity (Kd = 2.2 nM, Bmax = 1.2 pmol/mg protein) and a lower affinity component (Kd = 15.9 nM, Bmax = 4.4 pmol/mg protein). Diazepam and flurazepam (3 microM) increases significantly the specific binding of 40 nM but not of 2 nM [3H]muscimol. This stimulation was attributed to an increase in the affinity of the lower affinity component for GABA receptors. The time course of heat inactivation of GABA receptors revealed rapidly and then slowly denaturating Phases. These observations would suggest that there are multiple GABA receptors with different sensitivities to the heat treatment. Diazepam depressed remarkably the slowly denaturating phase(s). After heat treatment for 50 min, the single component of GABA receptors with Kd of 14.3 nM and Bmax of 0.6 pmol/mg protein survived, whereas in the membranes preincubated with 3 microM diazepam, the Kd and Bmax of the still viable GABA receptors were 14.8 nM and 1.14 pmol/mg protein, respectively. In light of these findings, the stimulation of the lower affinity component of GABA receptors may be related to the protective effects of these drugs against heat inactivation.