Preferential protection of cortical type II benzodiazepine receptors by γ-aminobutyric acid during heat inactivation

Pharmacological and biochemical evidence supports the existence of a heterogeneous population of benzodiazepine (BDZ) receptors. One major tool which has been used to identify distinct receptor subtypes is a novel series of triazolopyridazines (TPZ) that recognize two subpopulations (types I and II) of BDZ binding sites. Earlier studies demonstrated that γ-aminobutyric acid (GABA) enhanced specific BDZ binding and protected a fraction of BDZ binding sites against heat inactivation. GABA/BDZ interactions were further investigated by studying the properties of those BDZ binding sites that are thermostable in the presence of 1.0 mM GABA when cortical membrane fragments are heated at 60°C. Saturation isotherms of ³H-flunitrazepam (³H-Flu) binding revealed a 90% and 60% diminution of binding sites in the absence and presence of 1 mM GABA, respectively. In the presence of 1 mM GABA, displacement of ³H-Flu binding with varied concentrations of CL 218,872, a TPZ, in heated cortical membrane preparations indicated a significant increase in the IC₅₀ value, with no effect on the Hill coefficient when compared to parallel control experiments with unheated membranes. In contrast, no significant differences in either Hill coefficients or IC₅₀ values were obtained when similar displacement experiments were performed utilizing diazepam, an agent that does not distinguish between BDZ receptor subtypes. Hofstee plots of CL 218,872 displacement of ³H-Flu binding were curvilinear in both heated and unheated membranes and could be resolved into two components by computer analysis. The relative affinity of CL 218,872 at the high-affinity (K_D1) or lowaffinity (K_D2) recognition sites was unchanged after heating the membrane preparations. However, after heating, the density of type I receptors (B_max1) was reduced 75%, while the number of type II receptors (B_max2) was decreased only 38%. These results are indicative of a preferential GABA-mediated protective effect on the thermal inactivation of type II BDZ recognition sites.