Molecular size of benzodiazepine receptor in rat brain in situ: evidence for a functional dimer?

Abstract

Benzodiazepine tranquillizers such as diazepam and chlordiazepoxide interact with high-affinity binding sites in nervous tissue^1,2. The correlation between the affinities of various benzodiazepines for these sites with their clinical potencies and activity in behavioural and electrophysiological tests in animals suggests that the sites represent the functional ‘receptor’ whereby benzodiazepines exert their effects³. The intimate involvement of benzodiazepines with γ-aminobutyric acid (GABA) and chloride channels raised the possibility that the benzodiazepine binding site (BDZ-R) may be a protein in the GABA receptor–effector complex^4,5. GABA agonists enhance the affinity of BDZ-R for benzodiazepines⁶, although BDZ-R is distinct from the GABA receptor itself³. However, electrophysiological evidence suggests that the action of benzodiazepines is chloride channel, rather than receptor, directed^7–10. Several attempts have been made to measure the molecular weight (M_r) of BDZ-R after solubilization from brain membranes: treatment with 1% Triton X-100 followed by assay of binding activity in solute fractions separated according to molecular weight suggested¹¹ a value of ∼200,000, photoaffinity labelling of BDZ-R with ³H-flunitrazepam (³H-FNZ) followed by more rigorous solubilization and gel chromatography indicated^12,13 an apparent M_r of ∼55,000 and a third approach¹⁴ a value of ∼100,000. The measured molecular weight seems to depend critically on the solubilization procedure used. Chang et al.¹⁵ recently described the use of radiation inactivation to determine the size of BDZ-R in situ in calf brain membranes, and estimated a M_r, of 216,000. We have also used this approach; the results reported here indicate a M_r of between 90,000 and 100,000, but this is reduced to 60,000–63,000 in membranes pretreated with GABA, suggesting the disaggregation of a normally dimeric form.