Chloride-dependent enhancement by barbiturates of γ-aminobutyric acid receptor binding

Barbiturates enhance GABA receptor (sodium-independent) binding at 0°C to bovine brain membranes about 2-fold in chloride-containing buffer but not in chloride-free phoshate or Tris/citrate buffer. This effect is observed in frozen and thawed, thoroughly washed membranes from various regions of bovine brain as well as washed membranes prepared from unfrozen rat cerebral cortex; it is destroyed by brief treatment of the membranes with the detergent Triton X-100. The enhancement is concentration-dependent, with approximately 250 μM pentobarbital giving 50% of the maximal effect seen with saturating 2 mM barbiturate. This effect is reversed by the GABA chloride channel-blocking drug picrotoxinin in a concentration-dependent manner, with 50% inhibition at 0.6 μM, and also by GABA receptor antagonists, such as bicuculline, which inhibit base line GABA receptor binding. Under similar conditions, benzodiazepines do not perturb GABA binding. The barbiturate enhancement varies with brain region in a manner which does not correlate with overall GABA receptor binding; for example, cerebellum shows poor barbiturate enhancement despite a high level of GABA binding. The anions which support barbiturate interactions with GABA receptor binding in vitro are the same ones shown to be required for barbiturate interactions with benzodiazepine receptor binding and correlated with the permeation of GABA-regulated, barbiturate-enhanced inhibitory ion channels measured in vivo. Likewise, the activity of a series of barbiturates to enhance GABA binding shows a specificity and stereospecificity agreeing with that shown for enhancing benzodiazepine binding and correlating with pharmacological activity as nervous system depressants and enhancers of GABA responses. However, the activity of excitatory barbiturates remains to be clarified. These results suggest that there is a receptor site for barbiturates on the GABA·benzodiazepine receptor·chloride ion channel complex, consistent with physiological enhancement of GABA postsynaptic responses by barbiturates. The enhancement of GABA binding by barbiturates appears to involve an increase in the number of binding sites under most assay conditions and using several labeled receptor ligands. The amount of GABA bound to both high and low affinity subpopulations is increased by barbiturates in both equilibrium and kinetic measurements. It is possible that the effect of barbiturates involves an increase in affinity for normally undetectable low affinity (K(D) ≥ 1 μM) GABA sites. Low affinity sites are detected under some conditions, and the barbiturate enhancement is then consistent with an increased affinity. Barbiturates enhance GABA binding in a similar manner at 37°C an 0°C, but the effect is much greater at 37°C than at 0°C for the analogue piperidine-4-sulfonic acid. This may be related to the differential interactions of GABA analogues with benzodiazepine receptors at different temperatures. The more physiological conditions of temperature and saline appear to be more suitable for physiologically relevant receptor binding studies in vitro.