BINDING SITES ARE CONSTITUENTS OF THE y-AMINOBUTYRIC ACID BENZODIAZEPINE RECEPTOR COMPLEX

t-Butylbicyclophosphorothionate (TBPS), a derivative of potent GABA antagonistic cage convulsants, has recently been introduced (Squires, R. F., J. E. Casida, M. Richardson, and E. Saederup (1983) Mol. Pharmacol. 13: 326-336) as ligand for a GABA-A receptor-linked drug receptor. Using conventionally prepared washed membrane fractions from rat cerebral cortex, we have confirmed that in the presence of 200 IIIM NaBr [““SITBPS binds to a high affinity population of binding sites (& 26 + 5 nM) and that muscimol inhibits [““SITBPS binding (I&, 0.32 PM) allosterically. In 200 mM NaCl the apparent affinity of [““SITBPS binding sites is lower (Kd 60 + 5 nM), and muscimol has biphasic effects with stimulation at low concentrations of muscimol (EC,, 0.023 PM) followed by inhibition at high concentrations (IC& 0.72 PM). Both base line [“5S]TBPS binding (in 200 mM NaCl) and muscimol inhibition of [‘“SITBPS binding (in 200 mM NaBr) are bidirectionally modulated by the occupancy of benzodiazepine receptors with its ligands. Benzodiazepine receptor agonists, regardless of their structure, enhance and inverse benzodiazepine receptor agonists inhibit base line [““SITBPS binding and muscimol inhibition of [“‘SITBPS binding. Fourteen ligands for benzodiazepine receptors display a similar in vitro profile as benzodiazepine receptor agonists or inverse benzodiazepine receptor agonists on [%]TBPS binding as their antior proconvulsive effects in uiuo suggest (Jensen, L. H., E. N. Petersen, and C. Braestrup (1983) Life Sci. 33: 393399). That [‘%]TBPS binding sites are constituents of a GABA benzodiazepine receptor complex is also suggested by a number of membrane pretreatments. After photoaffinity labeling of benzodiazepine receptors with flunitrazepam, [%]TBPS binding sites became insensitive to modulation by flunitrazepam but not by 6,7-dimethoxy-4-ethyl+carboline-3-carboxylic acid methyl ester. Pretreatment of membranes with Ag+ ions, which leads to the sole appearance of high affinity [“HI muscimol binding sites, to high affinity muscimol stimulation of [“Hlflunitrazepam binding, and to a loss of allosteric modulation of these binding sites by etazolate (SQ 20009) or isopropylbicyclophosphate (IPTBO), results in the disappearance of [35S]TBPS binding. Pretreatment of membranes with Triton X-100, which perturbs the GABA and benzodiazepine receptor binding in a mode similar to Ag’ ions, also leads to the disappearance of [35S]TBPS binding. Thus [35S]TBPS binding sites can be inactivated separately from GABA and benzodiazepine receptors, and this inactivation goes parallel with perturbation of GABA and benzodiazepine receptor binding. The CNS depressants etazolate and pentobarbital differ in their effects on [“‘SITBPS binding when compared to picrotoxinin and IPTBO. The latter compounds inhibit [3%]TBPS binding unperturbed by the occupancy of benzodiazepine receptors. Etazolate and pentobarbital (in 200 mM NaCl) have biphasic effects on [35S]TBPS binding with stimulation at low concentrations (EC,,, 0.4 pM and 60.4 pM) and inhibition (I&o 12.9 pM and 550 pM) at higher concentrations with Hill numbers of 1.5 and 2.7. The potencies of etazolate or pentobarbital as inhibitors of [“‘SITBPS binding (in 200 mM NaBr) are bidirectionally modulated by the occupancy of benzodiazepine receptors with agonists or with inverse agonists. These observations are not compatible with the concept that these CNS depressants and CNS convulsants act on an identical drug receptor. There is evidence from biochemical and electrophysipharmacological effects in the brain by enhancing the ological experiments that benzodiazepines exert their actions of the inhibitory neurotransmitter GABA (Costa -et al., 1975; Haefely et al., 1975). Later, benzodiazepines ’ To whom correspondence should be addressed. have been shown to interact with their brain-specific