The GABA-A/benzodiazepine receptor (GBZR) complex on vertebrate neurons plays a key role in inhibitory neurotransmission. The long-term goal of this project is to understand the neuronal regulation of GBZR receptor number and subcellular distribution. We have recently demonstrated that chronic exposure of chick cortical neurons to GABA produces a down-regulation of GBZR3. In order to study the underlying regulatory mechanisms, three specific objectives are developed in this proposal. 1. To test the hypothesis that exposure of cortical neurons to GABA and benzodiazepine agonists accelerates internalization of GBZRs. A novel membrane-impermeant benzodiazepine, SPTC-1012S, which is a potent displacer of [35S]flunitrazepam binding to intact neurons, will be used in an assay for receptor sequestration. This will allow us to study the effects of GABA and benzodiazepine agonists, both alone and in combination, on this process. Possible up-regulation by benzodiazepine antagonists and reverse agonists, as well as GABA antagonists, will also be evaluated. 2. To test the hypothesis that exposure of neurons to GABA and benzodiazepine agonists produces a reduction in ligand binding sites and GBZR peptides. Our approach involves metabolic labeling of GBZR peptides with [35S]methionine and determining their rates of degradation by pulse-chase techniques. In addition, the effects of down-regulation on ligand binding density, affinity, and-specificity will be determined. 3. To test the hypothesis that agonist exposure reduces the level of GBZR transcripts. The GBZR alphasubunit mRNAs which are subject to down-regulation will be identified by Northern hybridization and their rates of degradation quantified by DNA-excess solution hybridization. Furthermore, nuclear run-on transcription assays will be used to determine the rates of GBZR alpha-subunit mRNA synthesis and to examine the role of receptor agonists in repression. It is suggested that this combination of techniques from pharmacology, biochemistry, and molecular biology will provide new insights into pathways which modulate synaptic function. By means of these regulatory mechanisms, cell-cell communication and drug-cell interaction could produce persistent changes in neuronal excitability. Furthermore, these may represent molecular mechanisms which could establish tolerance and habituation to benzodiazepines.
