The objectives of this project are to determine the molecular mechanism of action of the mammalian brain inhibitory neurotransmitter substance gamma-aminobutyric acid (GABA). The approach is a biochemical study of the structure and function of the GABA receptor protein, its associated chloride ion channel, and its interaction with the modulatory drugs, benzodiazepines and barbiturates and picrotoxin-like convulsants. The receptor will be further defined and characterized in the brain homogenate membrane-bound state, solubilized in mild detergent, characterized and purified, and used for production of antibodies. The functional consequences of GABA and drug binding to these receptors will be analyzed by quantitative radioactive ligand binding, kinetics and equilibrium, and by radioactive chloride flux measurements in brain slices, cultured nerve cells, and in cell-free membrane vesicles. We will attempt to reconstitute functional membrane-bound proteins from defined purified soluble components. Binding and ion flux data will be compared in order to develop a model for the action of GABA and drugs. Protein purification will involve classical methods including lectin and affinity chromatography and affinity labels. Partially purified receptors now available will be used in attempts to make monoclonal antibodies, which will be screened for ability to bind to receptor proteins, inhibit binding activity in membranes or to precipitate soluble binding activity following secondary reaction with antibodies against mouse immunoglobulins and insoluble protein A. Monoclonal or conventional antibodies will be employed for (a.) collaborations in immunocytochemistry, (b.) studies on the turnover, development, and plastic changes in brain, (c.) structural probes of the receptors, (d.) investigation of the functional role of the various GABA and drug binding sites known to exist, (e.) total purification of the receptors if not already achieved. This basic research is relevant to clinical problems of muscle and nerve, especially epilepsy, Huntington's chorea, anxiety, and sleep disorders.
