ANESTHETIC MECHANISMS AND GABA-A RECEPTOR STRUCTURE

The GABA-A receptors are the major inhibitory post-synaptic receptors in the mammalian central nervous system. Many drugs used in the induction of general anesthesia, including benzodiazepines, volatile and intravenous anesthetics and barbiturates potentiate GABA-induced currents. The extent of potentiation depends on the specific subunit-subtype composition of the GABA-A receptors. Progress has been made towards identifying the binding sites for these drugs. Mutagenesis and photoaffinity labeling studies identified some amino acids that may form the binding sites for these drugs. Understanding the three dimensional structure of the drug binding sites and the conformational changes induced by drug binding, however, will require a high -resolution, x-ray crystal structure. The crystallization of integral membrane proteins has been a difficult problem and it is unlikely that a crystal structure of the entire GABA receptor will be obtained in the near future. A partial solution to obtaining high-resolution, three-dimensional structural information about integral membrane proteins has been to produce and crystallize the extra-membrane domains of the proteins as separate, water-soluble proteins. The extracellular domain of the GABA receptors contains residues that form the GABA and benzodiazepine binding sites. The major goals of this application are 1) to identify a suitable expression system that will overproduce the extracellular domain of the GABA receptor as a water-soluble protein, 2) to characterize the physical and functional properties of the extracellular domain protein and 3) to initiate crystallization trials with the protein. We have truncated the GABA rho-1 subunit just prior to the start of the first membrane-spanning domain. These studies have been initiated with the rho-l subunit because it forms homomeric channels. The resultant 259 amino acid protein is secreted from Drosophila S2 cells. Its mobility on a sucrose-density gradient is consistent with its forming a pentamer. One hundred milligram quantities can be produced and purified from E. coli inclusion bodies. Conditions to refold the guanidine-solubilized protein will be investigated. If successful the refolded protein will be characterized and used in crystallization trials. If refolding is unsuccessful over-expression in eukaryotic expression systems including yeast and mammalian and insect cells in culture will be tried. While the risk of this project is high the impact would also be high if it results in a high-resolution structure of the extracellular domain of an ionotropic GABA receptor subunit. If this approach is successful it will provide new understanding of the structure of the ligand binding sites within the extracellular domain. It will also provide insights into the conformational changes induced by agonist and drug binding and thereby provide an understanding of anesthetic action at a molecular level.