(taken from the application): The gamma-aminobutyric acid Type A (GABA-A) receptors form ligand-activated, anion-selective channels. They are the primary, fast-acting, post-synaptic receptors for GABA, the major inhibitory neurotransmitter in the central nervous system. Current hypotheses suggest that GABA-A receptors may be a primary target for the actions of many general anesthetics. At low concentrations general anesthetics, such as propofol, etomidate, barbiturates and enflurane, potentiate GABA-induced currents, whereas at higher concentrations these anesthetics directly activate GABA-A receptors but do not appear to bind in the GABA binding sites. In order to understand the molecular basis of anesthetic action it is necessary to define the binding sites for these drugs, the conformational changes that occur following binding and the structure of the binding site. Mutations in the alpha-1 subunit of Ser270 (M2) and Ala291 (M3), residues near the extracellular ends of the M2 and M3 membrane-spanning segments, altered the efficacy of the inhaled ether anesthetics (enflurane and isoflurane) to potentiate GABA-induced currents. Whereas, mutations of the aligned residues in the beta subunits altered the efficacy of intravenous anesthetics (etomidate, barbiturates and perhaps propofol) to potentiate GABA-induced. It is uncertain whether these residues are part of anesthetic binding sites or are part of the transduction pathway. Cysteine substituted for these residues in the alpha-1 subunit were accessible to react with the negatively charged, sulfhydryl-specific reagent, pCMBS, applied extracellularly indicating that they are on the water-accessible surface of the protein. If these residues form a binding site(s) for anesthetics then anesthetics should protect the Cys-substituted mutants from modification by pCMBS. The ability of anesthetics to protect these Cys-substitution mutants will be determined. It was previously shown that Cys substituted for six of seventeen residues in the M3 segment were accessible to react with pCMBS. Reaction at four of the six positions was state dependent, it only occurred in the presence of GABA. It will be determined whether potentiating or directly activating concentrations of anesthetics induce changes in the accessibility of M3 segment substituted Cys mutants similar to those induced by GABA. Finally, if the M2 and M3 membrane-spanning segments participate in forming an anesthetic binding site or interactions between them are important for transduction of anesthetic effects then they should be in close proximity. Disulfide bond formation will be used as a molecular ruler to determine the relative proximity, mobility and orientation of the M2 and M3 segments within a single subunit. The successful completion of this proposal could provide new insights into the binding and transduction of anesthetic effects in the GABA-A receptors.
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