The objective of this project is to understand the molecular basis of the modulation of the gamma-aminobutyric acid(A) (GABA/A) receptor by the volatile anesthetics, enflurane, halothane and isoflurane, using the techniques of electrophysiology and molecular biology. The project proposes to first extend pharmacological studies of anesthetic modulation of the GABA(A) receptors of hippocampal neurons, in order to evaluate the hypothesis that there exists a correlation between anesthetic potency and drug potency for modulation of hippocampal GABA(A) receptors. Non- anesthetic compounds will also be tested. Single channel studies of the mechanisms of volatile anesthetic modulation of the GABA(A) receptor will also be performed. The GABA(A) receptor is a member of the 'family' of ligand-gated chloride channels. To begin to investigate the molecular basis of GABA(A) receptor modulation by volatile anesthetics, the effects of these agents will be studied on recombinant human GABA(A) receptors of known subunit composition. We will study heterooligomeric receptors of alpha/Beta, alpha/Beta/gamma, alpha/gamma and, if possible, homomeric receptors consisting of alpha subunits. In addition, the possible modulation by volatile anesthetics of other members of the ligand-gated chloride channel 'family', including the structurally related human glycine receptor, the human p1 subunit ('GABA(C) receptor') will be investigated, to determine whether all members of the ligand-gated chloride channel family are university modulated by volatile anesthetics, i.e., whether there are specific structural requirements for volatile anesthetic action at the GABA(A) receptor. Chimeric receptors will be constructed in order to gain further information about the nature of the sites for volatile anesthetic action on the human GABA(A) and glycine receptor subunits. Finally, more detailed information will be provided by using the techniques of site-directed mutagenesis to alter individual amino acid residues within the ligand-gated chloride channel subunits. Analysis of the molecular site of action of volatile anesthetics may lead to the development of anesthetic 'antagonists' which would be invaluable to clinical practice.
