The objective of this project is to understand the molecular basis of the modulation of the gamma-aminobutyric acidA (GABAA) receptor by commonly used inhaled anesthetics such as isoflurane ('Forane') and sevoflurane. The approach involves combining the techniques of electrophysiology and site-directed mutagenesis. The project proposes to consolidate and extend our recent findings concerning specific mutations within the human GABAA receptor that abolish receptor regulation by the anesthetic enflurane. Mutant GABAA receptors made up of of alpha2beta1 subunits will be studied, in which either alpha2 or beta1 subunits carry key mutations (e.g. alphaSer 270 His, alphaAla 291 Trp). Isoflurane and sevoflurane effects will be investigated in these mutant receptors. The role of the gamma2 subunit will then be studied, first by including this subunit with a mutant alphabeta combination, and then by mutation of the gamma2 subunit itself. The effect of varying the beta subunit isoform will then be studied by mutating the neuronally expressed beta2 and beta3 subunits at residues Asn265 and Met286, to compare with data previously obtained on the glial beta1 subunit. The role of Ser 270 and Ala 291 in the alpha2 subunit will then be further investigated by performing extensive mutagenesis of these residues to determine whether hydrophobicity, hydrogen bonding ability, or molecular size is a key feature of these critical amino acid residues. Finally, detailed information about other critical residues within TM2 and TM3 will be provided by site-directed mutagenesis of neighboring residues of Ser 270 and Ala 291. In all mutant receptors, both efficacy and potency of each of the inhaled anesthetics will be measured. Elucidation of the molecular site of action of inhaled anesthetics will lead to improved understanding of the pharmacology of these agents and the role of the GABAA receptor in achieving their desirable and undesirable effects.
