The objective of this research is to understand the mechanisms of modulation of GABAA receptors by inhaled anesthetics, and the significance of these effects to the behavioral actions of these agents in animals. In this proposal, our experiments will be closely linked to the program carried out by Dr. Gregg Homanics at the University of Pittsburgh.
The specific aims are: 1) To investigate the effects of inhaled anesthetics on GABAA-Rs with mutations in the alpha1 subunit, in receptors of the form a1b2g2 (alpha1beta2gamma2). We will study the effects of isoflurane and halothane on GABA responses in the following mutant GABAA-Rs: a1b2g2, a1(S270H)b2g2, a1(S270W) b2g2 and a1(S270W; A291W)b2g2. The hypothesis to be tested is that the efects of the inhaled anesthetics in these mutants on the a1b2g2 GABAA-Rs background closely parallel results obtained in GABAA-Rs of the format a2b1. 2) To investigate the effects of inhaled anesthetics on GABAA-Rs with point mutations in the fourth transmembrane segment (TM4) of the a1 subunit. We will perform tryptophan scanning mutagenesis in TM4. The hypothesis to be tested is that one or more TM4 residues is involved in mediating the effects of isoflurane and/or halothane. 3) To investigate the effects on GABA sensitivity of double mutations at key residues in the GABAA-R a1 subunit that control agonist potency. We will study the following GABAA-Rs: a1b2g2 and a1 (S270H; L277A) b2g2. The hypothesis to be tested is that the double mutation will restore the GABA sensitivity of the GABAA-R, while retaining insensitivity to isoflurane. 4) To investigate the effects of analogous mutations in the GABAA-R a2 and a3 subunits. We will study the following GABAA-Rs: a2b2g2, a3b2g2, a2(S270H)b2g2, a3(S270H)b2g2, a2(S270H; L277A)b2g2 and a3(S270H; L277A)b2g2. The hypothesis to be tested is that the double mutation restores normal GABA sensitivity to these GABAA-Rs. 5) To investigate the effects on GABAA-R kinetics of mutations in the a1 subunit. We will study the kinetics of these GABAA-Rs: a1b2g2, a1(S270H)b2g2 and a1(S270H; L277A)b2g2. The hypothesis to tested is that the receptor kinetics will be abnormal in the S270H mutant GABAA-Rs, but not in the double mutants. 6 ) To study the physiology and anesthetic pharmacology of inhibitory synaptic transmission in the a1(S270H) and a1(S270H; L277A) 'knock-in' mice. The hypothesis to be tested is that these animals will exhibit a decrease in modulation of synaptic inhibition by isoflurane. The overall impact of this research will be to increase our understanding of inhaled anesthetic action, leading to the development of safer anesthetic agents in the future.
