The type A gamma amino butyric acid (GABAA) receptor is an important pharmacological target for general anesthetic drugs. Recent progress suggests highly specific effects of several general anesthetics at the GABAA receptor; the details of this mechanism, however, are unknown. Additionally there are many different forms of the subunits which make up the GABAA receptor. The interaction between GABAA receptor subunit composition and general anesthetic pharmacology remains relatively unexplored, and the significance of this interaction to the function of neurons is unknown. This project tests the hypothesis that the GABAA receptor beta-subunit isoform dictates etomidate modulation of GABA-induced current in neurons. The study consists of a series of deliberate steps beginning with a biophysical characterization of the effects of intravenous general anesthetics at GABAA receptors, followed by an examination of the effects of b-subunit gene-targeting on general anesthetic pharmacology and concluding with the creating of neurons with a reversible externally-inducible alteration in sensitivity to etomidate. Specifically, the recently discovered critical role of the beta-subunit and the specific amino acid at location 270 on the beta-subunit will be explored through expression, in HEK293 cells, of different beta-subunit isoforms and point-mutants in an a1bxg2 heteromeric combination. Whole cell patch clamp and rapid GABA perfusion will be used to arrive at a kinetic model of drug action. Next this mechanistic model will be used as a tool to investigate the effects of beta-subunit gene-targeting on general anesthetic pharmacology in retinoic-acid-induced P19 neurons. The P19 system offers an unprecedented opportunity to investigate the effect of gene-targeting without the complexities involved in creating a whole animal. Conventional b1-transgenic, b1-knockout, and conditional gene-targeted neurons will be examined. In the next phase of the research project, the best gene-targeting strategy found in these studies will be used to create a gene-targeted mouse with a reversible externally-inducible alteration in sensitivity to general anesthetics. Detailed understanding of how existing general anesthetics work is essential for the development of improved anesthetics without the very significant side effects of existing agents. The experiments proposed will add quantitative and mechanistic information to general anesthetic molecular pharmacology and move this field of investigation closer towards truly understanding how these clinically essential drugs work.
