Gamma-amino-butyric-acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and one subtype of this neurotransmitter receptor, the GABAA receptor is a likely pharmacological target for general anesthetics. It is clear that general anesthetics act on the GABAA receptor in a highly specific manner. However the precise loci of the general anesthetic action within the GABAA receptor polypeptide, or the """"""""sites-of-action"""""""", is unknown. Defining the sites-of-action of general anesthetics is only possible by a systematic study of the interactions between general anesthetics and expressed GABAA receptors composed of known subunits where the precise amino acid sequences are known. GABAA receptors consisting of only the beta3 or the rho1 subunits expressed in Xenopus oocytes show diametrically opposite electrophysiological responses to GABAA and general anesthetics, and therefore, are excellent model systems for the molecular dissection of the sites-of-action. General anesthetics induce currents, but GABA has no effect on the beta3 subunit receptors. Conversely, the rho1 subunit receptors respond to GABA, but not to general anesthetics. The goal of this study is to test the hypothesis that general anesthetics interact with the GABAA receptor in a domain specific manner at sites distinct from the GABA recognition site. This will be accomplished by electrophysiological experiments on recombinant native, mutated, or chimeric GABAA receptors expressed in Xenopus oocytes. The epitope-tag technique, in combination with immunohistochemistry and Western blot analysis, will be used to distinguish non-functional from non-expressed receptors. Through a systematic study of the amino acid similarities and differences between different subunits, the sites on the receptor polypeptide necessary for the direct opening of channels by general anesthetics, and the sites necessary for the potentiation of GABA induced currents, will be defined. Next, the physiological relevance of these identified sites-of-action will be tested in GABAA receptor subunit combinations most likely to be present in the brain. Understanding where general anesthetics work is essential for future development of general anesthetics without the side-effects which presently contribute to clinical morbidity and mortality. Furthermore, once the sites-of-action are defined. site specific reversal agents could possibly be developed. Such an addition to the pharmacological armamentarium available to clinical anesthesiologists is of immense significance, perhaps only secondary to the discovery of general anesthetics itself.
