The first goal of this project is to use purified GABA(A) receptors reconstituted in liposomes to determine the membrane composition required for optimal receptor function. The second goal is to use the receptor-liposome system to investigate the action of acute ethanol on GABA(A) receptors, with and without postulated cofactors. GABA(A) receptors are among the most sensitive neuronal signaling systems to ethanol and clearly play a role in the neural adaptation which underlies ethanol dependence. Many behavioral effects of ethanol are mimicked by GABA(A) receptor agonists, and behavioral effects of acute ethanol are sharply reduced by GABA(A) receptor antagonists. Changes in GABA(A)receptor function are associated with ethanol tolerance and dependence. The mechanism whereby acute ethanol exposure potentiates GABA(A)receptor activity remains controversial. Current proposals in the literature range from direct binding of ethanol to involement of cofactors such as neurosteroids or protein kinase C. Reconstitution studies have demonstrated that GABA(A) receptor activity is acutely sensitive to the composition of the membrane, although the specific requirements for optimal function have not been delineated. The first phase of this project will involve purification of GABA(A) receptor from bovine brain. We have begun to synthesize an agarose affinity resin which will be used to purify GABA(A) receptors from detergent-solubilized membranes. Conditions for optimal solubilization and reconstitution are under investigation. A stopped-flow fluorescence spectrometer will be used to measure single-turnover activity in real time. The second phase of this project will involve the purification and reconstitution of specific isoforms of human GABA(A) receptor expressed in insect (Sf-9) cells.
