The goal of this project is to determine the effects of membrane composition on the function of the GABA(A) receptor, particularly with respect to changes in membrane composition commonly associated with chronic ethanol exposure. The GABA(A) receptor is a member of the cys-loop super family of ligand-gated ion channels and conducts chloride ions in response to binding the neurotransmitter gamma-amino butyric acid (GABA). GABA(A) receptors are among the most sensitive neuronal signaling systems to ethanol and play a role in the neural adaptation which underlies ethanol dependence. Changes in GABA(A) receptor function are associated with ethanol tolerance and dependence and may be due to the altered neuronal membrane composition associated with chronic ethanol exposure. During the past year we completed two studies of the interaction between the synaptic membrane and the GABA(A) receptor, made significant progress towards purifying the receptor from native tissue in quantities suitable for biophysical measurements, and initiated a study that will allow direct examination of ligand-induced chloride flux. The first completed study was an investigation of the effects of membrane cholesterol content on ligand binding and the interaction of ligands with allosterically linked binding sites. We showed that depletion of membrane cholesterol reduces the affinity of several benzodiazepines (BZs), while enrichment of membrane cholesterol had no effect on BZ binding. The effects of cholesterol on BZ binding were completely reversible, regardless of the order in which membrane cholesterol was manipulated. This strongly suggests that the effect of depleted membrane cholesterol is not due to alteration of a cholesterol-dependent membrane structure such as a lipid raft, but is due to specific receptor- cholesterol interaction. This study is currently under review for publication. The second completed study showed that a diet deficient in long chain n-3 fatty acids compromises BZ binding in rat synaptosomes in a manner that varies with gender. Binding of BZ by endogenous GABA(A) receptor was examined in synaptosomes from the cerebra of seven and twelve week old Sprague-Dawley rats raised on diets that were either deficient or adequate in long chain (LC) n-3 fatty acids. For rats fed the adequate LC n-3 fatty acid (FA) diet, there were no gender-related differences in BZ binding at either seven or twelve weeks of age. Likewise, no differences in synaptosome fatty acid composition were observed between male and female rats on the adequate LC n-3 FA diet. The deficient LC n-3 FA diet produced significant differences in BZ binding between male and female rats. Males on the n-3 FA deficient diet lost DHA and total LC n-3 FA as they aged, while females increased their synaptosomal levels of both DHA and total LC n-3 FA. The divergence in synaptosomal fatty acid composition between n-3 FA deficient male and female rats as both aged was correlated with increasing gender-related differences in GABAA receptor function. This study is currently under review for publication. A new study initiated this year will enable direct, electrophysiological monitoring of GABA(A) chloride flux activity of receptors either expressed in Xenopus oocytes or injected into Xenopus oocytes. We are using the two-electrode voltage clamp technique to monitor ligand-induced chloride currents. Currently this technique is being used to examine the effects of membrane cholesterol on chloride flux by using methyl-beta-cyclodextrin (MBCD) to deplete and enrich oocytes with cholesterol. Preliminary results indicate that the cholesterol content of the outer oocyte membrane can be manipulated with MBCD using the same techniques we successfully applied to brain synaptosomes. The next stage in this study will be to examine the effects of cholesterol on the chloride flux behavior of several single-site mutants of GABA(AAA) where putatively membrane-exposed residues have been altered. In preparation for this work we have acquired the DNA for several single-site GABA(A) mutants.
