The GABA(A) receptor family of ligand-gated ion channels consists of heteroligomeric complexes of five subunits from six different classes with each class consisting of several isoforms, however, only ten subunit combinations have been reported. GABA(A) receptors contain binding sites for GABA, barbituates, benzodiazepines, and neurosteroids. 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 the changes in membrane composition commonly associated with chronic exposure to ethanol. 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 induced by GABA(A) receptor agonists, and behavioral effects of acute ethanol are sharply reduced by GABA(A) receptor antagonists. In addition, its sensitivity to barbituates and benzodiazapenes means that compromised GABA(A) receptor function is a hallmark of people who simultaneously abuse drugs and alcohol. 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. GABA(A) receptor function is altered by membrane phospholipid composition because purification and reconstitution of fully active receptor depends on the inclusion of native brain lipids. A second indication of functional interaction with lipids is the highly selective effect of free fatty acids. Two highly polyunsaturated fatty acids, docosahexaenoic acid and arachidonic acid, increase the GABA-induced peak chloride ion current when applied at concentrations below 1 micromolar. At higher concentrations, both fatty acids gradually suppressed the peak amplitude. Interestingly, two other polyunsaturated fatty acids, docosapentaenoic acid and docosatetraenoic acid, had no effect at any concentration. Currently we are investigating the effects of membrane cholesterol content on ligand binding and the interaction of ligands with apparently overlapping binding sites. This work has emphasized assays of ligand binding saturation and competition using radio-labeled compounds and filter binding assays. Initial results indicate that both enrichment and depletion of membrane cholesterol reduce the affinity of several important ligands. This would suggest that the native membrane has an optimal cholesterol content with respect to GABA(A) receptor function. This hypothesis will be tested using purified receptor and membranes consisting of defined phospholipids and variable levels of cholesterol. Purification of the receptor free of native lipid has been accomplished on a limited scale. Our purification method involves solubilization of synaptosomal membranes in detergent and purification via affinity chromatography using a high affinity ligand linked to sepharose. Initially, the optimal ligand for this process, Ro 7-1986, was only available in limited quantity as a gift from a pharmaceutical company. Thus, the scale of the purification procedure was quite limited. Recently we were able to obtain a large quantity of Ro 7-1986 via contract with an organic synthesis laboratory. It is anticipated that this will allow the purification procedure to be scaled up to produce quantities of purified receptor suitable for reconstitution into phospholipid bilayers of defined composition. This will make it possible to conduct detailed investigations of receptor interaction with the lipid bilayer, and interaction with ethanol under controlled and defined conditions. A distinct advantage of working with the receptor in recombinant membranes is the ability to directly monitor, in real time, all aspects of receptor activity; agonist/antagonist binding, activation, and passage of chloride ions. This will be accomplished via a variety of non-invasive spectroscopic techniques. The binding of fluorescent ligands will be monitored via stopped-flow. Both fluorescence and CD assays of agonist binding will be conducted with a recently acquired stopped flow instrument that is capable of simultaneously collecting CD and fluorescence as well as fluorescence polarization stopped flow data. Both the fluorescence and CD techniques have the added advantage that they require very little sample in order to make accurate measurements.
