The goal of this project is to determine the effects of membrane composition and fatty acids on the function of the GABA(A) receptor, particularly with respect to changes in membrane composition associated with chronic ethanol exposure. The GABA(A) receptor is a 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 these changes may be due to the altered neuronal membrane composition associated with chronic ethanol exposure. ? Dietary n-3 fatty acid (n-3FA) deficiency impairs normal development and learning, and causes a significant reduction in docosahexaenoic acid (DHA, 22:6n-3), which is normally present at high concentrations in the mammalian CNS. GABA(A) receptor function may be mediated in part by DHA through effects on membrane composition or via direct interactions with transmembrane regions of the protein. We examined the consequences of DHA phospholipid depletion resulting from an n-3FA deficient diet on GABA(A) receptor function in rats raised on an n-3 FA deficient diet. The predominant change in brain FA composition for rats fed an n-3 FA deficient diet was a 60% depletion of brain DHA compared to controls. Purified rat brain synaptosomes were microtransplanted into Xenopus oocytes for electrophysiological assessment of GABAergic function. This technique yielded surface expression of functional GABA(A) recptors that elicited current in response to GABA. This current could be inhibited by the competitive antagonist SR-95531 and potentiated by the benzodiazepine flurazepam. The GABA EC50 for oocytes injected with adult female n-3 FA deficient rat brain synaptosomes was significantly increased compared to controls (280 and 100?YM, respectively). Maximum current elicited by GABA was also increased in these oocytes compared to controls (60 and 35nA, respectively). These results suggest that GABAergic function is sensitive to the presence of DHA-containing phospholipids in the synaptic plasma membrane. ? Neuronal membranes are abundant in polyunsaturated fatty acids (FAs), particularly the n-3 FA docosahexaenoic acid (DHA, 22:6n-3) and the n-6 FA arachidonic acid (AA, 20:4n-6). AA and/or DHA influence the function of several important neuronal transmembrane proteins such as G-protein coupled receptors and some voltage-gated ion channels. We determined the effect of direct application of PUFAs on the function of the inhibitory LGICs, the alpha1,beta2,gamma 2 GABA(A) receptor and the alpha1 homomeric glycine receptor. These receptors are pentameric transmembrane proteins with a total of twenty transmembrane regions, thus it is likely that membrane components play important roles in modulating their function. Receptors were expressed in Xenopus laevis oocytes and two-electrode voltage clamp was used to measure neurotransmitter concentration response curves in the absence and presence of the free FAs DHA, AA, docosapentanoic acid (DPAn-6, 22:5n-6) and oleic acid (OA, 18:1n9-) (10 microM). Neither OA nor DPAn-6 altered the function of either of the receptors, demonstrating that effects of the other FAs are not simply due to the prescence of a hydrophobic compound. DHA altered the function of both the GABA(A) and glycine receptors. DHA significantly decreased the GABA and glycine EC-50 values for their respective receptors by approximately 75%. In addition, application of DHA decreased the maximum currents elicited by GABA by approximately 60%. Thus, while DHA application improved the receptors!| affinities for the neurotransmitters, it decreased the GABA(A) receptors!| efficacy of response to the neurotransmitter. This suggests that DHA improves ligand-receptor interactions while negatively affecting channel gating processes. Both receptors showed remarkable molecular specificity as they had no response to a 22-carbon n-6 fatty acid and while exhibiting distinct responses to a 20-carbon n-6 fatty acid and a 22-carbon n-3 fatty acid.
