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, 22:6n3), 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 microM, 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, 22:6n3) and the n-6 FA arachidonic acid (AA, 20:4n6). 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 (DPAn6, 22:5n6), DPAn-3 (22:5n3) and oleic acid (OA, 18:1n9) (10 microM). OA, DPAn3 and DPAn6 had no effect on the function of either receptor, demonstrating that effects of the other FAs are not simply due to the prescence of a hydrophobic compound. For the GABA(A) receptor the maximum current was decreased by more than 50% by DHA (22:6n3), AA (20:4n6) and EPA (20:5n3), but OA (18:1n9), DPAn-6 (22:5n6) and DPAn-3 had no effect. The same pattern of effect was observed for inhibition of the GABA EC50. For the glycine receptor only AA and EPA reduced the maximum current by more than 25%, while all 5 PUFAs reduced the EC50 for glycine. The reduction in EC50 values indicates that the effective PUFAs enhance ligand-receptor interactions, while the reduced maximum currents indicate a reduced efficiency of the channel gating processes.
