Functional Modulation of Human Recombinant γ-Aminobutyric Acid Type A Receptor by Docosahexaenoic Acid

Abstract

Human ␥-aminobutyric acid type A (GABA A ) receptors were expressed in the baculovirus/Sf-9 insect cell expression system using recombinant cDNA of ␣ 1 ␤ 2 ␥ 2s subunits. ؊6 M or higher, gradually suppressed the peak amplitude of GABA response. A protein kinase A inhibitor, a protein kinase C inhibitor, and a Ca 2؉ chelator did not modify the effects of DHA on GABA-induced chloride ion current. Six unsaturated fatty acids other than DHA were examined. Arachidonic acid mimicked the effect of DHA while e.g. oleic acid had no effect.The inhibition of the GABA response in the presence of DHA was also observed in cells expressing GABA A receptors of ␣ 1 and ␤ 2 subunit combinations. The data show that the ␥ subunit is essential for DHA and arachidonic acid to potentiate the GABA-induced Cl ؊ channel activity and to affect the desensitization kinetics of the GABA A receptor.Polyunsaturated fatty acids are essential fatty acids that are abundantly bound in the phospholipids of brain membranes. The fatty acids influence the membrane fluidity of the neuronal plasma membrane as well as the functional properties of integral membrane protein (1). Arachidonic acid (AA) 1 and docosahexaenoic acid (DHA) are polyunsaturated fatty acids localized in the 2-position of membrane phospholipids and are liberated by various intracellular enzymes such as phospholipase A 2 and diacylglycerol lipase (2).Recently, various actions of these free fatty acids on the functional properties of neuronal plasma membrane neurotransmitter receptors have been reported, such as N-methyl-Daspartic acid receptor (3, 4), kainate receptor (4), and potassium channel receptor (5, 6). As for the effect of unsaturated fatty acids on ␥-aminobutyric acid type A (GABA A ) receptors, an electrophysiological study demonstrated that AA and DHA potently suppressed the GABA A receptor-mediated current response in the rat substantia nigra neurons (7). Receptor binding studies have shown marked effects of unsaturated fatty acids on brain GABA A receptor complexes in vitro (8 -11), as well as on human recombinant GABA A receptors expressed in Sf-9 insect cells (12). These findings suggest a variety of actions of fatty acids on the GABA A receptor-channel complex. The diversity of effects induced by free fatty acids might be due to ontogenetic, phylogenetic, and regional differences of the GABA A receptor subunit composition in the brain regions studied (13). Furthermore, a recent study on recombinant human GABA A receptors (12) reported that the effect of unsaturated fatty acids was dependent on the subunit composition of the GABA A receptor complexes. In addition, various combinations of GABA A receptor subunits might be expressed in the same neuron, complicating the interpretation of data on the effect of free fatty acids on the GABA A receptor complex obtained at the level of single cells, as, for example, by whole cell patch clamp techniques.The insect cell Sf-9/baculovirus expression system in combination with electrophysiological techniques provide the means for t...

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