Molecular and Cellular Biology Regulation of α4β2δ GABAA Receptor Cell Surface Expression.

Abstract

The extrasynaptically expressed α4βδ subtype of the ligand-gated chloride channel receptor GABAA regulates tonic inhibition in the hippocampus, dentate gyrus, and thalamus. It has exemplified a level of plasticity and physiological response under conditions of stress or models of related disorders such as premenstrual dysphoric disorder (PMDD), pre-menstrual syndrome (PMS), and puberty when circulating levels of THP (allopregnenalone), a stress-related neurosteroid, are fluctuating. These fluctuations in THP levels lead to increases in α4 and δ subunit expression in the pyramidal cells of the CA1 hippocampus where expression is normally undetectable. Little is known about the fluctuations in cell surface expression and the mechanism underlying these alterations. Therefore, the purpose of this study was to establish putative mechanisms for up-regulation of surface α4β2δ receptor in response to specific agonists and modulators with the use of a novel 3XFLAG-tagged α4 subunit reporter in recombinant in vitro systems. I proposed that: i) the efficacy of a ligand to α4β2δ influences its expression and physiology, ii) receptor insertion and internalization are affected by specific ligand interactions, and iii) the changes in the levels of α4β2δ cell surface receptors are influenced by chloride (Cl-) transporters. I used Human Embryonic Kidney (HEK-293) and cultured hippocampal cells to demonstrate that the expression of α4β2δ recombinant receptors increased after 48 hours of treatment with gamma-aminobutyric acid (GABA) plus THP, or with gaboxadol or β-alanine, GABA agonists which have high efficacy for δ-containing GABAA receptors. This increase in cell surface expression was inhibited by the benzodiazepine antagonist flumazenil, which was found to act as a negative modulator at α4β2δ GABAA receptors. Internalization and lysosomal degradation affected the level of surface receptor expression as down-regulation of α4βδ caused by flumazenil was prevented upon treatment of cells with specific blockers of these mechanisms. We observed that the level of cell surface receptor during down-regulation was due to dynamin-mediated endocytosis, but that initial up-regulation was a result of receptor insertion from ER trafficking. In addition to flumazenil, bumetanide, the Na+-K+-Cl- (sodium-potassium- chloride) co-transporter blocker, also was found to reduce α4βδ expression, suggesting a role for Cl- levels in triggering receptor expression. Information gathered from this study suggests that receptor insertion underlies the increase in surface expression of α4βδ produced by high efficacy states. In addition, these data suggest that flumazenil may be useful therapeutically in treating clinical conditions which are related to α4β2δ receptor function.