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Regulation of the surface expression of 42 GABAA receptors by high efficacy states

Kuver, A;Shen, H;Smith, SS;

4 GABA(A) receptors (GABARs) have low CNS expression, but their expression is increased by 48h exposure to the neurosteroid THP (3-OH-5[]-pregnan-20-one). THP also increases the efficacy of -containing GABARs acutely, where GABA is a partial agonist. Thus, we examined effects of THP (100 nM) and full GABA agonists at 42 (gaboxadol, 10 M, and -alanine, 10 M-1mM), on surface expression of 42. To this end, we used an 4 construct tagged with a 3XFLAG (F) epitope or measured expression of native 4 and . HEK-293 cells or cultured hippocampal neurons were transfected with 4F2 and treated 24h later with GABA agonists, THP, GABA plus THP or vehicle (0.01% DMSO) for 0.5 h-48 h. Immunocytochemistry was performed under both non-permeabilized and permeabilized conditions to detect surface and intracellular labeling, respectively, using confocal microscopy. The high efficacy agonists and GABA (1 or 10 M) plus THP increased 42 surface expression up to 3-fold after 48h, an effect first seen by 0.5h. This effect was not dependent upon the polarity of GABAergic current, although expression was increased by KCC2. Intracellular labeling was decreased while functional expression was confirmed by whole cell patch clamp recordings of responses to GABA agonists. GABA plus THP treatment did not alter the rate of receptor removal from the surface membrane, suggesting that THP-induced 42 expression is likely via receptor insertion. Surface expression of 42 was decreased by rottlerin (10 M), suggesting a role for PKC-. These results suggest that trafficking of 42 GABARs is regulated by high efficacy states.