Control of GABAA Receptor α4 Subunit Expression In a Human Neuroblastoma Cell Line

Abstract

The GABAA receptor (GABAR) is a pentameric ligand-gated Cl- channel, normally composed of two α, two β and one γ subunit. α4 expression has been shown to increase in response to extended periods of in vivo administration of positive modulators of the GABAR, such as neurosteroids, benzodiazepines (BZs) and ethanol. However, the mechanism of α4 upregulation is not clear. Therefore, the purpose of this study was to establish an in vitro model to study neurosteroid regulation of α4 expression. I proposed two potential initial triggers for α4 upregulation: i.) increased current produced by a positive GABA modulator or ii.) allosteric events produced by ligand-receptor interactions. To this end, I used the human neuroblastoma IMR-32 cell line to demonstrate increases in α4 expression by 48 h administration of the GABA-modulatory steroid 3α,5β-THP (3α -OH-5β -pregnan-20-one) following differentiation by nerve growth factor (NGF) in serum-free medium. Functional expression of α4βγ2 GABAR was validated by the unique pharmacology characteristic of this GABAR isoform, which include a near complete insensitivity to BZ agonists, but potent positive modulation by the BZ antagonist flumazenil and the BZ partial inverse agonist RO15-4513. THP induction of α4 expression was a result of its enhancement of GABA-gated current because co-application of the GABAR blocker picrotoxin prevented this effect. Moreover, 48 h administration of compounds which decrease GABA-gated current, such as submaximal concentrations of the GABAR open channel blocker penicillin G or the competitive GABAR antagonist gabazine, decreased α4 expression, as did the BZ inverse agonist DMCM. However, 48 h exposure to the BZ antagonist flumazenil, which allosterically binds to the GABAR but has no effect on GABA-gated current, did not change α4 expression. These findings suggest that α4 expression is correlated with the change in GABA-gated current. The results from my study may have implications for alterations in α4 expression across naturally occurring fluctuations in endogenous steroids.