Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures.

Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

Changes in gamma-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro.

The amounts of mRNAs encoding alpha 1, alpha 6, beta 2, beta 3, gamma 2, and delta subunits of gamma-aminobutyrate type A (GABAA) receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal rat granule cells in primary culture. We investigated possible correlations (i) between temporal changes in mRNA content and expression density of their respective translation products and (ii) between the quantitative changes of receptor subunit expression, the GABA EC50 for Cl- channel activation, and diazepam efficacy in modulating GABA action on the Cl- channels. At 3 days in vitro, the amount of GABAA receptor subunit mRNAs and the expression of their respective translation products were very low. During the next 2 weeks both parameters for every subunit studied increased asynchronously; moreover, at 14 days in vitro the sum of gamma 2 and delta subunit expression was smaller than the expression of the alpha 1 or alpha 6 or beta 2/beta 3 subunits. This suggests that during in vitro maturation each subunit may be regulated independently and invites speculation as to possible changes in specific GABAA receptor subtype abundance during development in vitro. The maximal current intensity elicited by GABA failed to increase from 5 to 14 days in vitro, though the amount of mRNA encoding various subunits and the expression density of their respective translation products increased. Thus, qualitative changes in the GABAA receptor subtypes expressed and/or abnormalities in the subunit assembly very likely account for the uniformity of the maximal current intensity elicited by GABA during in vitro development. Also, during maturation of neuronal cultures from 5 to 20 days in vitro the extent of the positive modulation of GABA action by diazepam decreased dramatically. This finding might be related to an increase in the abundance of GABAA receptors including the alpha 6 subunit and/or to the expression, during granule cell maturation in vitro, of GABAA receptors devoid of gamma 2 subunits.