Molecular mechanisms of benzodiazepine-induced downregulation of GABA(A) receptor alpha(1) subunit protein in rat cerebellar granule cells

1 Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acid(A) (GABA(A)) receptor alpha(1) subunit protein, that was dose-dependent (1 nM - 1 mu M) and prevented by the benzodiazepine antagonist flumazenil (1 mu M). After 2 days of treatment with 1 mu M flunitrazepam the alpha(1) subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABA(A) receptor alpha(6) subunit protein over the 2-12 day period. 2 GABA treatment for 2 days down-regulates the alpha(1) subunit protein in a dose-dependent (10 mu M - 1 mM) manner that was prevented by the selective GABA(A) receptor antagonist bicuculline (10 mu M). At 10 mu M and 1 mM GABA the reduction in alpha(1) subunit expression compared to controls was 31% and 66%, respectively. 3 The flunitrazepam-induced decrease in alpha(1) subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABA(A) receptor channel activity. 4 Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha(1) subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5 The flunitrazepam-induced down-regulation of alpha(1) subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 mu M). 6 This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABA(A) receptor alpha(1) subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect is independent of GABA, and that the benzodiazepine and GABA sites may not be equally coupled to the down-regulation process, with the benzodiazepine site being the more dominant. The biochemical mechanism underlying the benzodiazepine-mediated down-regulation of the alpha(1) subunit protein seems to involve the activity of staurosporine-sensitive protein kinases.