Activation of protein kinase C reduces benzodiazepine potency at GABAA receptors in NT2-N neurons.

Phosphorylation of GABA(A) receptors (GABARs) by protein kinase C (PKC) modulates GABAR function and allosteric enhancement by benzodiazepines and barbiturates. However, the effects of phosphorylation have been inconsistent, possibly due to variability in neuron or GABAR populations. We used NT2-N neurons to address this issue in a more homogeneous cell population. Whole-cell and gramicidin "perforated-patch" recordings were used to analyze changes in GABAR currents induced by preincubation with 4beta-phorbol-12,13-dibutyrate (PDBu), the inactive 4alpha-phorbol-didecanoate (4alpha-PDD) or bisindolylmaleimide (BIM, a PKC inhibitor). PDBu, but not 4alpha-PDD, caused a rightward shift of the concentration-response curve (C/R) for diazepam enhancement, without affecting maximal enhancement. BIM blocked the rightward shift of the diazepam C/R induced by PDBu. PDBu did not alter the GABA C/R or the current reversal potential. The PKC effect was specific to the benzodiazepine site, as PDBu did not alter potentiation of GABAR currents by pentobarbital. Exposure to diazepam (10 microM) for 7 days reduced maximal diazepam enhancement without affecting the EC(50); PDBu also caused a small rightward shift of the diazepam EC(50) in these cells. PKC activation reduced the apparent affinity of diazepam at NT2-N GABARs without altering maximal enhancement, suggesting decreased allosteric coupling of the benzodiazepine and GABA sites.