title: Activation of protein kinase C reduces benzodiazepine potency at GABAA receptors in NT2-N neurons.
abstract: 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.
authors.affiliation: Cellular and Molecular Neurobiology Program and Department of Neurology, Medical College of Ohio, 3120 Glendale Avenue, Toledo, OH 43614, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Gao
authors.forename: L John
authors.initials: LJ
authors.lastname: Greenfield
mesh_terms: D001569:Benzodiazepines / Q000494:pharmacology; D045744:Cell Line, Tumor; D003975:Diazepam / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D058785:GABA-A Receptor Agonists; D006801:Humans; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D011493:Protein Kinase C / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000378:metabolism*
chemical_list: D058785:GABA-A Receptor Agonists; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D011493:Protein Kinase C; D003975:Diazepam