title: The benzodiazepine diazepam potentiates responses of α1β2γ2L γ-aminobutyric acid type A receptors activated by either γ-aminobutyric acid or allosteric agonists.
abstract: BACKGROUND
  The γ-aminobutyric acid (GABA) type A receptor is a target for several anesthetics, anticonvulsants, anxiolytics, and sedatives. Neurosteroids, barbiturates, and etomidate both potentiate responses to GABA and allosterically activate the receptor. We examined the ability of a benzodiazepine, diazepam, to potentiate responses to allosteric agonists.
  
  
  METHODS
  The GABA type A receptors were expressed in human embryonic kidney 293 cells and studied using whole-cell and single-channel patch clamp. The receptors were activated by the orthosteric agonist GABA and allosteric agonists pentobarbital, etomidate, and alfaxalone.
  
  
  RESULTS
  Diazepam is equally potent at enhancing responses to orthosteric and allosteric agonists. Diazepam EC50s were 25 ± 4, 26 ± 6, 33 ± 6, and 26 ± 3 nm for receptors activated by GABA, pentobarbital, etomidate, and alfaxalone, respectively (mean ± SD, 5-6 cells at each condition). Mutations to the benzodiazepine-binding site (α1(H101C), γ2(R144C), γ2(R197C)) reduced or removed potentiation for all agonists, and an inverse agonist at the benzodiazepine site reduced responses to all agonists. Single-channel data elicited by GABA demonstrate that in the presence of 1 μm diazepam the prevalence of the longest open-time component is increased from 13 ± 7 (mean ± SD, n = 5 patches) to 27 ± 8% (n = 3 patches) and the rate of channel closing is decreased from 129 ± 28 s(-1) to 47 ± 6 s(-1) (mean ± SD) CONCLUSIONS: We conclude that benzodiazepines do not act by enhancing affinity of the orthosteric site for GABA but rather by increasing channel gating efficacy. The results also demonstrate the presence of interactions between allosteric activators and potentiators, raising a possibility of effects on dosage requirements or changes in side effects.
authors: Department of Anesthesiology and Taylor Family Institute for Innovative Psychiatric Research, Washington University School of Medicine, St. Louis, Missouri, USA.
authors: Ping
authors: P
authors: Li
authors: Megan M
authors: MM
authors: Eaton
authors: Joe Henry
authors: JH
authors: Steinbach
authors: Gustav
authors: G
authors: Akk
mesh_terms: D000777:Anesthetics / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003975:Diazepam / Q000494:pharmacology*; D004357:Drug Synergism; D005045:Etomidate / Q000494:pharmacology*; D018757:GABA Modulators / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D018408:Patch-Clamp Techniques / Q000379:methods; D010424:Pentobarbital / Q000494:pharmacology*; D011277:Pregnanediones / Q000494:pharmacology*; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
chemical_list: D000777:Anesthetics; D018757:GABA Modulators; D011277:Pregnanediones; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; C006477:alphaxalone; D010424:Pentobarbital; D003975:Diazepam; D005045:Etomidate