title: Characteristics of concatemeric GABA(A)  receptors containing α4/δ subunits expressed in Xenopus oocytes.
abstract: OBJECTIVE
  GABA(A) receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABA(A) receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems.
  
  
  METHODS
  We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes.
  
  
  RESULTS
  δ-β2 -α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn(2+) . We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations.
  
  
  CONCLUSIONS
  The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors.
authors.affiliation: Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Hong-Jin
authors.initials: HJ
authors.lastname: Shu
authors.forename: John
authors.initials: J
authors.lastname: Bracamontes
authors.forename: Amanda
authors.initials: A
authors.lastname: Taylor
authors.forename: Kyle
authors.initials: K
authors.lastname: Wu
authors.forename: Megan M
authors.initials: MM
authors.lastname: Eaton
authors.forename: Gustav
authors.initials: G
authors.lastname: Akk
authors.forename: Brad
authors.initials: B
authors.lastname: Manion
authors.forename: Alex S
authors.initials: AS
authors.lastname: Evers
authors.forename: Kathiresan
authors.initials: K
authors.lastname: Krishnan
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Covey
authors.forename: Charles F
authors.initials: CF
authors.lastname: Zorumski
authors.forename: Joe Henry
authors.initials: JH
authors.lastname: Steinbach
authors.forename: Steven
authors.initials: S
authors.lastname: Mennerick
mesh_terms: D000818:Animals; D005260:Female; D018755:GABA Agonists / Q000378:metabolism / Q000494:pharmacology; D018756:GABA Antagonists / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D018377:Neurotransmitter Agents / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015202:Protein Engineering; D021122:Protein Subunits; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D018755:GABA Agonists; D018756:GABA Antagonists; D018377:Neurotransmitter Agents; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins