title: Hydrophobic anions potently and uncompetitively antagonize GABA(A) receptor function in the absence of a conventional binding site.
abstract: OBJECTIVE
  A 'lock-and-key' binding site typically accounts for the effect of receptor antagonists. However, sulphated neurosteroids are potent non-competitive antagonists of GABA(A) receptors without a clear structure-activity relationship. To gain new insights, we tested two structurally unrelated hydrophobic anions with superficially similar properties to sulphated neurosteroids.
  
  
  METHODS
  We used voltage-clamp techniques in Xenopus oocytes and hippocampal neurons to characterize dipicrylamine (DPA) and tetraphenylborate (TPB), compounds previously used to probe membrane structure and voltage-gated ion channel function.
  
  
  RESULTS
  Both DPA and TPB potently antagonized GABA(A) receptors. DPA exhibited an IC₅₀ near 60 nM at half-maximal GABA concentration and antagonism with features indistinguishable from pregnenolone sulphate antagonism, including sensitivity to a point mutation in transmembrane domain 2 of the α1 subunit. Bovine serum albumin, which scavenges free membrane-associated DPA, accelerated both capacitance offset and antagonism washout. Membrane interactions and antagonism were explored using the voltage-dependent movement of DPA between membrane leaflets. Washout of DPA antagonism was strongly voltage-dependent, paralleling DPA membrane loss, although steady-state antagonism lacked voltage dependence. At antagonist concentrations, DPA failed to affect inhibitory post-synaptic current (IPSC) amplitude or decay, but DPA accelerated pharmacologically prolonged IPSCs.
  
  
  CONCLUSIONS
  Neurosteroid-like GABA(A) receptor antagonism appears to lacks a conventional binding site. These features highlight key roles of membrane interactions in antagonism. Because its membrane mobility can be controlled, DPA may be a useful probe of GABA(A) receptors, but its effects on excitability via GABA(A) receptors raise caveats for its use in monitoring neuronal activity.
authors: Departments of Psychiatry Anatomy & Neurobiology, Washington University School of Medicine, St. Louis, MO, USA.
authors: M
authors: M
authors: Chisari
authors: K
authors: K
authors: Wu
authors: C F
authors: CF
authors: Zorumski
authors: S
authors: S
authors: Mennerick
mesh_terms: D000818:Animals; D000838:Anions / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D025802:Electric Capacitance; D005260:Female; D058787:GABA-A Receptor Antagonists / Q000737:chemistry / Q000494:pharmacology*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D020128:Inhibitory Concentration 50; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D010851:Picrates / Q000737:chemistry / Q000494:pharmacology*; D011284:Pregnenolone / Q000737:chemistry / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D055366:Synaptic Potentials / Q000187:drug effects; D009435:Synaptic Transmission / Q000187:drug effects; D013775:Tetraphenylborate / Q000737:chemistry / Q000494:pharmacology*; D014982:Xenopus laevis
chemical_list: D000838:Anions; D058787:GABA-A Receptor Antagonists; D010851:Picrates; D011963:Receptors, GABA-A; C018370:pregnenolone sulfate; C011454:dipicrylamine; D013775:Tetraphenylborate; D011284:Pregnenolone