title: Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.
abstract: GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Middendorp
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Maldifassi
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Roland
authors.initials: R
authors.lastname: Baur
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland. Electronic address: sigel@ibmm.unibe.ch.
authors.forename: Erwin
authors.initials: E
authors.lastname: Sigel
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D003975:Diazepam / Q000737:chemistry / Q000494:pharmacology; D005442:Flumazenil / Q000737:chemistry / Q000494:pharmacology; D018682:GABA Agents / Q000737:chemistry / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009154:Mutation; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D009865:Oocytes; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms; D011804:Quinolines / Q000378:metabolism / Q000494:pharmacology*; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D018682:GABA Agents; D020033:Protein Isoforms; D011804:Quinolines; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; C000605973:SJM-3 compound; D013844:Thiazoles; D001569:Benzodiazepines; D005442:Flumazenil; D005680:gamma-Aminobutyric Acid; D003975:Diazepam
keywords: Benzodiazepine; Chloride channels; Electrophysiology; GABA(A) receptors; Positive allosteric modulator; SJM-3 (PubChem CID: 49713517); Xenopus oocyte