title: Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs.
abstract: Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.
authors.forename: Roland
authors.initials: R
authors.lastname: Baur
authors.forename: Kelly R
authors.initials: KR
authors.lastname: Tan
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Lüscher
authors.forename: Anne
authors.initials: A
authors.lastname: Gonthier
authors.forename: Maurice
authors.initials: M
authors.lastname: Goeldner
authors.forename: Erwin
authors.initials: E
authors.lastname: Sigel
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000495:Allosteric Site / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D001923:Brain Chemistry / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D003975:Diazepam / Q000031:analogs & derivatives* / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008968:Molecular Conformation; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D020033:Protein Isoforms / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D018757:GABA Modulators; D008024:Ligands; D020033:Protein Isoforms; D021122:Protein Subunits; D011963:Receptors, GABA-A; D003975:Diazepam