Relative positioning of diazepam in the benzodiazepine-binding-pocket of GABA receptors.
GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket. With this aim, we mutated residues located in the benzodiazepine-binding site individually to cysteine. These mutated receptors were combined with benzodiazepine site ligands carrying a cysteine reactive group in a defined position. Proximal apposition of reaction partners will lead to a covalent reaction. We describe here such proximity-accelerated chemical coupling reactions of alpha(1)S205C and alpha(1)T206C with a diazepam derivative modified at the C-3 position with a reactive isothiocyanate group (-NCS). We also provide new data that identify alpha(1)H101C and alpha(1)N102C as exclusive sites of the reaction of a diazepam derivative where the -Cl atom is replaced by a -NCS group. Based on these observations we propose a relative positioning of diazepam within the benzodiazepine-binding site of alpha(1)beta(2)gamma(2) receptors.
Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse, Switzerland.
Kelly R
KR
Tan
Roland
R
Baur
Sébastien
S
Charon
Maurice
M
Goeldner
Erwin
E
Sigel
D000494:Allosteric Regulation / Q000235:genetics; D000818:Animals; D014151:Anti-Anxiety Agents / Q000494:pharmacology*; D001569:Benzodiazepines / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D001703:Biophysics; D002461:Cell Line, Transformed; D003545:Cysteine / Q000378:metabolism; D003975:Diazepam / Q000494:pharmacology*; D016291:Dizocilpine Maleate / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D005442:Flumazenil / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D009865:Oocytes; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D014151:Anti-Anxiety Agents; D018757:GABA Modulators; D011963:Receptors, GABA-A; D001569:Benzodiazepines; C082821:3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine; D005442:Flumazenil; D005680:gamma-Aminobutyric Acid; D016291:Dizocilpine Maleate; D003545:Cysteine; D003975:Diazepam