Identification of a residue in the gamma-aminobutyric acid type A receptor alpha subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding.
GABAA receptors that contain either the alpha4- or alpha6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including beta-carbolines, bind to these diazepam-insensitive receptor subtypes. Some beta-carbolines [e.g. ethyl beta-carboline-3-carboxylate (beta-CCE) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)] display a higher affinity for alpha4- compared to alpha6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric alpha6/alpha4 subunits and co-expressed these with wild-type rat beta2 and gamma2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (alpha6N204/alpha4I203). Substitutions at alpha6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive alpha1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the beta-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the alpha1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by approximately 19-fold and approximately 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the alpha subunit in diazepam-sensitive and -insensitive receptors.
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Jason M C
JM
Derry
Susan M J
SM
Dunn
Martin
M
Davies
D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism* / Q000493:pharmacokinetics; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics*; D002243:Carbolines / Q000378:metabolism / Q000493:pharmacokinetics; D002460:Cell Line; D003975:Diazepam / Q000378:metabolism* / Q000494:pharmacology; D018014:Gene Transfer Techniques; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D002243:Carbolines; D008024:Ligands; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D001569:Benzodiazepines; D003975:Diazepam