title: Characterization of [(3)H]-CGP54626A binding to heterodimeric GABA(B) receptors stably expressed in mammalian cells.
abstract: Functional human GABA(B(1a,2)) and GABA(B(1b,2)) receptors have been stably expressed in mammalian CHO K1 cells. Detailed characterization of GABA(B) ligand binding at each of the receptors has been compared using [(3)H]-CGP54626A. In cell membranes fractions, [(3)H]-CGP54626A bound to a single site with a K(D) of 1. 51+/-1.12 nM, B(max) of 2.02+/-0.17 pmoles mg protein(-1) and 0. 86+/-0.20 nM, B(max) of 5.19+/-0.57 pmoles mg protein(-1) for GABA(B(1a,2)) and GABA(B(1b,2)) respectively. In competition binding assays the rank order was identical for both GABA(B) receptors. For known GABA(B) agonists the rank order was CGP27492>SKF97541=CGP46381>GABA>Baclofen and for GABA(B) antagonists the rank order was CGP54262A>CGP55845>CGP52432>SCH 50911>CGP51176>CGP36742=CGP35348 > or =2-OH Saclofen > or =ABPA. The allosteric effect of calcium cations was also investigated. The effect of removal of CaCl(2) from the binding assay conditions was ligand dependent to either cause a decrease in ligand affinity or to have no significant effect. However, these effects were similar for both GABA(B) receptors. A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABA(B) receptors. In competition assays, the rank order was the same for both GABA(B(1a,2)) and GABA(B(1b,2)) and consistent with that seen with cell membrane fractions. These data suggest that, in terms of ligand binding, the currently identified isoforms of the GABA(B) receptor are pharmacologically indistinguishable.
authors.affiliation: Receptor Systems Unit, Glaxo Wellcome Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY. AG9085@Glaxowellcome.co.uk
authors.forename: A
authors.initials: A
authors.lastname: Green
authors.forename: S
authors.initials: S
authors.lastname: Walls
authors.forename: A
authors.initials: A
authors.lastname: Wise
authors.forename: R H
authors.initials: RH
authors.lastname: Green
authors.forename: A K
authors.initials: AK
authors.lastname: Martin
authors.forename: F H
authors.initials: FH
authors.lastname: Marshall
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002118:Calcium / Q000494:pharmacology; D006224:Cricetinae; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D018755:GABA Agonists / Q000378:metabolism / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D015870:Gene Expression; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011869:Radioligand Assay; D018080:Receptors, GABA-B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014316:Tritium
chemical_list: D018755:GABA Agonists; D018756:GABA Antagonists; D009943:Organophosphorus Compounds; D018080:Receptors, GABA-B; D014316:Tritium; C107353:CGP 54626; D006165:Guanylyl Imidodiphosphate; D002118:Calcium