Article date: December 2000
By: Andrew Green, Steven Walls, Alan Wise, Richard H Green, Amanda K Martin, Fiona H Marshall in Volume 131, Issue 8, pages 1766-1774
Functional human GABAB(1a,2) and GABAB(1b,2) receptors have been stably expressed in mammalian CHO K1 cells.
Detailed characterization of GABAB ligand binding at each of the receptors has been compared using [3H]‐CGP54626A. In cell membranes fractions, [3H]‐CGP54626A bound to a single site with a KD of 1.51±1.12 nM, Bmax of 2.02±0.17 pmoles mg protein−1 and 0.86±0.20 nM, Bmax of 5.19±0.57 pmoles mg protein−1 for GABAB(1a,2) and GABAB(1b,2) respectively.
In competition binding assays the rank order was identical for both GABAB receptors. For known GABAB agonists the rank order was CGP27492>SKF97541=CGP46381>GABA>Baclofen and for GABAB antagonists the rank order was CGP54262A>CGP55845>CGP52432>SCH 50911>CGP51176>CGP36742=CGP35348 2‐OH Saclofen ABPA.
The allosteric effect of calcium cations was also investigated. The effect of removal of CaCl2 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 GABAB receptors.
A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABAB receptors. In competition assays, the rank order was the same for both GABAB(1a,2) and GABAB(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 GABAB receptor are pharmacologically indistinguishable.
Functional human GABAB(1a,2) and GABAB(1b,2) receptors have been stably expressed in mammalian CHO K1 cells.
Detailed characterization of GABAB ligand binding at each of the receptors has been compared using [3H]‐CGP54626A. In cell membranes fractions, [3H]‐CGP54626A bound to a single site with a KD of 1.51±1.12 nM, Bmax of 2.02±0.17 pmoles mg protein−1 and 0.86±0.20 nM, Bmax of 5.19±0.57 pmoles mg protein−1 for GABAB(1a,2) and GABAB(1b,2) respectively.
In competition binding assays the rank order was identical for both GABAB receptors. For known GABAB agonists the rank order was CGP27492>SKF97541=CGP46381>GABA>Baclofen and for GABAB antagonists the rank order was CGP54262A>CGP55845>CGP52432>SCH 50911>CGP51176>CGP36742=CGP35348 2‐OH Saclofen ABPA.
The allosteric effect of calcium cations was also investigated. The effect of removal of CaCl2 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 GABAB receptors.
A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABAB receptors. In competition assays, the rank order was the same for both GABAB(1a,2) and GABAB(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 GABAB receptor are pharmacologically indistinguishable.
British Journal of Pharmacology (2000) 131, 1766–1774; doi:10.1038/sj.bjp.0703755
DOI: 10.1038/sj.bjp.0703755
View this article