Characterization of [H-3]-CGP54626A binding to heterodimeric GABA(B) receptors stably expressed in mammalian cells

1 Functional human GABA(B(1a,2)) and GABA(B(1b,2)) receptors have been stab ly expressed in mammalian CHO K1 cells. 2 Detailed characterization of GABAB ligand binding at each of the receptor s has been compared using [H-3]-CGP54626A.In cell membranes fractions, [H-3 ]-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 p moles mg protein(-1) for GABA(B(1a,2)) and GABA(B(1b,2)) respectively. 3 In competition binding assays the rank order was identical for both GABA( B) receptors. For known GABA(B) agonists the rank order was CGP27492 > SKF9 7541 = CGP46381 > GABA > Baclofen and for GABA(B) antagonists the rank orde r was CGP54262A> CGP55845>CGP52432> SCH 50911 > CGP51176 > CGP36742 = CGP35 348 greater than or equal to 2-OH Saclofen greater than or equal to ABPA. 4 The allosteric effect of calcium cations was also investigated. The effec t of removal of CaCl2 from the binding assay conditions was ligand dependen t to either cause a decrease in ligand affinity or to have no significant e ffect. However, these effects were similar for both GABA(B) receptors. 5 A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABA(B) receptors. In compet ition 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. 6 These data suggest that, in terms of ligand binding, the currently identi fied isoforms of the GABA(B) receptor are pharmacologically indistinguishab le.