Characterisation and partial purification of the GABA(B) receptor from therat cerebellum using the novel antagonist [H-3]CGP 62349

The novel GABA(B) receptor antagonist [H-3]CGP 62349 binds rat cerebellar s ynaptosomal membranes with high affinity at a single population of sites (K -d = 0.9 nM, B-max = 760 fmol/mg protein). Solubilisation with 1% Triton X- 100/0.5 M NaCl/10% glycerol resulted in a marked increase in [H-3]CGP 62349 binding (K-d = 0.5 nM, B-max = 1285 fmol/mg protein). Competition of [H-3] CGP 62349 binding with a range of GABAergic ligands yielded the same rank o rder at membrane-bound and soluble receptors: CGP 54626 = CGP 55845 >> GABA > (-)-baclofen > CGP 35348 = CGP 36742. The GABA(A) ligand isoguvacine did not displace [H-3]CGP 62349 binding. Partial purification of [H-3]CGP 6234 9 binding sites was obtained by sucrose density centrifugation and a predom inant protein in the peak binding fraction was recognised by an anti-GABA(B ) receptor antibody and had a molecular weight similar to the recombinant e xpressed GABA(B)R1a. These results demonstrate that [H-3]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABA(B) receptor. (C) 1999 Elsevier Science B.V. All rights reserved.