Characterisation and partial purification of the GABAB receptor from the rat cerebellum using the novel antagonist [[formula omitted]]CGP 62349

Abstract

The novel GABAB receptor antagonist [3H]CGP 62349 binds rat cerebellar synaptosomal membranes with high affinity at a single population of sites (Kd=0.9 nM, Bmax=760 fmol/mg protein). Solubilisation with 1% Triton X-100/0.5 M NaCl/10% glycerol resulted in a marked increase in [3H]CGP 62349 binding (Kd=0.5 nM, Bmax=1285 fmol/mg protein). Competition of [3H]CGP 62349 binding with a range of GABAergic ligands yielded the same rank order at membrane-bound and soluble receptors: CGP 54626=CGP 55845≫GABA>(−)-baclofen>CGP 35348=CGP 36742. The GABAA ligand isoguvacine did not displace [3H]CGP 62349 binding. Partial purification of [3H]CGP 62349 binding sites was obtained by sucrose density centrifugation and a predominant protein in the peak binding fraction was recognised by an anti-GABAB receptor antibody and had a molecular weight similar to the recombinant expressed GABABR1a. These results demonstrate that [3H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABAB receptor.