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

The novel GABA(B) receptor antagonist [3H]CGP 62349 binds rat cerebellar synaptosomal 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 [3H]CGP 62349 binding (K(d) = 0.5 nM, B(max) = 1285 fmol/mg protein). Competition of [3HCGP 35348 = CGP 36742. The GABA(A) 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-GABA(B) receptor antibody and had a molecular weight similar to the recombinant expressed GABA(B)R1a. These results demonstrate that [3H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABA(B) receptor.