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

• Published in: Brain research. Molecular brain research. Vol. 71; no. 2; pp. 279 - 289
• Main Authors: Keir, MJ, Barakat, MJ, Dev, K K, Bittiger, H, Bettler, B, Henley, J M
• Format: Journal Article
• Language: English
• Published: 25.08.1999
• ISSN: 0169-328X
• DOI: 10.1016/S0169-328X(99)00199-0

The novel GABA sub(B) receptor antagonist [ super(3)H]CGP 62349 binds rat cerebellar synaptosomal membranes with high affinity at a single population of sites (K sub(d)=0.9 nM, B sub(max)=760 fmol/mg protein). Solubilisation with 1% Triton X-100/0.5 M NaCl/10% glycerol resulted in a marked increase in [ super(3)H]CGP 62349 binding (K sub(d)=0.5 nM, B sub(max)=1285 fmol/mg protein). Competition of [ super(3)H]CGP 35348=CGP 36742. The GABA sub(A) ligand isoguvacine did not displace [ super(3)H]CGP 62349 binding. Partial purification of [ super(3)H]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 sub(B) receptor antibody and had a molecular weight similar to the recombinant expressed GABA sub(B)R1a. These results demonstrate that [ super(3)H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABA sub(B) receptor.