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.
Keir
M J
MJ
Department of Anatomy, Medical School, University Walk, Bristol University, Bristol, UK.
Barakat
M J
MJ
Dev
K K
KK
Bittiger
H
H
Bettler
B
B
Henley
J M
JM
D000818:Animals; D001565:Benzoates / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D019921:Biotinylation; D019556:COS Cells; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002531:Cerebellum / Q000737:chemistry* / Q000187:drug effects; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D058788:GABA-B Receptor Antagonists; D007539:Isonicotinic Acids / Q000378:metabolism; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D018080:Receptors, GABA-B / Q000302:isolation & purification*; D014162:Transfection
D001565:Benzoates; C114540:CGP 62349; D003902:Detergents; D058788:GABA-B Receptor Antagonists; D007539:Isonicotinic Acids; D009943:Organophosphorus Compounds; D018080:Receptors, GABA-B; C015300:isoguvacine