SOLUBILIZATION AND CHARACTERIZATION OF GABA(B) RECEPTOR-BINDING SITESFROM PORCINE BRAIN SYNAPTIC-MEMBRANES

1 The characteristics of membrane bound GABA(B) receptors in pig brain are similar to those in rat brain as judged by in vitro binding exper iments and sensitivity to GTP. The rank order of affinity of GABA(B) r eceptor ligands was CGP 54626 > GABA approximately (-)-baclofen >> CGP 35348 = CGP 36742 > (+)-baclofen in membranes from both species. 2 Fo r solubilization of GABA(B) receptors from pig brain, washed membranes were preincubated with 5 mm MgSO4 and subsequently incubated with var ious detergents. 3-[(3-Cholamidopropyl)dimethyl-ammoniol]-1-propane su lphonate (CHAPS) (0.5%) proved to be the most successful, solubilizing 22.7 +/- 4.7% (mean +/- s.e.mean, n = 6) of GABA(B) receptors. 3 Bind ing of [H-3]-GABA to GABA(B) receptors solubilized with 0.5% CHAPS exh ibited similar characteristics to the binding at membrane bound recept ors since, firstly, the K(d) and B(max) values (around 30 nm and 450 f mol mg-1 protein, respectively) were comparable; secondly, stereospeci fic binding for baclofen was obtained in both forms; thirdly, the affi nity for the agonists GABA and (-)-baclofen and the antagonists CGP 35 348, CGP 36742 and CGP 54626 were the same; fourthly, comparable sensi tivity to Ca2+ (2.5 mm) was observed and finally, a similar sensitivit y to GTP was apparent. 4 Saturation experiments performed with the GAB A(B) antagonist, [H-3]-CGP 54626, indicated a higher K(d) value and a lower B(max) value for solubilized (7.7 +/- 2.6 nm and 1033 +/- 41 fmo l mg-1 protein, mean +/- s.e.mean, n = 3) than for membrane bound rece ptors (1.35 +/- 0.08 nm, 1171 +/- 20 fmol mg-1 protein, n = 3).