Characterization of the Binding of the GABA Agonist [3H]Piperidine‐4‐Sulphonic Acid to Bovine Brain Synaptic Membranes

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Abstract
The binding of radioactive piperidine-4-sulphonic acid ([3H]P4S) to thoroughly washed, frozen and thawed membranes isolated from cow and rat brains was studied. Quantitative computer analysis of the binding curves for 4 regions of bovine brain revealed the general presence of 2 binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing 1 or 3 binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0.degree. C the 2 affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: KD = 17 .+-. 7 nM (Bmax [maximum binding capacity] = 0.15 .+-. 0.07 pmol/mg protein) and KD = 237 .+-. 100 nM (Bmax = 0.80 .+-. 0.20 pmol/mg protein). Heterogeneity was observed for association and dissociation rates of [3H]P4S. The slow binding component (kon = 5.6 .times. 107 or 8.8 .times. 107 M-1 min-1, koff = 0.83 min-1 and KD = 14.7 or 9.4 nM, determined by 2 different methods in phosphate buffer containing KCl) corresponds to the high-affinity component of the equilibrium binding curve (KD = 11 nM, Bmax = 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [3H]P4S appears to involve the same 2 populations of sites with Bmax values similar to those for [3H]GABA binding to the same tissue, although the kinetic parameters for the 2 ligands differ. Comparative studies on the inhibition of [3H]P4S and [3H]GABA binding by various GABA analogs strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.