Abstract

N,N′-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and structurally related compounds are described as novel allosteric enhancers of GABA_B receptor function. They potentiate GABA-stimulated guanosine 5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to membranes from a GABA_B(1b/2)-expressing Chinese hamster ovary cell line at low micromolar concentrations, but do not stimulate [³⁵S]GTPγS binding by themselves. Similar effects of GS39783 are seen on native GABA_B receptors in rat brain membranes. Concentration-response curves with GABA in the presence of different fixed concentrations of GS39783 reveal an increase of both the potency and maximal efficacy of GABA at the GABA_B(1b/2) heterodimer. In radioligand binding experiments, GS39783 reduces the kinetic rate constants of the association and dissociation of [³H]3-aminopropylphosphinic acid, resulting in a net increase in affinity for the agonist radioligand. In equilibrium binding experiments (displacement of the antagonist ligand [³H]CGP62349), GS39783 increases agonist affinities. Agonist displacement curves are biphasic, probably reflecting the G protein-coupled and uncoupled states of the receptor. The proportion of the high-affinity component is increased by GS39783, suggesting that the G protein coupling of the receptor is also promoted by the positive modulator. We also show that GS39783 has modulatory effects in cellular assays such as GABA_B receptor-mediated activation of inwardly rectifying potassium channels in Xenopus oocytes and Ca²⁺ signaling in human embryonic kidney 293 cells. In a more physiological context, GS39783 is shown to suppress paired pulse inhibition in rat hippocampal slices. This effect is reversed by the competitive GABA_B receptor antagonist CGP55845A and is produced most likely by enhancing the effect of synaptically released GABA at presynaptic GABA_B receptors.