Kainate Receptors Coupled to G_i/G_oProteins in the Rat Hippocampus

Abstract

Kainate receptors are a subtype of ionotropic glutamate receptors, permeable to cations and thus expected to have an excitatory depolarizing action on neurons. However, kainate receptor activation inhibits γ-aminobutyric acid release in the hippocampus through activation of protein kinase C in a pertussis toxin-dependent manner, suggesting a coupling of kainate receptors to G proteins. Thus, we directly investigated the G protein coupling of kainate receptors in the rat hippocampus by using a selective kainate receptor agonist, [³H](2S,4R)-4-methylglutamate ([³H]MGA). [³H]MGA bound to a single site to hippocampal membranes with a K_D value of 32 nM and a B_max value of 1024 fmol/mg protein. This binding likely represents kainate receptors because it was displaced by domoate (K_i = 4 nM), kainate (K_i = 11 nM), and 6-cyano-7-nitroquinoxaline-2,3-dione (K_i = 1.4 μM), but not by α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (K_i > 10 μM), (RS)-α-methyl-4-phosphonophenylglycine (K_i > 10 μM), or (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (K_i > 10 μM). Guanylylimidodiphosphate (30 μM), which uncouples all G protein-coupled receptors, shifted to the right the saturation curve of [³H]MGA (K_D = 133 nM). This effect was mimicked by pretreatment of hippocampal membranes with modifiers of G_i/G_o proteins [30 μMN-ethylmaleimide (K_D = 98 nM) or 25 μg/ml pertussis toxin (K_D = 95 nM)] but not by a modifier of G_s proteins [50 μg/ml cholera toxin (K_D = 32 nM)]. Treatment of solubilized hippocampal membranes with pertussis toxin (25 μg/ml) decreased [³H]MGA affinity (K_D = 105–113 nM), which was recovered by reconstitution of these pretreated solubilized hippocampal membranes with G_i/G_o proteins (K_D = 41–76 nM). These results indicate that hippocampal kainate receptors are coupled to G_i/G_o proteins.