Vol. 56, Issue 2, 429-433, August 1999

Kainate Receptors Coupled to G_i/G_o Proteins in the Rat Hippocampus

Rodrigo A. Cunha, João O. Malva, and J. A. Ribeiro

Laboratory of Neurosciences, Faculty of Medicine (R.A.C., J.A.R.), and Department of Chemistry and Biochemistry, Faculty of Sciences (R.A.C.), University of Lisbon, Portugal; and Center for Neurosciences of Coimbra and Faculty of Medicine, University of Coimbra, Coimbra, Portugal (J.O.M.)

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 -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 -amino-3-hydroxy-5-methyl-4-isoxazole-propionate (K_i > 10 µM), (RS)--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 µM N-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.