A novel kainate receptor ligand [3H]-(2S,4R)-4-methylglutamate: pharmacological characterization in rabbit brain membranes.

Since kainate evokes large non-desensitizing currents at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, kainate is of limited use in discriminating between AMPA and kainate receptors. Following recent reports that (2S,4R)-4-methylglutamate is a kainate receptor-selective agonist, we have radiolabelled and subsequently characterized the binding of [3H]-(2S,4R)-4-methylglutamate to rabbit whole-brain membranes. [3H]-(2S,4R)-4-methylglutamate binding was rapid, reversible and labelled two sites (KD1 = 3.67+/-0.50 nM/Bmax1 = 0.54+/-0.03 pmol/mg protein and KD2 = 281.66+/-12.33 nM/ Bmax2 = 1.77+/-0.09 pmol/mg protein). [3H]-(2S,4R)-4-methylglutamate binding was displaced by several non-NMDA receptor ligands: domoate > kainate >> L-quisqualate > or = L-glutamate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) >> (S)-AMPA = (S)-5-fluorowillardiine > NMDA. Neither the metabotropic glutamate receptor agonists (1S,3R)-ACPD or L-AP4, together with the L-glutamate uptake inhibitor L-trans-2,4-PDC, influenced binding when tested at 100 microM. We conclude that [3H]-(2S,4R)-4-methylglutamate is a useful radioligand for labelling kainate receptors. It possesses high selectivity, and possesses a pharmacology similar to that for rat cloned low-affinity (Glu5 and 6) kainate receptor subunits.