[3H]-(2S,4R)-4-methylglutamate: a new ligand for glial glutamate transporters?

Full text: [3H]-Labelled L-glutamate (Glu), L-aspartate and D-aspartate are used for radioligand studies of Glu transporters (GluTs). Recently, compounds including (2 S ,4 R )-4-methylglutamate (4MG) have become available that possess high affinity for GluT binding sites (Bridges et al .,1999). [3H]4MG has been used to localise low affinity kainate receptors in mammalian brain (Carroll et al .,1998), but 4MG is also known to inhibit the astrocytic transporters GLT1 and GLAST (Bridges et al , 1999). Studies were undertaken to determine whether [3H]4MG could label these GluTs in rat brain. Forebrain tissue from female Sprague Dawley rats (anaesthetised with halothane) was homogenised in HEPES buffer containing NaCl, and membranes were washed twice. Specific binding (>90%total binding)of [3H]4MG to membrane homogenates at 4 deg C was saturable with an association t1/2 of ∼15min, and dissociated rapidly (t1/2 ∼ 3min) following addition of 1mM Glu. Binding of 20nM [3H]4MG was completely displaced by natural substrates for GluTs, e.g. Glu (IC 50 6 μM)and non-selective transporter inhibitors, such as (2 S ,3 S ,4 R )-2- (carboxycyclopropyl) glycine (IC50 1 μM). Selective GLT1 ligands,such as dihydro-kainate, kainate or 3-methylglutamate partially displaced [3H]4MG binding (35-50%). Binding was not displaced by the low affinity kainate receptor antagonist 6-cyano-7-nitroquinoxaline and other Glu receptor agonists and antagonists. These studies demonstrate that [3H]4MG and selective GluT inhibitors may serve as novel ligands to further study the properties of glial GluTs. Copyright (2001) Australian Neuroscience Society