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Kainate receptors (KARs) modulate synaptic transmission and plasticity and their dysfunction has been linked to several disease states such as epilepsy and chronic pain. KARs are tetramers formed from five different subunits. GluK1-3 are low affinity kainate binding subunits whereas GluK4/5 bind kainate with high affinity. A number of these subunits can be present in any given cell-type and different combinations of subunits confer different properties to KARs. Here we report the characterisation of a new GluK1 subunit selective radiolabelled antagonist [(3)H]UBP310 using human recombinant KARs. [(3)H]UBP310 binds to GluK1 with low nanomolar affinity (K(D) = 21 ± 7 nM) but shows no specific binding to GluK2. However, [(3)H]UBP310 also binds to GluK3 (0.65 ± 0.19 μM) but with ~30-fold lower affinity than that observed for GluK1. Competition [(3)H]UBP310 binding experiments on GluK1 revealed the same rank order of affinity of known GluK1-selective ligands as previously reported in functional assays. Non-conserved residues in GluK1-3 adjudged in modelling studies to be important in determining the GluK1 selectivity of UBP310 were point mutated to switch residues between subunits. None of the mutations altered the expression or trafficking of KAR subunits. While GluK1-T503A mutation diminished [(3)H]UBP310 binding, GluK2-A487T mutation rescued it. Similarly, while GluK1-N705S/S706N mutation decreased, GluK3-N691S mutation increased [(3)H]UBP310 binding activity. These data show that A487 in GluK2 and N691 in GluK3 are important determinants in reducing the affinity of UBP310 for these subunits. Insights from these modelling and point mutation studies will aid the development of new subunit selective KAR antagonists.
Bibliographical notePublisher: American Society for Pharmacology and Experimental Therapeutics
Other: PMID: 20837679
Atlason, PT., Scholefield, CL., Eaves, RJ., Mayo-Martin, MB., Jane, DE., & Molnar, E. (2010). Mapping the Ligand Binding Sites of Kainate Receptors: Molecular Determinants of Subunit-selective Binding of the Antagonist [3H]UBP310. Molecular Pharmacology, 78, 1036 - 1045. https://doi.org/10.1124/mol.110.067934