Structure and Affinity of Two Bicyclic Glutamate Analogues at AMPA and Kainate Receptors

Ionotropic glutamate receptors (iGluRs) are involved in most of the fast excitatory synaptic transmission in the central nervous system. These receptors are important for learning and memory formation, but are also involved in the development of diseases such as Alzheimer's disease, epilepsy and depression. To understand the function of different types of iGluRs, selective agonists are invaluable as pharmacological tool compounds. Here, we report binding affinities of two bicyclic, conformationally restricted analogues of glutamate (CIP-AS and LM-12b) at AMPA (G1uA2 and GIuA3) and kainate receptor subunits (GIuK1-3 and GluK5). Both CIP-AS and LM-12b were found to be GIuK3-preferring agonists, with Ki of 6 and 22 nM, respectively, at recombinant GIuK3 receptors. The detailed binding mode of CIP-AS and LM-12b in the ligand-binding domains of the AMPA receptor subunit GIuA2 (GluA2-LBD) and the kainate receptor subunits GluK1 (GIuK1-LBD) and GIuK3 (GluK3-LBD) was investigated by X-ray crystallography. CIP-AS stabilized all three receptor constructs in conformations similar to those with kainate. Remarkably, whereas LM-12b bound in a similar manner to CIP-AS in GluA2-LBD and GluK3-LBD, it introduced full closure of the ligand-binding domain in GIuKI-LBD and formation of a D1-D2 interlobe hydrogen bond between Glu441 and Ser721, as also observed with glutamate. As the binding affinity of LM-12b at GIuK1 is,-8-fold better than that for CIP-AS (K-i of 85 and 656 nM, respectively), it shows that small changes in agonist structure can lead to prominent differences in structure and function.