Modulation of Glutamate Receptors by Auxiliary Proteins a Structural Investigation

Ionotropic glutamate receptors (iGluRs) are the primary neurotransmitter receptors involved in excitatory synaptic transmission. iGluRs are tetrameric proteins comprised of extracellular amino terminal domains (ATD) and ligand binding domains (LBD), transmembrane domains (TM) and intercellular C-terminal domains. The LBD is a clamshell-like structure divided into segments termed S1 and S2 that form a cleft. Upon glutamate binding the LBD cleft closes and propagates its conformational change to the TM domains activating the cation-selective pore. Mutagenic disruptions in receptor structural organization clearly show distinct functional effects. Therefore, these structural changes serve as obvious targets for the modulation of receptor function. iGluR receptors are classified into subtypes according to their selective agonist; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate receptors (KARs). AMPA and KARs have the most similar structural homology. Additionally they both have auxiliary subunit proteins shown to modulate receptor function, Gamma-2 and Neto2 respectively. Studies have confirmed the interaction and modulatory effect of the auxiliary proteins with their receptors; however, little is known concerning their structural influence and its translation to functional changes. In this study, we investigated the cleft closer and inter-subunit distances of AMPA (GluR2) and KAR (GluK2) with and without Gamma-2 and Neto2 using luminescence energy resonance transfer (LRET). The LRET data shows the auxiliary proteins alter the structure of kainate and AMPA receptor in similar ways. The addition of auxiliary protein moves GluK2 and GluR2 from a relaxed to a more constricted conformation. The auxiliary proteins act by supporting the receptor extracellular domains thereby cradling them into closer inter-domain proximity while keeping them more structurally organized.