Functional and Structural Characterization of the GluK2/GluK5 Heterotetramer Gating Mechanism

Ionotropic glutamate receptors (iGluRs) mediate most of the excitatory synaptic neurotransmission, controlling synaptic strength and plasticity, aspects which are thought to underlay learning and memory. These ligand-gated ion channels are classified into three major families; the NMDA, AMPA and kainate receptors. The kainate receptor family is composed of five different subunits, GluK1-GluK5. GluK1-K3 can co-assemble to form both homomeric and heteromeric channels. GluK4 and GluK5 however, can only form heteromeric channels with GluK1-GluK3 subunits.The most widely expressed kainate receptor proteins are GluK2 and GluK5, and in co-expressing cells they are known to preferentially assemble together with a 2:2 stoichiometry, probably due to the high affinity of their amino-terminal domains. Although in vivo the GluK2/GluK5 heterotetramer is abundant, to date the most extensively studied kainate receptor construct has been the GluK2 homomer. In order to gain insights into this more physiologically relevant receptor, we have combined in-silico structural models and molecular dynamics (MD) simulations with functional studies of the GluK2/GluK5 heterotetramer. Several repeats of long-timescale atomistic MD simulations in the presence and absence of modulatory ions were used to assess the viability of each of the two possible combinations of GluK2 and GluK5 proteins. We find that the different conductance pattern observed in the heterotetramer with respect to the homomer might be accounted for by structural differences in the GluK5 ligand-binding domain, which in turn affect ligand binding affinity and the channel opening rate. Moreover, we also observe that the GluK5 protein is more robust than GluK2 to the absence of external modulating ions. Thus, despite belonging to the same family, GluK5 possesses unique features that help to broaden the functional and pharmacological profile of kainate receptors.