Functional Mechanisms of Desensitization in AMPA Receptors

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand gated ion channels that mediate excitatory synaptic transmission in the brain. Desensitization of AMPA receptors following glutamate binding is critical for brain function, and involves rearrangement of the ligand binding domains (LBDs). Recently, a couple of full-length structures of desensitized iGluRs were revealed. One of them, the cryo-EM structure of the GluK2 kainate receptor showed that the ligand binding domains (LBDs) adopt a quasi four-fold symmetric arrangement1. Contrastingly, the GluA2 full-length crystal structure with Fluorowillardine bound showed a disrupted, asymmetric orientation of the LBDs and disrupted ATDs2. We reasoned that these LBD arrangements should be detectable by crosslinking experiments during receptor desensitization. We noticed that cysteine mutants at the interface between subunits A and C, which cross-link receptors in partially-active states when desensitization is blocked3, can also trap desensitized receptors. When we exposed receptors to 100 µM glutamate, crosslinks formed that were much more stable than those seen in the absence of desensitization. Consistent with stabilizing a desensitized state, introduction of bulky amino acids at this subunit interface produced a receptor with slow recovery from desensitization. Using a mutation that strongly stabilized the desensitized state (E713T, Y768R), we were able to change the stability of crosslinks or, in one case, block their formation, indicating that there are multiple desensitized arrangements. Homology modelling guided by these functional data suggests that the stable trapping arises from an arrangement distinct from that observed in the partially-active state. Bibliography 1. Meyerson, J. R. et al. Structural mechanism of glutamate receptor activation and desensitization. Nature (2014). 2. Dürr, K. L. et al. Structure and Dynamics of AMPA Receptor GluA2 in Resting, Pre-Open, and Desensitized States. Cell (2014). 3. Lau, A. Y. et al. A Conformational Intermediate in Glutamate Receptor Activation. Neuron (2013).