Modulation of Heteromeric AMPA-Subtype iGluRs by Auxiliary Subunits

PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to determine molecular mechanisms of heteromeric ionotropic glutamate receptor (iGluR) gating and modulation by ligands and auxiliary subunits. iGluRs are ligand-gated, tetrameric ion channels that mediate fast neurotransmission in the central nervous system (CNS). iGluRs are critical for learning and memory, and their mis-regulation is implicated in neurological disorders like epilepsy and Alzheimer’s disease. The α-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid receptor (AMPAR), the fastest subtype of the iGluR family, assembles as a tetrameric combination of four distinct, principal subunits, GluA1 to GluA4. AMPARs can be further classified into GluA2-containing, Ca2+-impermeable (CI) receptors, and GluA2-lacking, Ca2+-permeable (CP) receptors, which are involved in Ca2+ influx-mediated excitotoxicity. While it is known that AMPAR formation is primarily heteromeric in the CNS, most research has focused on homomeric assemblies. Furthermore, AMPARs co-assemble with a broad range of structurally diverse auxiliary subunits that regulate their synaptic localization, trafficking, pharmacology, and ion permeation. Structural and functional characterization of heteromeric AMPARs, CP-AMPARs and their complexes with auxiliary subunits, which regulate the vast majority of excitatory neurotransmission in glutamatergic synapses and are involved in devastating neurological diseases, thus representing promising pharmacological targets, will provide novel insights into iGluR function in physiological conditions as well as aid in the rational design of therapeutics targeting these receptors in disease states. We will study AMPAR structure and function with the following Specific Aims: Aim 1. Determine the high-resolution cryo-EM structure of heteromeric AMPARs. Aim 2. Characterize structure and function of Ca2+-permeable AMPARs in complex with auxiliary subunit transmembrane AMPAR regulatory protein (TARP) ɣ7. To achieve these goals, we will use a combination of biophysical and biochemical methods, including the fluorescence-detection size-exclusion chromatography (FSEC), whole-cell patch-clamp and single-channel recordings, and cryo-electron microscopy (cryo-EM). Our results will uncover the molecular basis of heteromeric iGluR gating and inhibition, provide new insight into translational pharmacology and aid in the development of treatment of devastating neurological disorders.