Structure Determination of Heterologous and Native AMPA Receptors and their Complexes with Auxiliary Subunits - PROJECT SUMMARY/ABSTRACT The goal of this project is to determine the mechanisms of activation of ionotropic glutamate receptors (iGluRs) in complex with various auxiliary subunits, as well as elucidate compositional heterogeneity of iGluR subunit assembly between humans and other mammalian species. iGluRs are tetrameric ligand-gated ion channels responsible for the majority of excitatory neurotransmission in the central nervous system. Within neurons, iGluRs play key roles in brain function, including memory and learning, and their dysregulation leads to neurological and neurodevelopmental impairments, such as epilepsy, ischemia, and intellectual disability. The fastest iGluR subtype, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), assemble in the postsynaptic densities as homomeric or heteromeric complexes consisting of four primary subunits, GluA1 to GluA4. In addition to the core subunits, AMPARs co-assemble with many transmembrane cofactor proteins, or auxiliary subunits, that modulate the kinetics, trafficking, localization, permeation, and pharmacology of the receptor. Current structures of AMPARs in complex with several auxiliary proteins, specifically transmembrane AMPAR regulatory proteins (TARPs), germline-specific gene-1-like protein (GSG1L), and cornichons (CNIH2/3) lack details about the regulation of activation. Moreover, AMPAR structures have only been resolved from recombinant overexpression or extracted from rodent brains, while the composition of AMPAR synaptic complexes in human brains remains understudied. The proposed functional and structural studies of AMPAR synaptic complexes with auxiliary subunits in the open conducting state, a key conformational state for therapeutic intervention, and the delineation of human AMPAR assemblies will provide necessary, novel insights toward future drug design for the targeted treatment of psychiatric and neurological disorders linked to AMPAR dysfunction. AMPAR structure and function will be studied following two Specific Aims. Aim 1: Determine activation mechanisms of AMPARs bound to TARPs, GSG1L, and CNIHs. Aim 2: Structurally characterize subunit composition of human AMPARs. To achieve these aims, a combination of fluorescence-detection size- exclusion chromatography (FSEC) screening, cryo-electron microscopy (cryo-EM), whole-cell patch-clamp electrophysiology, and single-channel recordings will be used. The results of these studies will expand our understanding of AMPAR gating modulation by auxiliary subunits, provide insight into the brain-region-specific AMPAR subunit organization in humans, and foster the development of specialized therapies for clinical treatment of AMPAR-related neurological disorders.
