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 a-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) ¿7. 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.