PROJECT SUMMARY
Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission, underlie high cognitive
processes, and contribute to nearly all aspects of the central nervous system (CNS) development and function.
The fastest iGluRs, AMPA receptors (AMPARs) and kainate receptors (KARs), play key roles in generation of
excitatory postsynaptic potentials (EPSPs), regulation of synaptic plasticity, neurotransmitter release, and
nervous system development. They are also implicated in numerous psychiatric, neurological, and
neurodegenerative diseases, including epilepsy, amyotrophic lateral sclerosis, schizophrenia, obsessive-
compulsive, mood and bipolar disorders, mental retardation, depression, autism, Huntington's and Alzheimer’s
diseases, ischemia, and pain. However, development of drugs targeting AMPARs and KARs has been
hindered by limited knowledge about their structure and function. For example, most published structures of
AMPARs and KARs are structures of homotetrameric receptors, while the overwhelming majority of iGluRs in
the CNS are heterotetramers. Several noncompetitive inhibitors and ion channel blockers of AMPARs and
KARs have been identified as promising candidates for drug development but structural mechanisms of their
action remain poorly understood. This missing information is absolutely critical for the future structure-based
rational drug design. We plan to study the structure and function of AMPARs and KARs using a combination of
biophysical and biochemical approaches, including modern cryo-electron microscopy (cryo-EM) techniques,
fluorescence-based methods, mutagenesis, and electrophysiology. Our specific aims are to (1) determine
structures of heteromeric iGluRs, (2) uncover the molecular basis of noncompetitive inhibition, and (3)
establish the structural mechanisms of ion channel block. To reach our research goals, we will optimize
AMPAR and KAR constructs for cryo-EM experiments, develop protocols of their expression and purification
and solve structures of heterotetrameric receptors, with or without auxiliary subunits, in complex with
noncompetitive inhibitors or ion channel blockers. We will test our models using a combination of mutagenesis
and whole-cell patch-clamp recordings. We will combine the results of structural, functional and mutagenesis
experiments to propose molecular models of iGluR heteromeric assembly, noncompetitive inhibition, and ion
channel block. Reaching our research goals will provide molecular level knowledge essential for the design of
new molecules that can become safe and efficacious drugs for treatment of epilepsy and other disorders
associated with excitatory neurotransmission.