Structure and Function of Ionotropic Glutamate Receptors

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.