Single-Particle Cryo-EM Characterization of AMPA Receptor Functional States

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PROJECT SUMMARY AMPA-subtype ionotropic glutamate receptors mediate fast signaling between neurons and contribute to high cognitive processes. Since AMPA receptors are also implicated in numerous neurological disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, epilepsy, and ischemia, the ability to regulate them represents an important clinical goal. However, there is an unmet need for drugs to regulate AMPA receptor activity in pathological conditions, which highlights a profound gap in our knowledge of AMPA receptor structure and function. Our long-term goal is to understand how AMPA receptor molecular machinery operates at the atomic level. We plan to study AMPA receptor structure and function using single-particle cryo-electron microscopy (cryo-EM). Advances in cryo-EM that were signified by the “resolution revolution” brought us a number of AMPA receptor structures in different gating conformations and in complex with several small molecules and regulatory proteins. Nevertheless, only a little fraction of AMPA receptor conformational states and binding partners have been characterized structurally and the resolution of the reported structures remains relatively low, limiting our ability to see the atomic details. We, therefore, plan to use cryo-EM advances to fill up this knowledge gap and to focus on the following Specific Aims: (1) unravel structural principles of AMPA receptor regulation by auxiliary subunits, (2) reveal molecular determinants of AMPA receptor activation and conductance, and (3) determine molecular mechanisms of AMPA receptor desensitization. To reach our research goals, we will use cryo-EM to obtain structures of AMPA receptors alone or in complex with different auxiliary proteins, in the presence of agonists, competitive antagonists, positive or negative allosteric modulators, and in conditions favoring various conformational states. We will use Fluorescence-detection Size Exclusion Chromatography (FSEC) and thermostability assays to assess protein expression, assembly, homogeneity, and stability. We will also employ site-directed mutagenesis combined with single-channel and whole-cell patch-clamp electrophysiological recordings to study functional mechanisms and to critically test our structural models. Reaching our research goals will have a significant impact on understanding the mechanisms of excitatory neurotransmission and will provide molecular-level knowledge essential to facilitate the design of new drugs for the treatment of neurological disorders.