Theses Doctoral

Structural Determinants of Ionotropic Glutamate Receptor Function Revealed by Cryo- electron Microscopy

Twomey, Edward Charles

Fast excitatory neurotransmission is critical for learning and memory, and its dysregulation is linked to numerous neurological diseases. These include developmental diseases such as fragile X syndrome, psychiatric disorders like schizophrenia, and chronic neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Throughout the central nervous system, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors (AMPARs) mediate the fastest excitatory neurotransmission. In response to the neurotransmitter glutamate, AMPARs open their ion channels and allow cation flux through the post-synaptic membrane. This initiates rapid depolarization and signaling in the post-synaptic neuron. Nearly all AMPARs exist as complexes with auxiliary subunits, which are regulatory proteins that modulate receptor assembly, trafficking, pharmacology and function. These auxiliary subunits determine brain region-specific AMPAR signaling, and aberrancies in complex formation or function lead to neuropathologies. Despite their importance for CNS signaling and implication in neurologic disorders, the structural bases underlying the function of AMPARs and AMPAR complexes remain ambiguous, representing a critical barrier to our understanding of excitatory neurotransmission. As a consequence, structure-based design of neuro-therapeutics is largely undeveloped: there is only a single FDA-approved drug targeting AMPARs.
To address these problems, I wanted to dedicate my thesis work to study AMPAR synaptic complexes across an array of functional states and provide a new foundation for our structural understanding of AMPAR signaling. First, I designed a covalent-fusion construct approach to guarantee assembly and expression of AMPAR synaptic complexes in heterologous cells (HEK293). Then, I developed purification protocols allowing me to obtain chemically homogenous and pure complex protein. Since synaptic signaling is highly dynamic, complexes of AMPARs with auxiliary subunits are conformationally heterogeneous and are not amenable to X-ray crystallography.
Cryo-electron microscopy (cryo-EM) enabled me to approach these complexes structurally, where I could collect data and parse out heterogeneity through image classification. With cryo-EM, I solved the structure of an AMPAR bound to the auxiliary subunit stargazin, which promotes AMPAR activation. This work provided the first structural information on how AMPARs form complexes with regulatory subunits. In a following study, I solved the structure of an AMPAR in complex with a functionally distinct auxiliary subunit, GSG1L. In contrast to stargazin, GSG1L promotes inactivation and desensitization of AMPARs, thus having a neuroprotective effect. To further characterize the function of these auxiliary subunits, I designed chimeras between stargazin and GSG1L and examined their function electrophysiologically. This experiment revealed that AMPAR auxiliary subunits have a modular design, where variable extracellular domain regions, supported by a conserved transmembrane α-helical bundle, distinctly regulate function of the core AMPAR. This study provided the first evidence of how brain region-specific expression patterns of similarly-structured auxiliary subunits may contribute to unique AMPAR functions.
More recently, I’ve taken advantage of the modulatory effects of stargazin on AMPARs and I applied cryo-EM to an AMPAR-stargazin complex. This study determined how AMPARs are activated by the neurotransmitter glutamate, and revealed a novel mechanism by which glutamate binding induces opening of AMPAR ion channels. Our data show that two-fold symmetric kinking of ion channel helices allows cation flux into neurons, which triggers neurotransmission. Importantly, this study also provides insights into how mRNA editing and patient-derived disease mutations in the transmembrane (i.e., resulting in aberrantly firing of receptors during epilepsy) reshape AMPAR function and excitatory neurotransmission.
Collectively, the findings from my thesis work provide a new paradigm for the molecular-level understanding of glutamatergic neurotransmission throughout the CNS. These studies lay the groundwork for new directions in precision-medicine design of therapeutics targeting brain region-specific AMPAR synaptic complexes in neurological diseases.


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More About This Work

Academic Units
Cellular, Molecular and Biomedical Studies
Thesis Advisors
Sobolevsky, Alexander I.
Ph.D., Columbia University
Published Here
April 13, 2018