Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY The AMPA type ionotropic glutamate receptors (AMPARs) are ligand gated ion channels activated by the neu- rotransmitter glutamate. They mediate the majority of excitatory neurotransmission in the brain and the signals transduced by these complexes are critical for synaptic plasticity, learning and memory. AMPAR auxiliary sub- units regulate trafficking and gating modulation of AMPARs. In this proposal we will investigate the mechanism of AMPAR regulation by their auxiliary subunits. The core AMPAR auxiliary subunits are TARPs, GSG1L, and cornichons (CNIHs). The TARPs are extensively studied and therapeutic compounds to alleviate seizure are already available to target hippocampus enriched TARP gamma-8. GSG1L is a negative modulator of AM- PARs, while TARPs and CNIHs serve as positive modulators. In humans, various residues located at the inter- action interface between AMPAR and auxiliary subunits are intolerant to missense mutations, indicating their critical roles in brain function. We hypothesize that different auxiliary subunits can co-assemble with the chan- nel and produce a rich variety of gating modulations, which are fundamental in regulating synaptic transmission and plasticity. To establish the structural and mechanistic basis, we will study complex AMPAR assemblies that have high physiological relevance. In Aim 1 we hypothesize that fine structural differences among AMPAR assemblies are fundamental for producing characteristic gating modulation and propose to reveal the architec- tures of heterotetrameric AMPARs containing up to two types of auxiliary subunits at different functional states in detergent using cryo-EM. By comparing the structures, new mechanistic models that could explain how aux- iliary subunits control the time course and magnitude of gating are likely to emerge, which will be validated us- ing electrophysiology. Next, currently available cryo-EM structures revealed the presence of lipids surrounding the complex. We hypothesize that these lipids play important function in AMPAR gating modulation, which will be tested in Aim2. Finally, we suggest that AMPAR/auxiliary subunit complex prepared in near physiological conditions void of detergent must be studied to build more precise mechanistic models of its allosteric gating modulation. In Aim 3, we propose to solve high resolution cryo-EM structures of AMPAR/auxiliary subunit complex embedded in a lipid bilayer mimetic environment to resolve the known discrepancies between struc- tures obtained in detergent and electrophysiology data. The role of auxiliary subunits in tuning ion channel gat- ing kinetics is predicted to have significant impact on circuit dynamics. In summary, the outcomes of this study are expected to advance our mechanistic understanding of AMPAR function and assist developing new thera- peutic compounds that can alleviate dysregulation of AMPARs seen in neurological and psychiatric disorders, such as Alzheimer’s disease, stroke, autism, Rasmussen’s and limbic encephalitis, and seizure.
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