3D Structure and Function of Ligand-Gated Ion Channels

Project Summary Our nervous system relies on the transmission of signals from one nerve cell to another. The primary mechanism by which this occurs is via the release of neurotransmitter from the presynaptic nerve cell, binding of the transmitter to receptors, primarily located on the postsynaptic nerve cell, and opening of neurotransmitter-gated receptors, transforming the chemical signal of the neurotransmitter into an electrical signal in the postsynaptic neuron. Glutamate is the major neurotransmitter in the nervous system and glutamate receptors mediate most of the so-called ‘fast’ or sub second, neurotransmission. Over the past 3 decades, we and other researchers have revealed the overall structure and mechanism of AMPA, kainate and NMDA receptors. There are a large number of structures of receptor domains and full-length receptors and this structural information, in combination with biophysical, computational and biochemical studies has illuminated the mechanism(s) by which the binding of neurotransmitter results in the receptor transiting multiple conformational and functional states, including the open, ion conducting conformation. However, nearly all of the structural studies on glutamate receptors have involved the use of recombinant assemblies, sometimes with multiple modifications, produced in heterologous expression systems. Because AMPA receptors, in particular, assemble with a large number of auxiliary subunits, many of which modulate the activity of the receptor, it has proven difficult, if not impossible, to recapitulate the assembly of native-like receptor complexes in a heterologous expression system setting. A major goal of the research proposed in this application is to isolate native, or endogenous, receptor complexes from brain tissue, thus allowing us to elucidate the structure, and probe the function, of bona fide native receptor assemblies. To do this, we have developed an ensemble of receptor subunit specific antibodies that allow us to capture particular receptor assemblies, thus enabling, for the first time, capture of the AMPA receptor, GluA4-containing receptor from the cerebellum, a key receptor assembly implicated in motor learning and behavior. We have further employed a monoclonal antibody to capture the elusive GluN3A subunit containing NMDA receptor, allowing us to elucidate the subunit stoichiometry of native GluA1/Glu3A receptors and setting the stage for the structure determination of the receptor complex, in multiple functional states. Lastly, there is little knowledge, at the nanometer resolution, of how receptors are organized at synapses. To address this question, we have employed mouse models, gold particle-labeled antibody fragments, and cryo-electron tomography, to map the localization of AMPA and NMDA receptors at glutamatergic synapse, thus showing how the receptors are clustered, relative to presynaptic vesicles, and enabling the reconstruction of AMPA receptor assemblies in situ. Our work, overall, will elucidate the molecular assemblies and mechanisms of endogenous receptor assemblies, thus illuminating the principles by which the receptors function in the context of endogenous synapses.