Ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission in the human nervous system and are absolutely essential to its normal development and function. Furthermore, iGluRs underpin a vast spectrum of behaviors - including learning and memory - and are the target of several drug candidates in clinical trials for treatment of mild cognitive impairment and depression. There are three major subtypes of iGluRs - AMPA, kainate and NMDA receptors. Whereas AMPA and kainate receptors generally harbor calcium impermeable ion channel pores and act on a millisecond time-scale, NMDA receptors are calcium permeable and gate on a time-scale of hundreds of milliseconds, thus allowing ample time for calcium to flow into the cell. Moreover, AMPA and kainate receptors are activated solely by glutamate yet NMDA receptors require relief of voltage-dependent magnesium block and the binding of both glycine and glutamate, thus serving as Hebbian-like coincident detectors. A further distinction between AMPA, kainate and NMDA receptors is that NMDA receptors are obligate heteromeric assemblies, typically requiring glycine- and glutamate binding subunits, and whose ion channel gating activities are modulated by a wide range of ions and small molecules. The research proposed in this application is focused on determining molecular structures for the GluA2 AMPA receptor in an activated, open channel state and, following activation, bound with glutamate yet in an inactive desensitized state. Furthermore, because AMPA receptors in native synapses are typically found in complex with auxiliary proteins called TARPs, we aim to elucidate the structure of an isolated TARP and a receptor - TARP complex. A third major effort of the proposed research is to understand relationships between molecular structure and function in NMDA receptors by studying selected functional states of the GluN1/GluN2B receptor and, in particular, to determine how allosteric antagonists that bind within the most distal domain modulate ion channel activity. The molecular structures of both AMPA and NMDA receptors will be elucidated by either x-ray crystallographic or single particle electron cryo-microscopy (cryo-EM) methods, and specific mechanistic hypotheses will be tested by introduction of cysteine residues to form redox sensitive cross-links. We will additionally study the mechanism by which small molecules block the ion channel pore. Taken together, the proposed experiments will provide structural principles for understanding the biological function and mechanism of AMPA and NMDA receptors, laying the foundation for the potential development of new therapeutic agents.
A fundamental mechanism by which cells in the nervous system communicate with one another is by the release and detection of neurotransmitters. Neurotransmitter receptors bind released neurotransmitter and, as a consequence, open a transmembrane ion channel, initiating an electrical signal, thus propagating the initial stimulus. The research described in this grant application is focused on understanding the atomic structure and mechanism of neurotransmitter receptors, with a particular emphasis on how small molecules of biomedical importance modulate receptor activity.
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