The NMDA-receptor (NMDAR) subtype of ionotropic glutamate receptors (iGluRs) has fundamental roles in the processing and encoding of information, and in humans, defects in NMDAR signaling are a hallmark of many neurological disorders. A recent surprising and unexpected discovery using a C. elegans genetic screen for modifiers of NMDAR function has revealed that in this organism the NMDAR does not function autonomously as previously thought, and is instead part of a larger signaling complex, requiring for its activation the binding of a presynaptically secreted protein, NRAP-1. In C. elegans, NRAP-1 is rate limiting for NMDAR function, and thus likely controls the magnitude of NMDAR signaling. Therefore, the mechanisms regulating NRAP-1 secretion, such as the changes in neuronal activity that regulate synapse plasticity, are likely to have fundamental roles in synaptic transmission and NMDAR-mediated changes in synapse strength.
Aim 1 of this proposal will determine how neuronal activity regulates the secretion of NRAP-1. To accomplish this, genetic modification and elimination strategies combined with chemical and light controlled ion channels to selectively silence or activate neuronal populations will be employed. In vivo live imaging will be used to directly assess NRAP-1 secretion in response to chronic and acute changes in neuronal activity. The results of this aim will provide new mechanistic insights into acute and homeostatic control of NMDAR-mediated signaling and synaptic plasticity. Although NRAP-1 is of critical importance in the gating of NMDARs, we currently lack a mechanistic understanding of this essential process.
Aim 2 of this proposal will define NRAP-1's mechanism of action in gating NMDAR-mediated currents. To accomplish this I will use application of purified recombinant NRAP-1 and NRAP-1 mutants to electrophysiological NMDAR preparations. These constructs will be additionally used in in vitro protein-protein binding assays to define the functional domains of NRAP-1 as well as how and where it binds to the NMDAR. The results of this aim will provide insight into the structure and function of the newly identified C. elegans NMDAR signaling complex. Because of the deep evolutionary conservation of iGluRs and glutamatergic signaling, we anticipate that similar signaling mechanisms might also contribute to the regulation of vertebrate NMDAR signaling. Additionally, comparisons of the evolutionary divergence of NMDAR gating made possible by the results of this study will provide fundamental insights into the design and function of NMDARs. Thus, our studies will provide new conceptual framework for drug discovery that could ultimately motivate new approaches for therapeutic intervention in neurological disease characterized by disrupted NMDAR function.
The NMDA-type glutamate receptor (NMDAR) is essential for the processing and storage of information in the brain, and in humans, disruption of NMDAR-mediated signaling is associated with many neurological disorders. This proposal will examine the mechanism and regulation of NRAP-1, a novel presynaptically secreted auxiliary protein necessary for NMDAR function that was recently identified in C. elegans. The results of this study will provide fundamental insights into the regulation, design, and function of NMDARs that could ultimately motivate new approaches for therapeutic intervention in neurological disease.