The majority of synaptic transmission in the brain relies upon the ionotropic glutamate receptors. This class of ion channels is comprised of four subfamilies, and of these, the N-methyl-D-aspartate receptors (NMDARs) are critical for long-term potentiation and learning and their misregulation has been implicated in Alzheimer's disease, stroke, and a number of neurological disorders. The NMDAR assembles as an obligate heterotetramer with each subunit consisting of four modular domains: an extracellular Amino Terminal Domain (ATD) and Ligand Binding Domain (LBD), a transmembrane domain (TMD) which forms the ion channel, and an unstructured intracellular Carboxyl Terminal Domain (CTD). Numerous factors influence NMDAR activity, including pH, zinc & magnesium binding, and protein-protein interactions, at sites widely distributed across the protein, yet how these interactions are translated into differential channel activity remains largely unknown. This projec seeks to define the structural basis for the allosteric regulation of NMDAR activity through X-ray crystallographic, biophysical, and biochemical methods with two primary aims: 1) first, I will solve the X-ray crystal structure of the NMDAR ATD bound to a series of small molecules homologous to the antagonist Ifenprodil, which has considerable off-target effects but novel variations of which have been shown to bind the NMDAR only in the low-pH environments found during ischemic events such as stroke. 2) In my second aim, I will crystallize the intact receptor containing the ATD, LBD, and TMD in complex with the agonist spermine. Interestingly, this compound appears to bind at the ATD/LBD interface and specifically potentiates a subset of NMDAR splice variants; by solving and comparing the structures of the spermine-sensitive and spermine- insensitive receptors, I will determine how ligand binding to the extracellular domains allosterically alters the activity of the TMD. The Furukawa laboratory has extensive experience with the NMDAR, and I am uniquely situated to answer fundamental questions regarding NMDA receptor regulation through the use of our optimized mammalian expression system, advanced macromolecular crystallization techniques including lipidic cubic phase methods, and calorimetric and electrophysiological validation equipment. This research will allow me to gain tremendous experience in multiple expression systems and modern methods in electrophysiology and the structural biology of large membrane protein assemblies, and by defining the allosteric regulation of the NMDAR, my work has the potential to pave the way for the development of novel, highly-targeted therapeutics in a range of neurological disorders and diseases. Together, the training and the scientific achievements of this project will prepare me for research independence in this field.
The NMDA receptor is an ion channel protein in the brain whose proper functioning is a central component in learning and memory. However, misregulation of this protein has been linked to a wide range of neurological disorders; in the case of Alzheimer's disease and stroke, hyperactivity of the NMDA receptor causes neurons to be flooded with calcium and can result in cell death. This work seeks to determine how the activity of the NMDA receptor is modulated by visualizing how chemical compounds binding at one site on the receptor can effect changes at distant sites. A firm understanding of these interactions could ultimately result in new, highly-specific therapeutics for stroke, Alzheimer's disease, depression, and schizophrenia.
| Regan, Michael C; Grant, Timothy; McDaniel, Miranda J et al. (2018) Structural Mechanism of Functional Modulation by Gene Splicing in NMDA Receptors. Neuron 98:521-529.e3 |
| Elegheert, Jonathan; Cvetkovska, Vedrana; Clayton, Amber J et al. (2017) Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins. Neuron 95:896-913.e10 |
| Amin, Johansen B; Salussolia, Catherine L; Chan, Kelvin et al. (2017) Divergent roles of a peripheral transmembrane segment in AMPA and NMDA receptors. J Gen Physiol 149:661-680 |
| Regan, Michael C; Furukawa, Hiro (2016) Deeper Insights into the Allosteric Modulation of Ionotropic Glutamate Receptors. Neuron 91:1187-1189 |
