| Description |
N-methyl-D-aspartate receptors (NMDARs) are vital components of the mammalian nervous system that intimately contribute to excitatory neurotransmission. Learning, memory formation, synaptic plasticity, and many neurodegenerative diseases are linked with NMDARs. In order to study these processes, there is need for scientific tools that can be used to examine NMDARs. Cone snail venom is a mixture of neurotoxins with specific targets in the nervous system. Conantokins are a family of conotoxin that inhibit NMDARs and can select for different NMDAR subtypes. Therefore, understanding mechanisms of conantokin selectivity and using selective conantokins as tools to identify NMDAR subtypes in the brain are important advancements for neurobiology. In this dissertation, Chapters 2 and 3 describe structural studies of conantokin Bk-B (conBk-B) and of conantokin R/-B (conR/-B). These conantokins exhibited novel NMDAR subtype selectivities and were consequently of interest for structural characterization. The ultra-short conBk-B was found to adopt helical conformation similar to that observed in other conantokins. ConR/-B was found to be a kinked helix that is unique among structurally characterized conantokins. Comparison of conantokin structures, paired with mutational analysis, highlighted that residues 5, 6, 8, and 10 determine subtype selectivity in conantokins. Chapter 4 describes biochemical studies of NMDAR ligand-binding domain (LBD) proteins for the NR1-2b, NR2A, NR2B, and NR2C NMDAR subtypes. Limited proteolysis assays showed agonist binding, indicating LBD proteins were correctly folded. Additional experiments found no strong interactions between LBD proteins and conantokins. The results suggest that additional protein domains, heteromeric NMDARs, or neuronal membranes may be required for conantokin-NMDAR binding. In Chapter 5, conantokins are used to define the NMDAR subtypes in neurons from mouse cerebellum. Agonist challenge assays revealed diversity in cerebellar neurons, and conantokin R/-B was used to show NR2B NMDAR subtypes in cerebellar neurons of young mice. In older mice, a population of neurons with non-NR2B NMDARs was discovered. This dissertation demonstrates the first use of conantokins to define NMDAR subtypes in live neurons, provides a foundation for understanding neuronal diversity in the brain, and establishes NMDAR subtype composition in individual neurons of the developing cerebellum. |
