Proper neuronal function relies on the tightly regulated expression and discrete localization of voltage-gated sodium ion channels (NaVs), large protein complexes that control the movement of ions across cell membranes. A desire to better understand the role of NaVs in axonal plasticity and signal conduction, and the relationship between their disregulation and specific human pathologies motivates the development of high precision methods for their study in living systems. Real-time investigations of NaVs in live neuronal cells, however, are limited by the lack of available methods with which to modulate the function of individual NaV subtypes and to 'mark' their cellular distribution. We are developing small molecule probes for NaV studies based on naturally occurring guanidinium toxins - saxitoxin, gonyautoxin, and zetekitoxin AB. These agents function as molecular 'corks' to occlude the extracellular mouth of the ion conductance pore. De novo chemical synthesis makes available modified forms of these toxins, which we will use in combination with protein mutagenesis and electrophysiology to gain insights into the three-dimensional structure of the toxin binding site. Such information is needed to advance a NaV homology model that we have constructed, and will empower the rational design of toxin derivatives that show selective inhibition of individual NaV isoforms. Our structural investigations of toxin binding are informingthe development of new fluorescent imaging and affinity-based tools for investigating dynamic events associated with NaV function. We are motivated to understand how modulation of NaV expression influences the input-output responsiveness of neuronal cells (i.e., cellular plasticity) Toxin conjugates will be employed in initial experiments to measure channel synthesis and turnover rates, and ultimately to analyze quantitatively the extent to which these kinetic data vary as a function of nerve cell stimulation and nerve cell injury. The temporal control afforded by small molecule agents and the minimally invasive nature of such probes offer significant advantages over biological methods for labeling endogenous NaV channels. As such, the availability of toxin derivatives for NaV imaging studies should offer unprecedented insight into the dynamic role of these channel proteins in electrogenesis.

Public Health Relevance

We are interested in understanding at a molecular level how nerve cells conduct electricity and how the process of electrical signaling is affected when a nerve is injured. Chemical synthesis makes possible access to selective reagents that can be used to investigate these complex biological phenomena. Results from these studies could help guide the development of new therapies for the treatment of acute and/or chronic pain.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
High Priority, Short Term Project Award (R56)
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Special Emphasis Panel (ZRG1-BCMB-B (02))
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Stewart, Randall R
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Stanford University
Schools of Arts and Sciences
United States
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