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    William Hazen Parsons.
    Voltage-gated sodium channels (NaV) serve an essential role in physiology. Accordingly, their dysfunction is associated with a number of human diseases and disorders, including epilepsy, cardiac arrhythmias, and chronic pain conditions. Designed by Nature as a chemical weapon, the naturally occurring channel blocker (+)-saxitoxin (STX) can be repurposed as a tool for studying these large integral membrane proteins. Access to new toxin derivatives through de novo synthesis offers a unique strategy to probe NaV structure and function, circumventing key limitations associated with existing methods for studying these proteins. The preparation and electrophysiological evaluation of an expanded library of N21-modified STX analogues is described herein. Characterization of the binding properties of these nanomolar NaV inhibitors has contributed to the development of an enhanced model for the structure of the inner pore of the channel. A select set of STX-fluorophore conjugates that bind reversibly to NaV with submicromolar potency serve as fluorescent markers of channels in living cells to study channel motility in the cell membrane. By contrast, maleimide-conjugated STX derivatives can be engineered to act as irreversible inhibitors of ion conductance when applied to wild-type NaV isoforms. The unique binding behavior of these derivatives has been leveraged to develop a new class of NaV probes for use in live cell imaging experiments and protein profiling studies. Maleimide-toxin conjugates with bioorthogonal reactive groups have been synthesized and can be employed for ligation of visualization and isolation tags to covalently modified channels. Appendage of fluorine-18 to a modified STX affords a probe for studying NaV expression in living subjects. An efficient synthetic route yields a derivative that binds with nanomolar affinity to several NaV isoforms. Biodistribution, autoradiography, and PET-MRI imaging studies demonstrate accumulation of the radiotracer at the site of injury in a rat model of neuropathic pain. This uptake correlates with the previously reported upregulation of NaV isoforms at this site, validating the utility of this probe as a NaV imaging agent. Collectively, these STX derivatives, uniquely available through chemical synthesis, represent a novel set of molecular probes for studying NaV function in vitro and in vivo.
    Digital Access   2013