PROJECT SUMMARY Neuronal excitability relies on the tightly regulated expression and discrete subcellular localization of voltage-gated sodium ion channels (NaVs). These large membrane protein complexes control the movement of sodium ions across cell membranes and are responsible for initiating and propagating action potentials. A desire to better understand the role of NaV subtypes in electrical signal conduction and the relationship between channel dysregulation and specific human pathologies (e.g., arrhythmia, epilepsy, skeletal muscle disorders, neuropathic pain) motivates the development of high precision reagents to facilitate NaV studies. Investigations of NaV physiology are currently limited by a lack of available methods with which to modulate the function of individual channel subtypes and to mark changes in cellular distributions, membrane expression levels, and structural modifications (i.e., protein post- translational modification) in live cells and in response to external cues. We are developing small molecule probes for NaV studies based on naturally occurring bis- guanidinium toxins, among which saxitoxin (STX) is the archetype. These agents function as molecular ‘corks’ to occlude the extracellular mouth of the ion conductance pore, a desirable feature for the types of tool compounds we wish to access. De novo chemical synthesis has enabled the engineering of modified forms of STX, which we will use in combination with protein mutagenesis and electrophysiology to investigate NaV activity on action potential dynamics. Understanding how NaV expression is regulated and altered by extrinsic factors—glial cells, inflammatory mediators, pH, nerve injury—and how such changes modulate action potentials is a long-term goal of our research. To address these questions, we will develop and validate three classes of tool compounds. These reagents will enable 1) acute, spatiotemporal inhibition of individual NaV subtypes; 2) selective fluorescent labeling of membrane NaVs; and 3) affinity purification of membrane NaVs for proteomics analysis. With the success of our research program, we will deliver a unique set of high precision chemical probes to help illuminate the complex physiology of NaVs that underlies bioelectrical signaling.