Abstract
Neuronal excitability relies on the tightly regulated expression and discrete subcellular localization of voltage-gated sodium channels (Na(v)s). 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 Na(v) subtypes in electrical signal conduction and the relationship between channel dysregulation and specific human pathologies (e.g., epilepsy, musculoskeletal disorders, neuropathic pain) motivates the development of high-precision pharmacological reagents to facilitate Na(v) studies. Investigations of Na(v) physiology and nerve cell conduction are limited by a lack of available methods with which to modulate acutely and reversibly the function of individual channel subtypes. Moreover, discriminating between Na(v)s expressed in different cell types is not possible even with potent and selective ligands that target specific channel homologues. We have capitalized on both chemical design and protein engineering to advance a chemogenetic tool to inhibit a single Na(v) isoform. A synthetic derivative of the bis-guanidinium toxin saxitoxin (STX) is paired with two unique outer pore-forming amino acid mutations to achieve ~100:1 selectivity for the engineered channel over wild-type Na(v)1.1-1.4, 1.6, and 1.7. The designer ligand is nanomolar potent against the mutant channel and acts within seconds to block sodium ion conduction; washing cells with buffer solution rapidly and completely restores channel function. This technology will empower studies of Na(v) physiology and have additional applications for manipulating action potential signals given the requisite role of Na(v)s in electrogenesis.