A chemogenetic ligand-receptor pair for voltage-gated sodium channel subtype-selective inhibition

Neuronal excitability relies on the tightly regulated expression and discrete subcellular localization of voltage-gated sodium 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., epilepsy, musculoskeletal disorders, neuropathic pain) motivates the development of high-precision pharmacological reagents to facilitate NaV studies. Investigations of NaV 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 NaVs 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 NaV 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 NaV1.1- 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 NaV physiology and have additional applications for manipulating action potential signals given the requisite role of NaVs in electrogenesis. SIGNIFICANCEVoltage-gated sodium channels are an obligatory component of the biochemical machinery that makes possible electrical signaling in cells. Malfunction of these large protein complexes underlies a number of debilitating human disorders including certain forms of epilepsy, cardiac arrhythmia, and neuropathic pain. A desire to better understand how sodium channels initiate, propagate, and integrate electrical signals in healthy and aberrant cells necessitates access to molecular tools that enable precise manipulation of channel function. This work describes the advancement of such technology, applications of which should facilitate discoveries in foundational and translational research.