Structure-guided unlocking of Na_X reveals a non-selective tetrodotoxin-sensitive cation channel.

Abstract

Unlike classical voltage-gated sodium (Na_V) channels, Na_X has been characterized as a voltage-insensitive, tetrodotoxin-resistant, sodium (Na⁺)-activated channel involved in regulating Na⁺ homeostasis. However, Na_X remains refractory to functional characterization in traditional heterologous systems. Here, to gain insight into its atypical physiology, we determine structures of the human Na_X channel in complex with the auxiliary β3-subunit. Na_X reveals structural alterations within the selectivity filter, voltage sensor-like domains, and pore module. We do not identify an extracellular Na⁺-sensor or any evidence for a Na⁺-based activation mechanism in Na_X. Instead, the S6-gate remains closed, membrane lipids fill the central cavity, and the domain III-IV linker restricts S6-dilation. We use protein engineering to identify three pore-wetting mutations targeting the hydrophobic S6-gate that unlock a robust voltage-insensitive leak conductance. This constitutively active Na_X-QTT channel construct is non-selective among monovalent cations, inhibited by extracellular calcium, and sensitive to classical Na_V channel blockers, including tetrodotoxin. Our findings highlight a functional diversity across the Na_V channel scaffold, reshape our understanding of Na_X physiology, and provide a template to demystify recalcitrant ion channels.