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

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.
(© 2022. The Author(s).)