TY - JOUR

T1 - Structure-guided unlocking of NaX reveals a non-selective tetrodotoxin-sensitive cation channel

AU - Noland, Cameron L.

AU - Chua, Han Chow

AU - Kschonsak, Marc

AU - Heusser, Stephanie Andrea

AU - Braun, Nina

AU - Chang, Timothy

AU - Tam, Christine

AU - Tang, Jia

AU - Arthur, Christopher P.

AU - Ciferri, Claudio

AU - Pless, Stephan Alexander

AU - Payandeh, Jian

N1 - Funding Information: We thank members of the Pless laboratory and Genentech colleagues in the Research Materials, BioMolecular Resources, Microchemistry, Proteomics and Lipidomics, and Structural Biology departments for their support of this project. We appreciate advice from E. Green, A. Rohou, C. Koth, W. Sandoval and the encouragement of S. Hymowitz, V. Dixit, and A. Chan. We are grateful to Harold Zakon (University of Texas at Austin) for insightful discussions. Members of the Pless group acknowledge the Novo Nordisk Foundation (NNF20OC0064550), the Carlsberg Foundation (CF16-0504), and the Independent Research Fund Denmark (7025-00097?A) for financial support. Funding Information: We thank members of the Pless laboratory and Genentech colleagues in the Research Materials, BioMolecular Resources, Microchemistry, Proteomics and Lipidomics, and Structural Biology departments for their support of this project. We appreciate advice from E. Green, A. Rohou, C. Koth, W. Sandoval and the encouragement of S. Hymowitz, V. Dixit, and A. Chan. We are grateful to Harold Zakon (University of Texas at Austin) for insightful discussions. Members of the Pless group acknowledge the Novo Nordisk Foundation (NNF20OC0064550), the Carlsberg Foundation (CF16-0504), and the Independent Research Fund Denmark (7025-00097 A) for financial support. Publisher Copyright: © 2022, The Author(s).

PY - 2022

Y1 - 2022

N2 - Unlike classical voltage-gated sodium (NaV) channels, NaX has been characterized as a voltage-insensitive, tetrodotoxin-resistant, sodium (Na+)-activated channel involved in regulating Na+ homeostasis. However, NaX remains refractory to functional characterization in traditional heterologous systems. Here, to gain insight into its atypical physiology, we determine structures of the human NaX channel in complex with the auxiliary β3-subunit. NaX 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 NaX. 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 NaX-QTT channel construct is non-selective among monovalent cations, inhibited by extracellular calcium, and sensitive to classical NaV channel blockers, including tetrodotoxin. Our findings highlight a functional diversity across the NaV channel scaffold, reshape our understanding of NaX physiology, and provide a template to demystify recalcitrant ion channels.

AB - Unlike classical voltage-gated sodium (NaV) channels, NaX has been characterized as a voltage-insensitive, tetrodotoxin-resistant, sodium (Na+)-activated channel involved in regulating Na+ homeostasis. However, NaX remains refractory to functional characterization in traditional heterologous systems. Here, to gain insight into its atypical physiology, we determine structures of the human NaX channel in complex with the auxiliary β3-subunit. NaX 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 NaX. 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 NaX-QTT channel construct is non-selective among monovalent cations, inhibited by extracellular calcium, and sensitive to classical NaV channel blockers, including tetrodotoxin. Our findings highlight a functional diversity across the NaV channel scaffold, reshape our understanding of NaX physiology, and provide a template to demystify recalcitrant ion channels.

U2 - 10.1038/s41467-022-28984-4

DO - 10.1038/s41467-022-28984-4

M3 - Journal article

C2 - 35301303

AN - SCOPUS:85126585477

VL - 13

JO - Nature Communications

JF - Nature Communications

SN - 2041-1723

IS - 1

M1 - 1416

ER -