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. 2022 Mar 17;13(1):1416.
doi: 10.1038/s41467-022-28984-4.

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

Cameron L Noland #  1   2 , Han Chow Chua #  3 , Marc Kschonsak #  1 , Stephanie Andrea Heusser  3 , Nina Braun  3   4 , Timothy Chang  5 , Christine Tam  5 , Jia Tang  6 , Christopher P Arthur  1 , Claudio Ciferri  7 , Stephan Alexander Pless  8 , Jian Payandeh  9   10
Affiliations

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

Cameron L Noland et al. Nat Commun. .

Abstract

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.

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Conflict of interest statement

C.L.N., M.K., T.C., C.T., J.T., C.A., C.C., and J.P. are or were employees of Genentech or Roche and own shares in the Genentech or Roche group. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Characterization of and overall structure of human NaX.
a Representative currents from Xenopus laevis oocytes expressing human NaX or NaV1.7. NaX: steps between +80 to −100 mV, in 20 mV increments from a HP of 0 mV; NaV1.7: depolarizing steps between −80 and +65 mV, in 5 mV increments, from a HP of −100 mV. b Representative currents from oocytes expressing NaX in response to extracellular application of indicated compounds (BDS-I Blood depressing substance I, ATX-II Neurotoxin 2). Voltage protocols as above. See Methods for concentrations of compounds tested. c Representative currents from oocytes expressing NaX and co-expression of NaV or CaV channel auxiliary-subunits, Na+/K+-ATPase subunits and synapse-associated protein 97 (SAP97), and in the presence of the indicated extracellular Na+ concentration. Voltage protocols as above. d Data summary of independent experiments performed as in parts a–c. Data are shown as mean ± SD; ns not significant; ****p < 0.0001; one-way ANOVA with Dunnett’s test (against NaX). Exact p-values and statistical parameters are provided in Source Data. Numbers of biological replicates (n) are indicated. e Representative currents from murine Neuro-2a cells expressing human NaX or NaV1.7 in response to changes of the extracellular Na+ concentration (HP = −60 mV), as indicated, or voltage: depolarizing steps between −60 to +60 mV, in 10 mV increments from a HP of −100 mV. f Data summary of independent experiments performed as in parts e. Data are shown as mean ± SD; ns not significant; *p < 0.05; **p < 0.01; ****p < 0.0001; one-way ANOVA with Dunnett’s test (against mock-transfected cells). Exact p-values and statistical parameters are provided in Source Data. Numbers of biological replicates (n) are indicated. g Western blots of total lysate and surface fraction of proteins extracted from Neuro-2a cells probed for the indicated proteins. Data represent three independent biological replicates. h Side and extracellular view of the β3-NaX channel complex. Approximate membrane boundaries are indicated. DI, DII, DIII, and DIV are colored in green, blue, orange, and pink, respectively, with the β3-subunit in gray surface representation.
Fig. 2
Fig. 2. NaX pore module structure reveals a nonconductive state.
a NaX pore volume shown as gray surface with DII and DIV in cartoon rendering (DI and DIII omitted for clarity). b View of the S6-helices with side-chains lining the activation gate shown. Orthogonal view provides a wider perspective with DIII and DIV colored orange and pink, and the IFI-motif (green) from the DIII–DIV linker shown in stick and semi-transparent surface representation. c Orthogonal views sliced through the pore module highlighting lateral fenestrations and bound lipids. The phosphatidylethanolamine that crosses the S6-gate is in purple stick representation. d Similar to middle panel c, but with cryo-EM map shown in blue mesh representation. e Location of S6-gate hydrophobic side-chains targeted by pore-wetting mutations. f Representative currents from Xenopus laevis oocytes expressing the NaX-QTT construct with voltage protocol indicated. g Data summary of independent experiments with indicated constructs (see Supplementary Fig. 8). Data are shown as mean ± SD; ns not significant; ****p < 0.0001; one-way ANOVA with Dunnett’s test (against NaX-QTT + H2O). Exact p-values and statistical parameters are provided in Source Data. Numbers of biological replicates (n) are indicated.
Fig. 3
Fig. 3. Structure and characterization of the NaX selectivity filter.
a NaX DENA-motif side-chains shown as sticks. b Residues in NaX that form a conserved interaction network around the selectivity filter in NaV channels (NaV1.7, PDB 6J8J). c Superimposed view of the DI-DIV interface comparing NaX and NaV1.7 (gray, PDB 6J8J). d View as in b, NaX and NaV1.7 selectivity filter electrostatic surface rending. Note, central cavity and activation gate excluded for clarity. e Representative currents from HEK293T cells expressing human NaX-QTT with a C-terminal GFP-Flag tag in a physiological (left) or NMDG+-only extracellular solution (middle). See methods for composition of intracellular (IC) and extracellular (EC) solutions. Steps between +80 to −100 mV, in 20 mV increments, from a HP of 0 mV. Right, shows I–V curve data summary from n = 6 cells over two independent experiments. Data are shown as mean ± SD. Numbers of biological replicates (n) are indicated. f Representative currents from HEK293T cells expressing human NaX-QTT with indicated monovalent cations in the extracellular solution. Voltage ramp from −80 to +80 mV was applied. Right, summary of reversal potentials and permeability ratios measured from three independent experiments. Data are shown as mean ± SD. Numbers of biological replicates (n) are indicated.
Fig. 4
Fig. 4. Pharmacology of the human NaX-QTT channel.
a Representative I–V curves from HEK293T cells expressing human NaX-QTT with indicated extracellular monovalent cations with or without indicated amounts of CaCl2 in the extracellular solution. Voltage ramp from −80 to +80 mV was applied. Right, percentage of block of outward Na+ by indicated concentrations of Ca2+ at 80 mV. Data are shown as mean ± SD of n = 5 cells over three independent experiments. b Representative currents from Xenopus laevis oocytes expressing human NaX-QTT in standard extracellular solution with or without indicated divalent and trivalent cations (unit in mM), when stepping from 0 to +80 mV. c Representative currents from oocytes expressing human NaX-QTT in standard extracellular solution with or without indicated blockers added, when stepping from 0 to +80 mV (left) or from 0 to −100 mV (right). Middle, summary of currents measured from two independent experiments. Data are shown as mean ± SD. Numbers of biological replicates (n) are indicated. See Methods for concentrations of compounds tested.
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