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Structure of the human sodium leak channel NALCN

Abstract

Persistently depolarizing sodium (Na+) leak currents enhance electrical excitability1,2. The ion channel responsible for the major background Na+ conductance in neurons is the Na+ leak channel, non-selective (NALCN)3,4. NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm4,5,6,7,8,9,10. NALCN activity is under tight regulation11,12,13,14 and mutations in NALCN cause severe neurological disorders and early death15,16. NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here we investigate this essential leak channel and determined the structure of NALCN in complex with a distinct auxiliary subunit, family with sequence similarity 155 member A (FAM155A). FAM155A forms an extracellular dome that shields the ion-selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Unusual voltage-sensor domains with asymmetric linkages to the pore suggest mechanisms by which NALCN activity is modulated. We found a tightly closed pore gate in NALCN where the majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6 gate and distinctive π-bulges. Our findings provide a framework to further study the physiology of NALCN and a foundation for discovery of treatments for NALCN channelopathies and other electrical disorders.

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Fig. 1: Overall structure and function of NALCN.
Fig. 2: NALCN pore, pharmacology and CTD interactions.
Fig. 3: NALCN voltage-sensor domains.
Fig. 4: NALCN disease-associated mutations.

Data availability

The NALCN–FAM155A coordinates are deposited at the PDB under accession number 6XIW. Cryo-EM data are deposited in the Electron Microscopy Data Bank under accession number EMD-22203. All other data are included in the paper and the supplementary information files. Source data are provided with this paper.

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Acknowledgements

We thank members of the Pless laboratory and Genentech colleagues in the Research Materials, BioMolecular Resources and Structural Biology departments for their support of this project; and appreciate the encouragement of A. Rohou, C. Koth, J. Kiefer, S. Hymowitz, V. Dixit and A. Chan. Members of the Pless group acknowledge the Carlsberg Foundation (CF16-0504), the Independent Research Fund Denmark (7025-00097A, 9124-00002B), the Novo Nordisk Foundation (NNF19OC0056438) and the Lundbeck Foundation (R252-2017-1671) for financial support. Reagents are available under a material transfer agreement with Genentech or the appropriate party.

Author information

Authors and Affiliations

Authors

Contributions

M.K., C.L.N. and T.C. established protein purification and reconstitution methods. Z.R.L. generated key protein expression reagents. M.K., C.L.N. and C.P.A. optimized cryo-EM sample preparation and data collection. M.K. determined the structure, with guidance from C.C. H.C.C. established methods to record function of the NALCN complex. H.C.C., C.W., O.Ø.B. and A.O.A. performed molecular biology, electrophysiology, biochemistry and pharmacology experiments. M.K., H.C.C., C.L.N., C.W., T.C., O. Ø.B., A.O.A, C.C., S.A.P. and J.P. analysed the data. M.K., H.C.C., C.C., S.A.P. and J.P. wrote the manuscript with input from all authors. C.C., S.A.P. and J.P. supervised the project and are co-senior authors.

Corresponding authors

Correspondence to Claudio Ciferri, Stephan Alexander Pless or Jian Payandeh.

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Competing interests

M.K., C.L.N., T.C., Z.R.L., C.P.A., C.C. and J.P. are employees of Genentech/Roche; the other authors declare no competing interests.

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Peer review information Nature thanks J. David Spafford and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 NALCN function in heterologous expressions systems.

a, b, Representative current traces from Xenopus laevis oocytes (a) and HEK293T cells (b) expressing NALCN alone or with FAM155A, UNC80 and UNC79. Holding potential 0 mV, hyperpolarizing steps from +80 mV to −100 mV in 20 mV increments; oocyte experiments were performed in ND96 recording buffer, HEK293T experiments were performed under symmetrical Na+ conditions. See source data for full experimental details. c, Western blot of total lysate and surface fraction proteins extracted from HEK293T cells expressing the indicated constructs, representative of three independent experiments. d, Representative current traces from oocytes expressing NALCN, FAM155A, UNC80 and UNC79 with NaVβ1 (n = 14), NaVβ2 (n = 10) or CaVα2δ-1 (n = 11). Protocol as above.

Source data

Extended Data Fig. 2 NALCN biochemistry and cryo-electron microscopy processing.

a, NALCN purification scheme. b, SDS–PAGE of select samples from the STREP and FLAG affinity purification steps (representative of three or more independent experiments). c, Size-exclusion chromatography of NALCN-FAM155A in lipid nanodiscs (MSP1E3D1), representative of two independent experiments. d, Example cryo-EM micrograph image of NALCN-FAM155A-MSP1E3D1 complex from 15,080 collected micrographs (see f). e, Representative 2D-class averages after 2 rounds of 2D classification from 200 classes and approximately 720,000 particles. f, Data collection and processing workflow. g, Heat map representation of the distribution of assigned particle orientations. h, FSC between two half datasets yields a global resolution estimate of approximately 2.8 Å resolution.

Extended Data Fig. 3 NALCN cryo-EM map.

a, Isosurface rendering of the 3D map with surface coloring according to the local resolution estimated by windowed FSCs. b, Example 3D map overlay for transmembrane regions across DI. c, Example 3D map overlay on representative lipids modelled within the transmembrane region.

Extended Data Fig. 4 Comparison of NALCN with NaV and CaV channel structures.

a, Helical S4-S5 linkers are highlighted by colour in NALCN, NaV1.7 (PDB: 6J8G), CaV1.1 (PDB: 6JPA), and CaV3.1 (PDB: 6KZP); intracellular view (left) and side views (right) are shown. b, Side view of NALCN, CaV1.1, NavPaS (PDB: 6A90) and NaV1.7 highlighting the DIII-DIV linker, CTD, and the β1a-subunit (of CaV1.1).

Extended Data Fig. 5 NALCN structure-based sequence alignment.

Selectivity filter with inner ring (IR, red box) and outer ring (OR, purple box) residues indicated.

Extended Data Fig. 6 Extracellular pore loops, FAM155A sequence alignment (partial), and auxiliary subunit comparisons.

a, Close-in view of the NALCN ECL loops from the DI, DIII and DIV PM, highlighting disulfide bonds (in stick representation), compared to the analogous ECLs from NaV1.7 (PDB: 6J8G), CaV1.1 (PDB: 6JPA), and CaV3.1 (PDB: 6KZP). b, Multi-sequence alignment of selected FAM155A-like proteins; for clarity, spanning only the parts of the cysteine-rich domain (CRD). N-terminal region (171-175) not shown. c, FAM155A-CRD and the MuSK Fzd-like CRD (PDB: 3HKL). d, Superposition of the NaV1.7-β1-β2 complex (PDB: 6J8G) onto NALCN. e, Superposition of the CaV1.1- α2δ-1-β1a-ɣ complex (PDB: 6JPA) onto NALCN.

Extended Data Fig. 7 NALCN pore volume and selectivity filter comparisons.

a, Pore volume rendering65 of NALCN (similar to Fig. 3a), NaV1.7 (PDB: 6J8G), CaV1.1 (PDB: 6JPA), and CaV3.1 (PDB: 6KZP). b, Comparison of selectivity filters of NALCN, NaV1.7 (PDB: 6J8G), CaV1.1 (PDB: 6JPA), and CaV3.1 (PDB: 6KZP). c, NaV1.7-tetrodotoxin (TTX; PDB: 6J8I) and NaV1.7-saxitoxin (STX; PDB: 6J8G) complexes shown alone and superimposed onto NALCN (where NaV1.7 has been omitted for clarity in the superposition).

Extended Data Fig. 8 S6 sequence alignment and NALCN π-bulge.

a, Multi-sequence alignment from select ion channels. Residues that support the S6 π-bulge in NALCN are outlined with a purple box and dashed line indicates the observed hydrogen bond to the backbone carbonyl of the i-4 residue. Residues with side-chains that fill the NALCN pore fenestrations are outlined with a blue box, and residues with side-chains lining the closed S6-activation gate in NALCN are outlined with a green box. b, Side-view of S6 with polar side-chains supporting the π-bulge shown as stick with putative hydrogen bonds (dashed line) to the i-4 backbone carbonyls (*). c, Close-in view of the S6 π-bulges in NALCN. d, TRPV3 channel S6 in apo and ligand bound states (PDB: 6DVW and 6DVZ, respectively), with location of the π-bulge indicated.

Extended Data Fig. 9 NALCN voltage-sensor domain comparisons and features.

ac, Comparison of the VSDs from KVchim (KV1.2/2.1; PDB: 3LNM), NALCN and NaV1.7 (PDB: 6J8G) with the S2 residue of the HCS shown in green sticks and the S4 gating charges sown in blue sticks. d, S3-S4 of NALCN VSD1-4 with the 310-helix regions indicated. e, Basic residues clustered around the intracellular vestibules of NALCN VSDs are shown as blue sticks; the S2 aromatic residue of the HCS is shown in green sticks.

Extended Data Fig. 10 Characterization of select CLIFAHDD and IHPRF1 mutations.

a, Position of CLIFAHDD (blue) and IHPRF1 (red) mutations on NALCN. b, Exemplar current traces from Xenopus laevis oocytes expressing WT or indicated mutants recorded in ND96 solution (dashed line indicates zero current level). c, ImaxV plots (normalized to averaged outward current at +80 mV of WT complex) from Xenopus laevis oocytes expressing WT (white) or indicated CLIFAHDD/IHPRF1 mutants (blue and red, respectively) recorded in ND96 solution. d, Left panel: western blot of total lysate and surface fraction proteins extracted from HEK293T cells expressing the indicated constructs. NALCN was co-expressed with UNC80, UNC79 and FAM155A (all tagged with C-terminal eGFP-2xFLAG). Right panel: surface expression of NALCN (relative to Na+/K+ATPase expression levels). Data shown as average ± s.d. (n = 3); ns = not statistically different in two-sided paired Student’s t-test.

Source data

Extended Data Fig. 11 Conservation analysis and potential protein interaction sites.

a, Conservation analysis66 of NALCN highlighting the FAM155A docking site (FAM155A in grey surface rendering). Sequences used for analysis: Homo sapiens, Mus musculus, Rattus norvegicus, Xenopus tropicalis, Ictalurus punctatus, Danio rerio, Gallus gallus, Columba livia, Drosophila melanogaster, Aedes albopictus, Caenorhabditis elegans, Anisakis simplex. b, Superposition of NALCN, NaV1.7 (PDB: 6J8G), CaV1.1 (PDB: 6JPA), CaV3.1 (PDB: 6KZP) and HCN1 (PDB: 5U6P) based on the S5 segment, with only the S5 and S4-S5 linkers shown for clarity. c, Conservation analysis (as in panel a) shown for the NALCN CTD-region.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

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Kschonsak, M., Chua, H.C., Noland, C.L. et al. Structure of the human sodium leak channel NALCN. Nature 587, 313–318 (2020). https://doi.org/10.1038/s41586-020-2570-8

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