Abstract
Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1,2,3,4,5,6,7,8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9,10,11,12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN–FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79–UNC80 subcomplex and the NALCN DI–DII and DII–DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.
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Data availability
The NALCN–FAM155A–UNC79–UNC80–CaM coordinates are deposited in the PDB (7SX3 and 7SX4) and cryo-EM data are deposited in the Electron Microscopy Data Bank (EMD-25492 and EMD-25493) for conformation 1 and conformation 2, respectively. Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE54 partner repository with the dataset identifier PXD027213 and details are included in the Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We thank members of the Pless and Ben-Johny laboratories, P. Picotti, and Genentech colleagues in the BioMolecular Resources and Structural Biology departments for their support, and appreciate the encouragement of A. Rohou, C. Koth, S. Hymowitz, V. Dixit and A. Chan. Members of the Ben-Johny group acknowledge support from the NIH National Institute of Neurological Disorders and Stroke (R01 NS110672). Members of the Pless group acknowledge the Carlsberg Foundation (CF16-0504), the Independent Research Fund Denmark (7025-00097A and 9124-00002B) and the Lundbeck Foundation (R252-2017-1671) for financial support. Reagents are available under a material transfer agreement with Genentech or the appropriate party.
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Contributions
M.K. established protein purification and reconstitution methods with input from C.L.N. T.C., C.T. and N.P. generated key protein expression reagents. M.K 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 the function of the NALCN complex. H.C.C., C.W. and K.S. performed the molecular biology and two-electrode voltage clamp electrophysiology experiments. A.L. performed and analysed the crosslinking mass spectrometry experiments. N.C. and M.B.-J. performed the single-channel experiments and analyses. M.K., H.C.C., C.W., N.C., K.S., A.L., M.B.-J., C.C., S.A.P. and J.P. analysed the data. H.C.C. performed and analysed the linker competition experiments with input from M.K., S.A.P. and J.P. M.K., H.C.C., C.C., S.A.P. and J.P. wrote the manuscript with input from all authors. A.L., M.B.-J., C.C., S.A.P. and J.P. supervised the project and are co-senior authors.
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M.K., C.L.N., T.C., C.T., N.P., C.P.A., C.C. and J.P. are or were employees of Genentech/Roche; all remaining authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Purification and structure determination of the NALCN channelosome.
a, NALCN channelosome protein expression and purification scheme. b, Example size exclusion chromatogram and SDS-PAGE of nanodisc-reconstituted NALCN channelosome sample. c, Example cryo-EM micrograph image of the NALCN channelosome-MSP1E3D1 complex. d, Representative 2D-class averages of selected particles from 200 classes and approximately 130,000 particles. e, Data collection and processing workflow. f, Conformation 1, FSC between two half datasets yields a global resolution estimate of approximately 3.1 Å resolution from the refinement using an overall mask of the NALCN channelosome, 3.2 Å resolution from the focused NALCN-CaM-FAM, 2.9 Å from the focused UNC crossover, 3.7 Å from the focused UNC N-C and 3.8 Å from the focused UNC C-N refinements. g, Conformation 1, heat map representation of the distribution of assigned particle orientations. h, Conformation 2, FSC between two half datasets yields a global resolution estimate of approximately 3.5 Å resolution. i, Conformation 2, heat map representation of the distribution of assigned particle orientations.
Extended Data Fig. 2 Select cryo-EM map regions of the NALCN channelosome.
a–g, Example 3D map overlay for indicated regions.
Extended Data Fig. 3 NALCN and FAM155A structures in the NALCN channelosome and NALCN-FAM155A subcomplex.
a, Comparison of the NALCN subunit in the NALCN-FAM155A subcomplex and NALCN channelosome (with UNC79 and UNC80 removed for clarity). b, Superposition of the FAM155A subunit in the NALCN-FAM155A subcomplex and NALCN channelosome. c, Pore radius of the ion conduction pathway in the NALCN channelosome or NALCN-FAM155A subcomplex structures (PDB 6XIW) calculated by HOLE55.
Extended Data Fig. 4 UNC79 and UNC80 are HEAT-repeat proteins.
a, Side- and top-views of UNC79. Position of disordered loops >50 residues in length are indicated. b, Side- and top-views of UNC80. Position of disordered loops >50 residues in length are indicated. c, Open-book view of UNC79-UNC80 interface showing contact surface. d, Same as part c, showing electrostatic surface. e, Same as part c, showing surface conservation across selected vertebrate species calculated by ConSurf56.
Extended Data Fig. 5 Structure-function analysis of UNC79 and UNC80.
a, Schematic of UNC79 and UNC80 with corresponding amino acid residues of fragments indicated. Example current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where various isolated ~500 residue fragments of either UNC79 or UNC80 are co-expressed in trans, with steps from +80 to −80 mV, 40 mV increments, in ND96 recording solution. Right shows summary of mean current amplitudes elicited at +80 mV (top bar graph) or −80 mV (bottom bar graph) from a holding potential of 0 mV for indicated construct combinations. Numbers of biological replicates (n) are indicated. Recordings were performed on four to five oocytes from two different batches. Data are shown as mean±SD. ****, * and ns represent p value < 0.0001, < 0.05 and >0.05, respectively; one-way analysis of variance (ANOVA), Dunnett’s test (against +H2O). P values are adjusted to account for multiple comparisons and are presented in Supplementary Table 5. For electrophysiological measurements, see Source Data. b, Schematic of UNC80 with corresponding truncated constructs indicated. Example current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where wild-type UNC80 or truncated constructs are expressed, with steps from +80 to −80 mV, 20 mV increments, in ND96 recording solution. Star (#) indicates constructs that contain fusion of a C-terminal GFP-Flag tag. Right shows summary of mean current amplitudes elicited at +80 mV (top bar graph) or −80 mV (bottom bar graph) from a holding potential of 0 mV for indicated construct combinations. Numbers of biological replicates (n) are indicated. Recordings were performed on four to six oocytes from two different batches. Data are shown as mean±SD. ****, **, and * represent p value < 0.0001, < 0.01 and < 0.05, respectively; one-way analysis of variance (ANOVA), Dunnett’s test (against UNC80#). P values are adjusted to account for multiple comparisons and are presented in Supplementary Table 5. For electrophysiological measurements, see Source Data. c, Schematic of UNC80 with corresponding nonsense mutation constructs indicated (note, R1265X, L2586X, and Y2796X mutations have been previously reported by us10 and representative data are shown here for comparison). Example current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where wild-type UNC80 or nonsense mutation constructs are expressed, with steps from +80 to −80 mV, 20 mV increments, in ND96 recording solution. Right shows summary of mean current amplitudes elicited at +80 mV (top bar graph) or −80 mV (bottom bar graph) from a holding potential of 0 mV for indicated construct combinations. Numbers of biological replicates (n) are indicated. Recordings were performed on three to six oocytes from two to three different batches. Data are shown as mean±SD. **** represents p value < 0.0001; one-way analysis of variance (ANOVA), Dunnett’s test (against UNC80). P values are adjusted to account for multiple comparisons and are presented in Supplementary Table 5. For electrophysiological measurements, see Source Data. d, Example current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where wild-type UNC80 or missense mutation constructs are expressed, with steps from +80 to −80 mV, 20 mV increments, in ND96 recording solution. Right shows summary of mean current amplitudes elicited at +80 mV (top bar graph) or −80 mV (bottom bar graph) from a holding potential of 0 mV for indicated construct combinations. Numbers of biological replicates (n) are indicated. Recordings were performed on two to eight oocytes from two to three different batches. Data are shown as mean±SD. **** and ns represent p value < 0.0001 and >0.05, respectively; one-way analysis of variance (ANOVA), Dunnett’s test (against UNC80). P values are adjusted to account for multiple comparisons and are presented in Supplementary Table 5. For electrophysiological measurements, see Source Data. e, Close-in views of select missense mutations previously identified in UNC80 mapped onto the UNC79-UNC80 subcomplex structure. Note, exact p values are presented in Supplementary Table 5.
Extended Data Fig. 6 Characterization of NALCN-NaV1.4 chimeras.
a, Schematic of human NALCN and human NALCN-rat NaV1.4 chimeric constructs. Example current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where wild-type or chimeric truncated NALCN constructs are expressed, with steps from +80 to −80 mV, 20 mV increments, in ND96 recording solution. Numbers of biological replicates (n) are indicated in b. Recordings were performed on two to five oocytes from two to five different batches. For electrophysiological measurements, see Source Data. b, Summary of mean current amplitudes elicited at +80 mV (top bar graph) or −80 mV (bottom bar graph) from a holding potential of 0 mV for indicated constructs. Data are shown as mean±SD. **** and ns represent p value < 0.0001 and >0.05, respectively; one-way analysis of variance (ANOVA), Dunnett’s test (against NALCN). P values are adjusted to account for multiple comparisons and are presented in Supplementary Table 5.
Extended Data Fig. 7 Structure-function of the NALCN DI-DII and DII-DIII linkers.
a, Current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where the NALCN subunit is wild-type or has indicated mutations, insertions or deletions in the DI-DII linker. Steps from +80 to −100 mV, 20 mV increments, in ND96 recording solution. Right shows summary of mean current amplitudes elicited at +80 mV (top bar graph) or −80 mV (bottom bar graph) from a holding potential of 0 mV for indicated constructs. b, Representative Western blot of total lysate and surface fraction proteins extracted from Xenopus oocytes expressing the indicated constructs where wild-type or mutant NALCN was co-expressed with wild-type UNC80, UNC79 and FAM155A. For gel source data, see Supplementary Fig. 3. c, Current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where the NALCN subunit is wild-type or has indicated insertions and deletions in the DII-DIII linker. Steps from +80 to −100 mV, 20 mV increments, in ND96 recording solution. Right shows summary of mean current amplitudes elicited at +80 mV (top bar graph) or −80 mV (bottom bar graph) from a holding potential of 0 mV for indicated constructs. a, c, Numbers of biological replicates (n) are indicated. Recordings were performed on four to five oocytes from two different batches. Data are shown as mean±SD. ****, ***, **, * and ns represent p value < 0.0001, < 0.001, < 0.01, < 0.05 and >0.05, respectively; one-way analysis of variance (ANOVA), Dunnett’s test (against WT). P values are adjusted to account for multiple comparisons and are presented in Supplementary Table 5.
Extended Data Fig. 8 Structure-function analysis of the NALCN C-terminal domain.
a, Current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79 and UNC80, where the NALCN subunit is wild-type or has indicated CTD-deletions or mutations. Steps from +80 to −100 mV, 20 mV increments, in ND96 recording solution. Right shows summary of mean current amplitudes elicited at +80 mV (top bar graph) or −80 mV (bottom bar graph) from a holding potential of 0 mV for indicated constructs. Numbers of biological replicates (n) are indicated. Recordings were performed on three to six oocytes from two different batches. Data are shown as mean±SD. * and ns represent p value < 0.05 and >0.05, respectively; one-way analysis of variance (ANOVA), Dunnett’s test (against WT). P values are adjusted to account for multiple comparisons and are presented in Supplementary Table 5. b, Current traces from Xenopus oocytes expressing NALCN, FAM155A, UNC79, UNC80 and control (H2O) or an isolated NALCN-CTD construct in trans. Steps from +80 to −100 mV, 20 mV increments, in ND96 recording solution. Right shows summary of mean current amplitudes elicited at +80 mV from a holding potential of 0 mV for indicated conditions. Numbers of biological replicates (n) are indicated. Recordings were performed on four to five oocytes from two different batches. Data are shown as mean±SD. Two-sided unpaired t-test, p = 0.1286 (+80 mV), p = 0.0037 (-80 mV). c, Current traces from Xenopus oocytes expressing wild-type NALCN (1-1738) or indicated C-terminal truncation constructs recorded in ND96 (top) and divalent cation (X2+)-free buffer (bottom). Steps from +80 to −100 mV, 20 mV increments. Right shows fold-increase in inward current elicited at −100 mV for wild-type NALCN and indicated truncation mutants in response to removal of divalent cations. Numbers of biological replicates (n) are indicated. Recordings were performed on three to four oocytes from two different batches. Data are shown as mean±SD. ns represents p value >0.05; one-way analysis of variance (ANOVA), Dunnett’s test (against WT). P values are adjusted to account for multiple comparisons and are presented in Supplementary Table 5. d, Current traces from Xenopus oocytes expressing wild-type NALCN, FAM155A, UNC79 and UNC80, where a “CaM-chelator” construct (which is composed of four tandem IQ motifs (IQ3-6: residues L766–K920) of mouse myosin Va (Uniprot Q99104) that diminishes free concentration of apo-CaM48,49) is co-expressed. Numbers of biological replicates (n) are indicated. Recordings were performed on four to five oocytes from three different batches. Data are shown as mean±SD. Two-sided unpaired t-test, p = 0.0002 (+80 mV), p = 0.0421 (−80 mV).
Extended Data Fig. 9 Cell-attached single channel recordings of the NALCN channelosome.
a, Exemplar traces from an individual patch show absence of channel openings when HEK293 cells are transfected with only UNC79, UNC80, and FAM155A. Gray slanted line, unitary current relationship for wild-type NALCN. b, Ensemble average current obtained from 154 sweeps for the individual patch shown in panel a. No detectible channel activity was observed in a total of n = 8 cells (852 sweeps). c, Exemplar traces show additional examples of single-channel openings of wild-type NALCN channelosome from the same patch shown in Fig. 4b. Gray slanted line, unitary current relationship. d, Ensemble average current obtained from 90 sweeps for the single-channel patch of NALCN channelosome as shown in panel c. e, Diary plot of single-trial PO values from the one-channel patch of wild-type NALCN, as shown in panel c, reveal distinct active (A; traces with brief and flickery openings) and quite (Q; traces with no discernible activity) gating modes. f, Exemplar traces show additional examples of single-channel openings of Y578S mutant channels from the individual patch shown in Fig. 4b. Gray slanted line, unitary current relationship. g, Ensemble average current obtained from 40 sweeps for the single-channel patch of Y578S mutant channel shown in panel f. h, Diary plot of single-trial PO for the one-channel patch for Y578S mutant displayed in panel f. Y578S mutant channel also show switching between active and quite gating modes.
Supplementary information
Supplementary Information
This file contains Supplementary Figures 1–3 and Supplementary Tables 1 and 3–5.
Supplementary Table 2
Complete cross-linking mass spectrometry of the NALCN channelosome.
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Kschonsak, M., Chua, H.C., Weidling, C. et al. Structural architecture of the human NALCN channelosome. Nature 603, 180–186 (2022). https://doi.org/10.1038/s41586-021-04313-5
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DOI: https://doi.org/10.1038/s41586-021-04313-5
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