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Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels

Nature Structural & Molecular Biology volume 30, pages 841–852 (2023)Cite this article

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Abstract

Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume-regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (SWELL1) and LRRC8B–E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents across cell types. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here we leverage a fiducial-tagging strategy to determine single-particle cryo-EM structures of heterohexameric LRRC8A:C channels in multiple conformations. Compared to homomers, LRRC8A:C channels show pronounced differences in architecture due to heterotypic LRR interactions that displace subunits away from the conduction axis and poise the channel for activation. Structures and functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. These results provide insight into determinants for heteromeric LRRC8 channel assembly, activity and gating by lipids.

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Fig. 1: Structures of heterohexameric LRRC8A:C channels.
Fig. 2: LRRC8C subunit structure and impact on channel architecture.
Fig. 3: Lipids block conduction in closed LRRC8A:C channels.
Fig. 4: Disrupting lipid interactions activates LRRC8A:C channels.
Fig. 5: Model for lipid gating of LRRC8 channels.

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Data availability

For LRRC8ABRIL:C conformation 1, the final models have been deposited in the PDB under nos. 8DS3, 8DRK, 8DRN, 8DRO and 8DRQ and the final maps are deposited in the EMDB under nos. EMD-27682, 27677, 27677, 27679 and 27681. For LRRC8ABRIL:C conformation 2, the final models have been deposited in the PDB under nos. 8DRE, 8DR8 and 8DRA, and the final maps are deposited in the EMDB under nos. EMD-27676, 27674 and 27675. For LRRC8ABRIL:C in lipid nanodiscs, the final models are deposted in the PDB under nos. 8DSA and 8DS9, and the final maps are deposited in the EMDB under nos. EMD-27687 and 27686. For LRRC8ABRIL(T48D):C conformation 1, the final models are deposited in the PBD under nos. 8F79, 8F7B, 8F7D, 8F7E and 8F7J, and the final maps are deposted in the EMDB under nos. 28898, 28901, 28903, 28904 and 28905. For LRRC8ABRIL(T48D):C conformation 2, the final models are deposited in the PDB under nos. 8F77, 8F74 and 8F75 and the final maps are in the EMDB under nos. EMD-28897, 28894 and 28895. Original micrograph videos and final particle stacks are deposited in the Electron Microscopy Public Image Archive (EMPIAR) under nos. 11283 and 11312 for LRRC8ABRIL:C in GDN and nanodiscs, respectively, and 11321 for LRRC8ABRIL(T48D):C in GDN. Source data are provided with this paper.

Change history

  • 15 June 2023

    In the version of this article initially published, an incorrect Peer review information file was posted, which has now been updated in the HTML version of the article.

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Acknowledgements

We thank J. Remis, D. Toso and P. Tobias for microscope and computational support at the Cal-Cryo facility. We thank the laboratories of A. Patapoutian and T. J. Jentsch for generous gifts of HeLa and HEK293 LRRC8 knockout cell lines. We thank members of the Brohawn laboratory and A. F. Kern, C. M. Hoel and R. A. Rietmeijer for discussions and feedback on the manuscript. S.G.B. is a New York Stem Cell Foundation-Robertson Neuroscience Investigator. This work was funded by The New York Stem Cell Foundation, a McKnight Foundation Scholar Award, and Sloan Research Fellowship to S.G.B.; NIGMS grant no. GM128263 to D.M.K.; NIGMS grant no. GM117372 to A.A.K.; and NIGMS grant no. GM117051 and NINDS grant no. NS119826 to E.Y.I.

Author information

Authors and Affiliations

  1. Department of Molecular & Cell Biology, University of California, Berkeley, CA, USA

    David M. Kern, Jennifer M. Hill, Ehud Y. Isacoff & Stephen G. Brohawn

  2. Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA

    David M. Kern, Julia Bleier, Ehud Y. Isacoff & Stephen G. Brohawn

  3. California Institute for Quantitative Biology (QB3), University of California, Berkeley, CA, USA

    David M. Kern, Ehud Y. Isacoff & Stephen G. Brohawn

  4. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA

    Somnath Mukherjee & Anthony A. Kossiakoff

  5. Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA

    Somnath Mukherjee & Anthony A. Kossiakoff

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  1. David M. Kern
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Contributions

D.M.K. performed and analyzed all biochemistry, cryo-EM and electrophysiology experiments. J.M.H. contributed to the development of fiducial tag constructs. S.M. and A.A.K. provided anti-BRIL Fab and anti-Fab Nb. J.B. performed subunit counting experiments and analyzed data under supervision from E.Y.I. D.M.K. and S.G.B. modeled and refined the structures. D.M.K. and S.G.B. conceived of the project. D.M.K. and S.G.B. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Stephen G. Brohawn.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Fiducial tagging strategy.

(a) Schematic of biochemical approach. A BRIL domain is inserted into LRRC8A extracellular loop 1 as a fiducial to facilitate computational alignment of pseudosymmetric LRRC8A and LRRC8C subunits in single particle cryo-EM reconstructions. Anti-BRIL Fab and anti-Fab Nb are added to purified samples to increase fiducial mass. (b-d) Function of LRRC8ABRIL:C channels. Representative voltage clamp recordings from LRRC8A:C, LRRC8ABRIL:C, LRRC8ABRIL:C (with added anti-BRIL Fab and anti-Fab Nb) expressing LRRC8A/B/C/D/E -/- cells in (left) isotonic and (center) hypotonic solution. Current-voltage relationship from each record is plotted on the right. (e) Comparison of fold activation at 60 mV by hypotonic solution from untransfected, wild-type LRRC8A:C, LRRC8ABRIL:C, or LRRC8ABRIL:C (with added anti-BRIL Fab and anti-Fab Nb) expressing LRRC8A/B/C/D/E -/- cells. Neither BRIL fusion nor anti-BRIL Fab and anti-Fab Nb show statistically significant differences from wild-type channels. Mean ± s.e.m. for n = 7,5,4,3 cells for LRRC8A:C, LRRC8ABRIL:C, LRRC8A/B/C/D/E -/-, and LRRC8ABRIL(Fab/Nb):C, respectively. Differences assessed with one-way analysis of variance (ANOVA) with Dunnett correction for multiple comparisons to LRRC8A:C (ns, not significant, **P = .0017). LRRC8A:C and LRRC8 -/- data are reproduced from Fig. 4 for comparison.

Source data

Extended Data Fig. 2 Purification and reconstitution of LRRC8A:C channels.

(a) Schematic of purification strategy. (b) Superose 6 gel filtration chromatogram of LRRC8ABRIL:C purified in GDN and 150 mM KCl before (left) and after (middle)) addition of anti-BRIL Fab and anti-Fab Nb with pooled fractions indicated in blue and Coomassie stained SDS–PAGE of final sample (right). (c) Superose 6 gel filtration chromatogram of LRRC8ABRIL:C purified in GDN and 75 mM KCl before (left) and after (middle)) addition of anti-BRIL Fab and anti-Fab Nb with pooled fractions indicated in blue and Coomassie stained SDS–PAGE of final sample (right). (d) Superose 6 gel filtration chromatogram of LRRC8ABRIL:C purified in DDM, after reconstitution in MSP2N2 lipid nanodiscs, and after addition of anti-BRIL Fab and anti-Fab Nb with pooled fractions indicated in blue and Coomassie stained SDS–PAGE of final sample (right). (e) Coomassie stained SDS–PAGE of samples from purification in GDN (left) and DDM/CHS (right) prior to gel filtration chromatography. Note presence of LRRC8C band in anti-GFP nanobody flowthrough indicating presence of homomeric LRRC8C channels.

Source data

Extended Data Fig. 3 Cryo-EM data, processing pipeline, and validation for LRRC8ABRIL:C in GDN.

(a,b) Representative micrographs (of 6228 final micrographs from each condition) of LRRC8ABRIL:C in GDN and (a) 150 mM or (b) 75 mM KCl. (c) Cryo-EM data processing steps for LRRC8ABRIL:C conformations 1 and 2. Data from the two datasets were pooled as indicated. (d,e) Selected 2D class averages and (f,g) validation for conformations 1 (d,f) and 2 (e,g).

Extended Data Fig. 4 Focused refinements and validation for LRRC8ABRIL:C in GDN.

(a,b) Focused refinement strategy, validation, and composite maps for conformation 1 and 2. (c) Local resolution for top masked region of conformation 1 and 2.

Extended Data Fig. 5 Cryo-EM data and validation for LRRC8ABRIL:C in lipid nanodiscs.

(a) Representative micrograph (of 3593 final micrographs), (b) selected 2D class averages, (c) data processing pipeline, and (d) validation and focused refinement strategy of LRRC8ABRIL:C in lipid nanodiscs.

Extended Data Fig. 6 Cryo-EM maps and validation of subunit assignment.

(a) Views of cryo-EM maps from the extracellular side at low contour illustrating position of density for BRIL-Fab-Nb (gray) above five LRRC8A subunits (blue) and one LRRC8C subunit (yellow). (b) Selected 2D Classes with labeled BRIL-Fab-Nb densities. (c) Selected regions of cryo-EM density around amino acids that are different in LRRC8A and LRRC8C subunits.

Extended Data Fig. 7 Validation of EGFP subunit counting strategy.

(a) Distribution of HVCN1-EGFP photobleaching steps compared to previously published data44 and a predicted binomial distribution with 80% functional EGFP. (b) Distribution of LRRC8A-EGFP photobleaching steps and predicted binomial distributions for EGFP-tagged homohexamers with varying ratios of mature and fluorescent EGFP. (c) Single molecule subunit counting of LRRC8A:C channels at the 1:1 transfection ratio with comparison to predicted distributions assuming 50% mature EGFP fluorophores and either only 3:3 or all possible LRRC8A:C stoichiometries within a hexameric channel. Error bars are plotted as mean ± s.e.m. The data in parts (a-c) are reproduced from Fig. 2 for this analysis. Each movie contained ≥ 50 analyzed particles. For heteromeric LRRC8A:C channel samples, n = 12 movies total from two independent transfections and purifications with 6 movies each. For LRRC8A-EGFP, n = 15 movies total from 3 independent preparations. For HCVN1-EGFP n = 24 movies from 5 independent preparations.

Source data

Extended Data Fig. 8 Functional characterization of LRRC8A:C mutants.

(a) Representative voltage clamp recording from each construct tested in (left) isotonic and (center) hypotonic solution. Current-voltage relationship from each record is shown on the right. LRRC8A:C, LRRC8A(T48D):C, and LRRC8 -/- data are reproduced from Fig. 4 for comparison. (b) Current density at 60 mV in isotonic (green) and hypotonic (blue) solution for all records analyzed. Lines connect data from a single cell. (c) Reversal potentials calculated by linear interpolation of current-voltage relationship from all records analyzed. Values are mean ± s.e.m. for n = 7 (LRRC8A:C), 6 (LRRC8A(T48D):C), 5 (LRRC8A(V47K):C, 4 (LRRC8A/B/C/D/E -/-, LRRC8A(T48W)A:C, LRRC8A(T48Y):C, LRRC8A(T48N):C), and 3 cells (LRRC8A-GFP:C-mCherry, LRRC8A(V40D):C, LRRC8A(T44D):C, LRRC8A(V47D):C, LRRC8A(V47N):C, LRRC8A(T48K):C). Data are from two or more independent transfections for each construct. Differences assessed with one-way analysis of variance (ANOVA) with Dunnett correction for multiple comparisons to LRRC8A:C (ns, not significant, ****P ≤ .0001).

Source data

Extended Data Fig. 9 Cryo-EM data and pipeline for LRRC8ABRIL(T48D):C in GDN.

(a,b) Representative micrograph (of 5934 final micrographs) of LRRC8ABRIL:C in GDN. (c,d) Selected 2D class averages for conformations 1 and 2. (e) Initial processing pipeline and (f) Cryo-EM data processing steps for separating and refining conformations 1 and 2.

Source data

Extended Data Fig. 10 Focused refinements and validation for LRRC8ABRIL(T48D):C in GDN.

(a,b) Focused refinement strategy, validation, and composite maps for conformation 1 and 2. (c) Local resolution for top masked region of conformations 1 and 2.

Supplementary information

Source data

Source Data Fig. 2

Data for panel i.

Source Data Fig. 4

Data for panel c.

Source Data Extended Data Fig. 1

Data for panel e.

Source Data Extended Data Fig. 2

Uncropped gels for panels b–e.

Source Data Extended Data Fig. 7

Data for panels a–c.

Source Data Extended Data Fig. 8

Data for panels b,c.

Source Data Extended Data Fig. 9

Uncropped gel for panel a.

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Kern, D.M., Bleier, J., Mukherjee, S. et al. Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels. Nat Struct Mol Biol 30, 841–852 (2023). https://doi.org/10.1038/s41594-023-00944-6

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