Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Nature Structural & Molecular Biology (2023)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

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.

References

  1. Nilius, B. et al. Properties of volume-regulated anion channels in mammalian cells. Prog. Biophys. Mol. Biol. 68, 69–119 (1997).

    Article  CAS  PubMed  Google Scholar 

  2. Hoffmann, E. K., Lambert, I. H. & Pedersen, S. F. Physiology of cell volume regulation in vertebrates. Physiol. Rev. 89, 193–277 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Voss, F. K. et al. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science 344, 634–638 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Qiu, Z. et al. SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel. Cell 157, 447–458 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jentsch, T. J. VRACs and other ion channels and transporters in the regulation of cell volume and beyond. Nat. Rev. Mol. Cell Biol. 17, 293–307 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Strange, K., Yamada, T. & Denton, J. S. A 30-year journey from volume-regulated anion currents to molecular structure of the LRRC8 channel. J. Gen. Physiol. 151, 100–117 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hyzinski-García, M. C., Rudkouskaya, A. & Mongin, A. A. LRRC8A protein is indispensable for swelling-activated and ATP-induced release of excitatory amino acids in rat astrocytes. J. Physiol. 592, 4855–4862 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Lutter, D., Ullrich, F., Lueck, J. C., Kempa, S. & Jentsch, T. J. Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels. J. Cell Sci. 130, 1122–1133 (2017).

    CAS  PubMed  Google Scholar 

  9. Yang, J. et al. Glutamate-releasing SWELL1 channel in astrocytes modulates synaptic transmission and promotes brain damage in stroke. Neuron 102, 813–827 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lahey, L. J. et al. LRRC8A:C/E heteromeric channels are ubiquitous transporters of cGAMP. Mol. Cell 80, 578–591 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Zhou, C. et al. Transfer of cGAMP into bystander cells via LRRC8 volume-regulated anion channels augments STING-mediated interferon responses and anti-viral. Immunity 52, 767–781 (2020).

    Article  CAS  PubMed  Google Scholar 

  12. Chen, X. et al. Regulation of anion channel LRRC8 volume-regulated anion channels in transport of 2′3′-cyclic GMP-AMP and cisplatin under steady state and inflammation. J. Immunol. 206, 2061–2074 (2021).

    Article  CAS  PubMed  Google Scholar 

  13. Zhang, Y. et al. SWELL1 is a regulator of adipocyte size, insulin signalling and glucose homeostasis. Nat. Cell Biol. 19, 504–517 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kang, C. et al. SWELL1 is a glucose sensor regulating β-cell excitability and systemic glycaemia. Nat. Commun. 9, 367 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Stuhlmann, T., Planells-Cases, R. & Jentsch, T. J. LRRC8/VRAC anion channels enhance β-cell glucose sensing and insulin secretion. Nat. Commun. 9, 1974 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Menegaz, D. et al. Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell. Nat. Metab. 1, 1110–1126 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gunasekar, S. K. et al. Small molecule SWELL1 complex induction improves glycemic control and nonalcoholic fatty liver disease in murine Type 2 diabetes. Nat. Commun. 13, 784 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lück, J. C., Puchkov, D., Ullrich, F. & Jentsch, T. J. LRRC8/VRAC anion channels are required for late stages of spermatid development in mice. J. Biol. Chem. 293, 11796–11808 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Chen, L., Becker, T. M., Koch, U. & Stauber, T. The LRRC8/VRAC anion channel facilitates myogenic differentiation of murine myoblasts by promoting membrane hyperpolarization. J. Biol. Chem. 294, 14279–14288 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kumar, A. et al. SWELL1 regulates skeletal muscle cell size, intracellular signaling, adiposity and glucose metabolism. eLife 9, e58941 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Alghanem, A. F. et al. The SWELL1-LRRC8 complex regulates endothelial AKT-eNOS signaling and vascular function. eLife 10, e61313 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Pervaiz, S., Kopp, A., Kleist, von, L. & Stauber, T. Absolute protein amounts and relative abundance of Volume-Regulated Anion Channel (VRAC) LRRC8 subunits in cells and tissues revealed by quantitative immunoblotting. Int. J. Mol. Sci. 20, 5879 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lee, C. C., Freinkman, E., Sabatini, D. M. & Ploegh, H. L. The protein synthesis inhibitor blasticidin S enters mammalian cells via leucine-rich repeat-containing protein 8D. J. Biol. Chem. 289, 17124–17131 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Planells-Cases, R. et al. Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. EMBO J. 34, 2993–3008 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Syeda, R. et al. LRRC8 proteins form volume-regulated anion channels that sense ionic strength. Cell 164, 499–511 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Deneka, D., Sawicka, M., Lam, A. K. M., Paulino, C. & Dutzler, R. Structure of a volume-regulated anion channel of the LRRC8 family. Nature 558, 254–259 (2018).

    Article  CAS  PubMed  Google Scholar 

  27. Kefauver, J. M. et al. Structure of the human volume regulated anion channel. eLife 7, e38461 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Kasuya, G. et al. Cryo-EM structures of the human volume-regulated anion channel LRRC8. Nat. Struct. Mol. Biol. 25, 797–804 (2018).

    Article  CAS  PubMed  Google Scholar 

  29. Kern, D. M., Oh, S., Hite, R. K. & Brohawn, S. G. Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs. eLife 8, e42636 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Nakamura, R. et al. Cryo-EM structure of the volume-regulated anion channel LRRC8D isoform identifies features important for substrate permeation. Commun. Biol. 3, 240 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Deneka, D. et al. Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains. Nat. Commun. 12, 5435 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gaitán-Peñas, H. et al. Investigation of LRRC8-mediated volume-regulated anion currents in Xenopus oocytes. Biophys. J. 111, 1429–1443 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Gaitán-Peñas, H., Pusch, M. & Estévez, R. Expression of LRRC8/VRAC currents in Xenopus oocytes: advantages and caveats. Int. J. Mol. Sci. 19, 719 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  34. König, B., Hao, Y., Schwartz, S., Plested, A. J. & Stauber, T. A FRET sensor of C-terminal movement reveals VRAC activation by plasma membrane DAG signaling rather than ionic strength. eLife 8, e45421 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Zhou, P., Polovitskaya, M. M. & Jentsch, T. J. LRRC8 N termini influence pore properties and gating of Volume-Regulated Anion Channels (VRACs). J. Biol. Chem. 293, 13440–13451 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yamada, T. & Strange, K. Intracellular and extracellular loops of LRRC8 are essential for volume-regulated anion channel function. J. Gen. Physiol. 150, 1003–1015 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yamada, T., Figueroa, E. E., Denton, J. S. & Strange, K. LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology. Am. J. Physiol. Cell Physiol. 320, C293–C303 (2021).

    Article  CAS  PubMed  Google Scholar 

  38. Chun, E. et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20, 967–976 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Tsutsumi, N. et al. Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling. eLife 9, e58464 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mukherjee, S. et al. Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins. Nat. Commun. 11, 1598 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ullrich, F., Reincke, S. M., Voss, F. K., Stauber, T. & Jentsch, T. J. Inactivation and anion selectivity of Volume-Regulated Anion Channels (VRACs) depend on C-terminal residues of the first extracellular loop. J. Biol. Chem. 291, 17040–17048 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ereño-Orbea, J. et al. Structural basis of enhanced crystallizability induced by a molecular chaperone for antibody antigen-binding fragments. J. Mol. Biol. 430, 322–336 (2018).

    Article  PubMed  Google Scholar 

  43. Hermans, W.J.J., ten Haaft, M.R. & Overweel, A. Method for affinity purification. US patent US20170369527A1 (2017); https://patents.google.com/patent/US20170369527A1/en

  44. Tombola, F., Ulbrich, M. H. & Isacoff, E. Y. The voltage-gated proton channel Hv1 has two pores, each controlled by one voltage sensor. Neuron 58, 546–556 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Brohawn, S. G., Campbell, E. B. & MacKinnon, R. Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel. Nature 516, 126–130 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kefauver, J. M., Ward, A. B. & Patapoutian, A. Discoveries in structure and physiology of mechanically activated ion channels. Nature 587, 567–576 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Romanenko, V. G., Davies, P. F. & Levitan, I. Dual effect of fluid shear stress on volume-regulated anion current in bovine aortic endothelial cells. Am. J. Physiol. Cell Physiol. 282, C708–C718 (2002).

    Article  CAS  PubMed  Google Scholar 

  48. Browe, D. M. & Baumgarten, C. M. Stretch of β1 integrin activates an outwardly rectifying chloride current via FAK and Src in rabbit ventricular myocytes. J. Gen. Physiol. 122, 689–702 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Best, L. & Brown, P. D. Studies of the mechanism of activation of the volume-regulated anion channel in rat pancreatic β-cells. J. Membr. Biol. 230, 83–91 (2009).

    Article  CAS  PubMed  Google Scholar 

  50. Abascal, F. & Zardoya, R. LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication. Bioessays 34, 551–560 (2012).

    Article  CAS  PubMed  Google Scholar 

  51. Syrjanen, J., Michalski, K., Kawate, T. & Furukawa, H. On the molecular nature of large-pore channels. J. Mol. Biol. 433, 166994 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Drożdżyk, K. et al. Cryo-EM structures and functional properties of CALHM channels of the human placenta. eLife 9, e55853 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Burendei, B. et al. Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids. Sci. Adv. 6, eaax3157 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Syrjanen, J. L. et al. Structure and assembly of calcium homeostasis modulator proteins. Nat. Struct. Mol. Biol. 27, 150–159 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kuzuya, M. et al. Structures of human pannexin-1 in nanodiscs reveal gating mediated by dynamic movement of the N terminus and phospholipids. Sci. Signal. 15, eabg6941 (2022).

    Article  CAS  PubMed  Google Scholar 

  56. Takahashi, H., Yamada, T., Denton, J. S., Strange, K. & Karakas, E. Structure of a LRRC8 chimera with physiologically relevant properties reveals heptameric assembly and pore-blocking lipids. Preprint at https://www.biorxiv.org/content/10.1101/2022.07.28.501913v2.full (2022).

  57. Rutz, S., Deneka, D., Dittmann, A., Sawicka, M. & Dutzler, R. Structure of a volume-regulated heteromeric LRRC8A/C channel. Nat Struct Mol Biol 30, 52–61 (2023).

    Article  CAS  PubMed  Google Scholar 

  58. Chu, R. et al. Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography. J. Mol. Biol. 323, 253–262 (2002).

    Article  CAS  PubMed  Google Scholar 

  59. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

    Article  CAS  PubMed  Google Scholar 

  60. Ritchie, T. K. et al. Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  62. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  66. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  PubMed  Google Scholar 

  67. Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Asarnow, D., Palovcak, E. & Cheng, Y. asarnow/pyem: UCSF pyem v0.5 (Zenodo, 2019); https://doi.org/10.5281/zenodo.3576630

  70. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. IUCr. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

  73. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  74. Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354-60–354-76 (1996).

    Article  PubMed  Google Scholar 

Download references

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

Authors
  1. David M. Kern
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julia Bleier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Somnath Mukherjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jennifer M. Hill
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anthony A. Kossiakoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ehud Y. Isacoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stephen G. Brohawn
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

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.

Additional information

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

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.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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 (2023). https://doi.org/10.1038/s41594-023-00944-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41594-023-00944-6

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing