Article | Published:

Structure of the receptor-activated human TRPC6 and TRPC3 ion channels

Cell Research (2018) | Download Citation

Abstract

TRPC6 and TRPC3 are receptor-activated nonselective cation channels that belong to the family of canonical transient receptor potential (TRPC) channels. They are activated by diacylglycerol, a lipid second messenger. TRPC6 and TRPC3 are involved in many physiological processes and implicated in human genetic diseases. Here we present the structure of human TRPC6 homotetramer in complex with a newly identified high-affinity inhibitor BTDM solved by single-particle cryo-electron microscopy to 3.8 Å resolution. We also present the structure of human TRPC3 at 4.4 Å resolution. These structures show two-layer architectures in which the bell-shaped cytosolic layer holds the transmembrane layer. Extensive inter-subunit interactions of cytosolic domains, including the N-terminal ankyrin repeats and the C-terminal coiled-coil, contribute to the tetramer assembly. The high-affinity inhibitor BTDM wedges between the S5-S6 pore domain and voltage sensor-like domain to inhibit channel opening. Our structures uncover the molecular architecture of TRPC channels and provide a structural basis for understanding the mechanism of these channels.

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Montell, C. & Rubin, G. M. Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2, 1313–1323 (1989).

  2. 2.

    Hardie, R. C. & Minke, B. The trp gene is essential for a light-activated Ca2+channel in Drosophila photoreceptors. Neuron 8, 643–651 (1992).

  3. 3.

    Li, H. TRP channel classification. Adv. Exp. Med. Biol. 976, 1–8 (2017).

  4. 4.

    Hofmann, T. et al. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259–263 (1999).

  5. 5.

    Okada, T. et al. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor. J. Biol. Chem. 274, 27359–27370 (1999).

  6. 6.

    Trebak, M., Vazquez, G., Bird, G. S. & Putney, J. W. Jr. The TRPC3/6/7 subfamily of cation channels. Cell Calcium 33, 451–461 (2003).

  7. 7.

    Hofmann, T., Schaefer, M., Schultz, G. & Gudermann, T. Subunit composition of mammalian transient receptor potential channels in living cells. Proc. Natl. Acad. Sci. USA 99, 7461–7466 (2002).

  8. 8.

    Zhou, J. et al. Critical role of TRPC6 channels in the formation of excitatory synapses. Nat. Neurosci. 11, 741–743 (2008).

  9. 9.

    Hartmann, J. et al. TRPC3 channels are required for synaptic transmission and motor coordination. Neuron 59, 392–398 (2008).

  10. 10.

    Reiser, J. et al. TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function. Nat. Genet. 37, 739–744 (2005).

  11. 11.

    Winn, M. P. et al. A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science 308, 1801–1804 (2005).

  12. 12.

    Davis, J., Burr, A. R., Davis, G. F., Birnbaumer, L. & Molkentin, J. D. A TRPC6-dependent pathway for myofibroblast transdifferentiation and wound healing in vivo. Dev. Cell 23, 705–715 (2012).

  13. 13.

    Yang, S. L., Cao, Q., Zhou, K. C., Feng, Y. J. & Wang, Y. Z. Transient receptor potential channel C3 contributes to the progression of human ovarian cancer. Oncogene 28, 1320–1328 (2009).

  14. 14.

    Tsvilovskyy, V. V. et al. Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo. Gastroenterology 137, 1415–1424 (2009).

  15. 15.

    Onohara, N. et al. TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy. EMBO J. 25, 5305–5316 (2006).

  16. 16.

    Heeringa, S. F. et al. A novel TRPC6 mutation that causes childhood FSGS. PLoS ONE 4, e7771 (2009).

  17. 17.

    Zhu, B. et al. Identification and functional analysis of a novel TRPC6 mutation associated with late onset familial focal segmental glomerulosclerosis in Chinese patients. Mutat. Res. 664, 84–90 (2009).

  18. 18.

    Gigante, M. et al. TRPC6 mutations in children with steroid-resistant nephrotic syndrome and atypical phenotype. Clin. J. Am. Soc. Nephrol. 6, 1626–1634 (2011).

  19. 19.

    D’Agati, V. D., Kaskel, F. J. & Falk, R. J. Focal segmental glomerulosclerosis. N. Engl. J. Med. 365, 2398–2411 (2011).

  20. 20.

    Ilatovskaya, D. V. & Staruschenko, A. TRPC6 channel as an emerging determinant of the podocyte injury susceptibility in kidney diseases. Am. J. Physiol. Ren. Physiol. 309, F393–397 (2015).

  21. 21.

    Beer, A., Mayer, G. & Kronbichler, A. Treatment strategies of adult primary focal segmental glomerulosclerosis: a systematic review focusing on the last two decades. Biomed. Res. Int. 2016, 4192578 (2016).

  22. 22.

    Dragovic, D. et al. Increasing incidence of focal segmental glomerulosclerosis and an examination of demographic patterns. Clin. Nephrol. 63, 1–7 (2005).

  23. 23.

    Fogo, A. B. Causes and pathogenesis of focal segmental glomerulosclerosis. Nat. Rev. Nephrol. 11, 76–87 (2015).

  24. 24.

    Kwon, Y., Hofmann, T. & Montell, C. Integration of phosphoinositide- and calmodulin-mediated regulation of TRPC6. Mol. Cell 25, 491–503 (2007).

  25. 25.

    Boulay, G. Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells. Cell Calcium 32, 201–207 (2002).

  26. 26.

    Zhang, Z. Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain. Proc. Natl. Acad. Sci. USA 98, 3168–3173 (2001).

  27. 27.

    Lepage, P. K. et al. Identification of two domains involved in the assembly of transient receptor potential canonical channels. J. Biol. Chem. 281, 30356–30364 (2006).

  28. 28.

    Paulsen, C. E., Armache, J. P., Gao, Y., Cheng, Y. & Julius, D. Structure of the TRPA1 ion channel suggests regulatory mechanisms. Nature 525, 552 (2015).

  29. 29.

    Guo, J. et al. Structures of the calcium-activated, non-selective cation channel TRPM4. Nature https://doi.org/10.1038/nature24997 (2017).

  30. 30.

    Winkler, P. A., Huang, Y., Sun, W., Du, J. & Lu, W. Electron cryo-microscopy structure of a human TRPM4 channel. Nature https://doi.org/10.1038/nature24674 (2017).

  31. 31.

    Autzen, H. E. et al. Structure of the human TRPM4 ion channel in a lipid nanodisc. Science https://doi.org/10.1126/science.aar4510 (2017).

  32. 32.

    Yin, Y. et al. Structure of the cold- and menthol-sensing ion channel TRPM8. Science https://doi.org/10.1126/science.aan4325 (2017).

  33. 33.

    Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013).

  34. 34.

    Huynh, K. W. et al. Structure of the full-length TRPV2 channel by cryo-EM. Nat. Commun. 7, 11130 (2016).

  35. 35.

    Zubcevic, L. et al. Cryo-electron microscopy structure of the TRPV2 ion channel. Nat. Struct. Mol. Biol. 23, 180–186 (2016).

  36. 36.

    Shen, P. S. et al. The structure of the polycystic kidney disease channel PKD2 in lipid nanodiscs. Cell 167, 763–773 e711 (2016).

  37. 37.

    Jin, P. et al. Electron cryo-microscopy structure of the mechanotransduction channel NOMPC. Nature 547, 118–122 (2017).

  38. 38.

    Chen, Q. et al. Structure of mammalian endolysosomal TRPML1 channel in nanodiscs. Nature 550, 415–418 (2017).

  39. 39.

    Hirschi, M. et al. Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3. Nature 550, 411–414 (2017).

  40. 40.

    Schmiege, P., Fine, M., Blobel, G. & Li, X. Human TRPML1 channel structures in open and closed conformations. Nature 550, 366–370 (2017).

  41. 41.

    Lichtenegger, M. et al. A novel homology model of TRPC3 reveals allosteric coupling between gate and selectivity filter. Cell Calcium 54, 175–185 (2013).

  42. 42.

    Zeng, W. et al. STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction. Mol. Cell. 32, 439–448 (2008).

  43. 43.

    Autzen, H. E. et al. Structure of the human TRPM4 ion channel in a lipid nanodisc. Science 359, 228–232 (2018).

  44. 44.

    Cao, E., Liao, M., Cheng, Y. & Julius, D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013).

  45. 45.

    Voolstra, O. & Huber, A. Post-translational modifications of TRP channels. Cells 3, 258–287 (2014).

  46. 46.

    Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

  47. 47.

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

  48. 48.

    Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2010).

  49. 49.

    Lynch, C. J. et al. Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels. Am. J. Physiol. Endocrinol. Metab. 302, E540–551 (2012).

  50. 50.

    Efremov, R. G., Leitner, A., Aebersold, R. & Raunser, S. Architecture and conformational switch mechanism of the ryanodine receptor. Nature 517, 39–43 (2015).

  51. 51.

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

  52. 52.

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

  53. 53.

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

  54. 54.

    Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).

  55. 55.

    Zhou, N., Wang, H. & Wang, J. EMBuilder: a template matching-based automatic model-building program for high-resolution cryo-electron microscopy maps. Sci. Rep. 7, 2664 (2017).

  56. 56.

    Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).

  57. 57.

    DiMaio, F. et al. Atomic-accuracy models from 4.5-A cryo-electron microscopy data with density-guided iterative local refinement. Nat. Methods 12, 361–365 (2015).

  58. 58.

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

  59. 59.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  60. 60.

    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, 354–360 (1996). 376.

  61. 61.

    Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–258 (2014).

  62. 62.

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

Download references

Acknowledgements

We thank all of Chen Laboratory members for their kind help. Cryo-EM data collection was supported by the National Center for Protein Science (Shanghai) with assistance of Liangliang Kong and Zhenglin Fu, Electron Microscopy Laboratory and Cryo-EM platform of Peking University with assistance of Xuemei Li and Daqi Yu, and Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Science with assistance of Zhenxi Guo. Part of structural computation was also performed on the Computing Platform of the Center for Life Science and High-performance Computing Platform of Peking University. The work is supported by grants from the Ministry of Science and Technology of China (National Key R&D Program of China, 2016YFA0502004 to L.C.), National Natural Science Foundation of China (31622021 and 31521062 to L.C.), Young Thousand Talents Program of China to L.C. and the China Postdoctoral Science Foundation (2016M600856 and 2017T100014 to J.-X.W.). J.-X.W. is supported by the postdoctoral foundation of the Peking-Tsinghua Center for Life Sciences, Peking University.

Author information

Author notes

  1. These authors contributed equally: Qinglin Tang, Wenjun Guo.

Affiliations

  1. State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, 100871, Beijing, China

    • Qinglin Tang
    • , Wenjun Guo
    • , Jing-Xiang Wu
    •  & Lei Chen
  2. Dizal Pharmaceutical Company, Jiangsu, China

    • Li Zheng
    • , Meng Liu
    • , Xindi Zhou
    •  & Xiaolin Zhang
  3. Peking-Tsinghua Center for Life Sciences, Peking University, 100871, Beijing, China

    • Jing-Xiang Wu
    •  & Lei Chen

Authors

  1. Search for Qinglin Tang in:

  2. Search for Wenjun Guo in:

  3. Search for Li Zheng in:

  4. Search for Jing-Xiang Wu in:

  5. Search for Meng Liu in:

  6. Search for Xindi Zhou in:

  7. Search for Xiaolin Zhang in:

  8. Search for Lei Chen in:

Contributions

X. Zhang and L.C. initiated the project. Q.T., W.G., L.Z., M.L., X. Zhou, X. Zhang, and L.C. designed the experiments. Q.T., W.G., J.-X.W., and L.C. prepared the EM sample, collected the EM data, performed image processing, and built the model. Q.T. and W.G. generated mutants. L.Z., M.L., X. Zhou, and X. Zhang provided BTDM and performed electrophysiology recording and FLIPR assay. Q.T., W.G., J.-X.W., and L.C. wrote the manuscript draft. All authors contributed to manuscript preparation.

Competing interests

L.Z., M.L., X. Zhou and X. Zhang are employees of Dizal Pharmaceutical Company. The other authors declare no competing interests.

Corresponding authors

Correspondence to Xiaolin Zhang or Lei Chen.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41422-018-0038-2

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.