Article | Published:

Crystal structure of the epithelial calcium channel TRPV6

Nature volume 534, pages 506511 (23 June 2016) | Download Citation

Abstract

Precise regulation of calcium homeostasis is essential for many physiological functions. The Ca2+-selective transient receptor potential (TRP) channels TRPV5 and TRPV6 play vital roles in calcium homeostasis as Ca2+ uptake channels in epithelial tissues. Detailed structural bases for their assembly and Ca2+ permeation remain obscure. Here we report the crystal structure of rat TRPV6 at 3.25 Å resolution. The overall architecture of TRPV6 reveals shared and unique features compared with other TRP channels. Intracellular domains engage in extensive interactions to form an intracellular ‘skirt’ involved in allosteric modulation. In the K+ channel-like transmembrane domain, Ca2+ selectivity is determined by direct coordination of Ca2+ by a ring of aspartate side chains in the selectivity filter. On the basis of crystallographically identified cation-binding sites at the pore axis and extracellular vestibule, we propose a Ca2+ permeation mechanism. Our results provide a structural foundation for understanding the regulation of epithelial Ca2+ uptake and its role in pathophysiology.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Data deposits

The structure coordinates have been deposited in Protein Data Bank (PDB) with accession numbers 5IWK, 5IWP, 5IWR and 5IWT for native, Ca2+, Ba2+ and Gd3+ data, respectively.

References

  1. 1.

    TRP channels as cellular sensors. Nature 426, 517–524 (2003)

  2. 2.

    , , & Permeation and selectivity of TRP channels. Annu. Rev. Physiol. 68, 685–717 (2006)

  3. 3.

    , , , & Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6. Nature Commun. 6, 8871 (2015)

  4. 4.

    , , & The epithelial calcium channels, TRPV5 & TRPV6: from identification towards regulation. Cell Calcium 33, 497–507 (2003)

  5. 5.

    et al. Functional TRPV6 channels are crucial for transepithelial Ca2+ absorption. Am. J. Physiol. Gastrointest. Liver Physiol. 303, G879–G885 (2012)

  6. 6.

    et al. Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene. J. Bone Miner. Res. 22, 274–285 (2007)

  7. 7.

    et al. Male fertility depends on Ca2+ absorption by TRPV6 in epididymal epithelia. Sci. Signal. 4, ra27 (2011)

  8. 8.

    , & in Mammalian Transient Receptor Potential (TRP) Cation Channels (eds & ) Ch. TRPV6 Channels, 359–384 (Springer, 2014)

  9. 9.

    , & The role of the TRPV6 channel in cancer. J. Physiol. (Lond.) 590, 1369–1376 (2012)

  10. 10.

    et al. TRPV6 calcium channel translocates to the plasma membrane via Orai1-mediated mechanism and controls cancer cell survival. Proc. Natl Acad. Sci. USA 111, E3870–E3879 (2014)

  11. 11.

    et al. In vivo detection of human TRPV6-rich tumors with anti-cancer peptides derived from soricidin. PLoS ONE 8, e58866 (2013)

  12. 12.

    , , & Tamoxifen inhibits TRPV6 activity via estrogen receptor-independent pathways in TRPV6-expressing MCF-7 breast cancer cells. Mol. Cancer Res. 7, 2000–2010 (2009)

  13. 13.

    et al. Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6. EMBO J. 22, 776–785 (2003)

  14. 14.

    , , & Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013)

  15. 15.

    , , & TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013)

  16. 16.

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

  17. 17.

    , , , & Structure of the TRPA1 ion channel suggests regulatory mechanisms. Nature 520, 511–517 (2015)

  18. 18.

    , , & Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450, 376–382 (2007)

  19. 19.

    , , & The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011)

  20. 20.

    et al. Cryo-electron microscopy structure of the Slo2.2 Na+-activated K+ channel. Nature 527, 198–203 (2015)

  21. 21.

    , , , & The β-glucuronidase klotho exclusively activates the epithelial Ca2+ channels TRPV5 and TRPV6. Nephrol. Dial. Transplant. 23, 3397–3402 (2008)

  22. 22.

    et al. Molecular determinants in TRPV5 channel assembly. J. Biol. Chem. 279, 54304–54311 (2004)

  23. 23.

    et al. Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking. J. Biol. Chem. 286, 32132–32139 (2011)

  24. 24.

    , , & Interplay between calmodulin and phosphatidylinositol 4,5-bisphosphate in Ca2+-induced inactivation of transient receptor potential vanilloid 6 channels. J. Biol. Chem. 288, 5278–5290 (2013)

  25. 25.

    , , , & Regulation of the mouse epithelial Ca2+ channel TRPV6 by the Ca2+-sensor calmodulin. J. Biol. Chem. 279, 28855–28861 (2004)

  26. 26.

    et al. Molecular mechanisms of calmodulin action on TRPV5 and modulation by parathyroid hormone. Mol. Cell. Biol. 31, 2845–2853 (2011)

  27. 27.

    et al. Heavy metal cations permeate the TRPV6 epithelial cation channel. Cell Calcium 49, 43–55 (2011)

  28. 28.

    et al. Structural basis for Ca2+ selectivity of a voltage-gated calcium channel. Nature 505, 56–61 (2014)

  29. 29.

    et al. Structure of the voltage-gated calcium channel Cav1.1 complex. Science 350, 1492–1501 (2015)

  30. 30.

    , , & Structural analysis, identification, and design of calcium-binding sites in proteins. Proteins 47, 344–356 (2002)

  31. 31.

    , , & Crystal structure of the calcium release-activated calcium channel Orai. Science 338, 1308–1313 (2012)

  32. 32.

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

  33. 33.

    , & FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996)

  34. 34.

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

  35. 35.

    , , , & Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells. Protein Sci. 15, 2008–2013 (2006)

  36. 36.

    Acta Crystallogr. D 66, 125–132 (2010)

  37. 37.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  38. 38.

    Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D 63, 32–41 (2007)

  39. 39.

    , , , & Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels. Biochemistry 47, 2476–2484 (2008)

  40. 40.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  41. 41.

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

  42. 42.

    et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

  43. 43.

    The PyMol Molecular Graphics System (DeLano Scientific, 2002)

  44. 44.

    et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol. 8, e1002708 (2012)

  45. 45.

    , , , & HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360, 376 (1996)

  46. 46.

    et al. Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2+-dependent inactivation. J. Physiol. (Lond.) 577, 31–44 (2006)

  47. 47.

    , , , & The ankyrin repeats of TRPV1 bind multiple ligands and modulate channel sensitivity. Neuron 54, 905–918 (2007)

  48. 48.

    , , & Differential regulation of TRPV1, TRPV3, and TRPV4 sensitivity through a conserved binding site on the ankyrin repeat domain. J. Biol. Chem. 285, 731–740 (2010)

Download references

Acknowledgements

We thank E. C. Twomey and J. M. Sampson for comments on the manuscript, members of the E. C. Greene laboratory for assistance with their fluorimeter, and the Columbia Protein Core facility for assistance with isothermal titration calorimetry (ITC) measurements. We also thank the personnel at beamlines 24-ID-C/E of APS, X25/X29 of NSLS and 5.0.1/5.0.2 of ALS. This work was supported by National Institutes of Health grants R01 NS083660 (A.I.S.) and T32 GM008281 (K.S.), by a Pew Scholar Award in Biomedical Sciences, a Schaefer Research Scholar Award, a Klingenstein Fellowship Award in the Neurosciences and an Irma T. Hirschl Career Scientist Award (A.I.S.).

Author information

Author notes

    • Kei Saotome
    •  & Appu K. Singh

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, New York 10032, USA

    • Kei Saotome
    • , Appu K. Singh
    • , Maria V. Yelshanskaya
    •  & Alexander I. Sobolevsky

Authors

  1. Search for Kei Saotome in:

  2. Search for Appu K. Singh in:

  3. Search for Maria V. Yelshanskaya in:

  4. Search for Alexander I. Sobolevsky in:

Contributions

K.S., A.K.S., M.V.Y. and A.I.S. designed the project. K.S., A.K.S. and M.V.Y. performed the experiments. K.S., A.K.S., M.V.Y. and A.I.S. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alexander I. Sobolevsky.

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature17975

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.