Advertisement

Expression, Purification, and Crystallization of the Transient Receptor Potential Channel TRPV6

  • Appu K. Singh
  • Luke L. McGoldrick
  • Alexander I. SobolevskyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1987)

Abstract

Transient receptor potential (TRP) channels are polymodal sensory transducers that respond to chemicals, temperature, mechanical stress, and membrane voltage and are involved in vision, taste, olfaction, hearing, touch, thermal perception, and nociception. TRP channels are implicated in numerous devastating diseases, including various forms of cancer, and represent important drug targets. The large sizes, low expression levels, and conformational dynamics of TRP channels make them challenging targets for structural biology. Here, we present the methodology used in structural studies of TRPV6, a TRP channel that is highly selective for calcium and mediates Ca2+ uptake in epithelial tissues. We provide a protocol for the expression, purification, and crystallization of TRPV6. Similar approaches can be used to determine crystal structures of other membrane proteins, including different members of the TRP channel family.

Key words

Transient receptor potential channel TRP channel Membrane protein Ion channel Protein structure X-ray crystallography Protein crystallization Protein purification Protein expression HEK 293 cells Sf9 cells Baculovirus 

References

  1. 1.
    Clapham DE (2003) TRP channels as cellular sensors. Nature 426(6966):517–524CrossRefPubMedGoogle Scholar
  2. 2.
    Montell C, Birnbaumer L, Flockerzi V (2002) The TRP channels, a remarkably functional family. Cell 108(5):595–598CrossRefPubMedGoogle Scholar
  3. 3.
    Schlingmann KP et al (2007) TRPM6 and TRPM7—Gatekeepers of human magnesium metabolism. Biochim Biophys Acta 1772(8):813–821CrossRefPubMedGoogle Scholar
  4. 4.
    Nilius B et al (2007) Transient receptor potential cation channels in disease. Physiol Rev 87(1):165–217CrossRefPubMedGoogle Scholar
  5. 5.
    Lehen’kyi V, Raphael M, Prevarskaya N (2012) The role of the TRPV6 channel in cancer. J Physiol 590(6):1369–1376CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wissenbach U et al (2004) TRPV6 and prostate cancer: cancer growth beyond the prostate correlates with increased TRPV6 Ca2+ channel expression. Biochem Biophys Res Commun 322(4):1359–1363CrossRefPubMedGoogle Scholar
  7. 7.
    Wissenbach U et al (2001) Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer. J Biol Chem 276(22):19461–19468CrossRefPubMedGoogle Scholar
  8. 8.
    Zhang SS et al (2016) TRPV6 plays a new role in predicting survival of patients with esophageal squamous cell carcinoma. Diagn Pathol 11(1):14CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    McCleverty CJ et al (2006) Crystal structure of the human TRPV2 channel ankyrin repeat domain. Protein Sci 15(9):2201–2206CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Yamaguchi H et al (2001) Crystal structure of the atypical protein kinase domain of a TRP channel with phosphotransferase activity. Mol Cell 7(5):1047–1057CrossRefPubMedGoogle Scholar
  11. 11.
    Jin X, Touhey J, Gaudet R (2006) Structure of the N-terminal ankyrin repeat domain of the TRPV2 ion channel. J Biol Chem 281(35):25006–25010CrossRefPubMedGoogle Scholar
  12. 12.
    Lishko PV et al (2007) The ankyrin repeats of TRPV1 bind multiple ligands and modulate channel sensitivity. Neuron 54(6):905–918CrossRefPubMedGoogle Scholar
  13. 13.
    Phelps CB et al (2008) Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels. Biochemistry 47(8):2476–2484CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Fujiwara Y, Minor DL Jr (2008) X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil. J Mol Biol 383(4):854–870CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Yu Y et al (2009) Structural and molecular basis of the assembly of the TRPP2/PKD1 complex. Proc Natl Acad Sci U S A 106(28):11558–11563CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Schumann F et al (2009) Ca2+−dependent conformational changes in a C-terminal cytosolic domain of polycystin-2. J Biol Chem 284(36):24372–24383CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Petri ET et al (2010) Structure of the EF-hand domain of polycystin-2 suggests a mechanism for Ca2+−dependent regulation of polycystin-2 channel activity. Proc Natl Acad Sci U S A 107(20):9176–9181CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Li M, Yu Y, Yang J (2011) Structural biology of TRP channels. Adv Exp Med Biol 704:1–23CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Moiseenkova-Bell VY, Wensel TG (2009) Hot on the trail of TRP channel structure. J Gen Physiol 133(3):239–244CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Cheng YF (2015) Single-particle cryo-EM at crystallographic resolution. Cell 161(3):450–457CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hendrickson WA (2016) Atomic-level analysis of membrane-protein structure. Nat Struct Mol Biol 23(6):464–467CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Grieben M et al (2017) Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2). Nat Struct Mol Biol 24(2):114–122CrossRefPubMedGoogle Scholar
  23. 23.
    Huynh KW et al (2016) Structure of the full-length TRPV2 channel by cryo-EM. Nat Commun 7:11130CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Jin P et al (2017) Electron cryo-microscopy structure of the mechanotransduction channel NOMPC. Nature 547(7661):118–122CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Shen PS et al (2016) The structure of the polycystic kidney disease channel PKD2 in lipid nanodiscs. Cell 167(3):763–773CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Zubcevic L et al (2016) Cryo-electron microscopy structure of the TRPV2 ion channel. Nat Struct Mol Biol 23(2):180–186CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Gao Y et al (2016) TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534(7607):347–351CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Liao M et al (2013) Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504(7478):107–112CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Hirschi M et al (2017) Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3. Nature 550(7676):411–414CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Zhou X et al (2017) Cryo-EM structures of the human endolysosomal TRPML3 channel in three distinct states. Nat Struct Mol Biol 24:1146–1154.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Li M et al (2017) Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel. Nat Struct Mol Biol 24(3):205–213CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Chen Q et al (2017) Structure of mammalian endolysosomal TRPML1 channel in nanodiscs. Nature 550(7676):415–418CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Schmiege P et al (2017) Human TRPML1 channel structures in open and closed conformations. Nature 550(7676):366–370CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Zhou YF et al (2001) Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 angstrom resolution. Nature 414(6859):43–48CrossRefPubMedGoogle Scholar
  35. 35.
    Hou X et al (2012) Crystal structure of the calcium release-activated calcium channel Orai. Science 338(6112):1308–1313CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Tang L et al (2014) Structural basis for Ca2+ selectivity of a voltage-gated calcium channel. Nature 505(7481):56–61CrossRefPubMedGoogle Scholar
  37. 37.
    Liu Q, Liu Q, Hendrickson WA (2013) Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data. Acta Crystallogr D Biol Crystallogr 69(Pt 7):1314–1332CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Liu Q, Hendrickson WA (2015) Crystallographic phasing from weak anomalous signals. Curr Opin Struct Biol 34:99–107CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Saotome K et al (2016) Crystal structure of the epithelial calcium channel TRPV6. Nature 534(7608):506–511CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Singh AK, Saotome K, Sobolevsky AI (2017) Swapping of transmembrane domains in the epithelial calcium channel TRPV6. Sci Rep 7(1):10669CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Peng JB et al (1999) Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 274(32):22739–22746CrossRefPubMedGoogle Scholar
  42. 42.
    Goehring A et al (2014) Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat Protoc 9(11):2574–2585CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14(4):673–681CrossRefPubMedGoogle Scholar
  44. 44.
    Cherezov V et al (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318(5854):1258–1265CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Nasr ML et al (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14(1):49–52CrossRefPubMedGoogle Scholar
  46. 46.
    Bayburt TH, Sligar SG (2010) Membrane protein assembly into nanodiscs. FEBS Lett 584(9):1721–1727CrossRefPubMedGoogle Scholar
  47. 47.
    Nikolaev M et al (2017) Integral membrane proteins can be crystallized directly from nanodiscs. Cryst Growth Des 17(3):945–948CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Appu K. Singh
    • 1
  • Luke L. McGoldrick
    • 1
    • 2
  • Alexander I. Sobolevsky
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA
  2. 2.Integrated Program in Cellular, Molecular and Biomedical StudiesColumbia UniversityNew YorkUSA

Personalised recommendations