Skip to main content

Advertisement

SpringerLink
Log in

N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

  • Original Article
  • Published:
Purinergic Signalling Aims and scope Submit manuscript

Abstract

The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a functionally active fusion protein.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Morandini AC, Savio LEB, Coutinho-Silva R (2014) The role of P2X7 receptor in infectious inflammatory diseases and the influence of ectonucleotidases. Biom J 37(4):169–177. https://doi.org/10.4103/2319-4170.127803

    Google Scholar 

  2. BJ G, Zhang WY, Bendall LJ et al (2000) Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors. Am J Physiol Cell Physiol 279:C1189–C1197

    Article  Google Scholar 

  3. Sperlagh B, Vizi ES, Wirkner K, Illes P (2006) P2X7 receptors in the nervous system. Prog Neurobiol 78(6):327–346. https://doi.org/10.1016/j.pneurobio.2006.03.007

    Article  CAS  PubMed  Google Scholar 

  4. Wang J, Liu S, Nie Y, Wu B, Wu Q, Song M, Tang M, Xiao L, Xu P, Tan X, Zhang L, Li G, Liang S, Zhang C (2015) Activation of P2X7 receptors decreases the proliferation of murine luteal cells. Reprod Fertil Dev 27(8):1262. https://doi.org/10.1071/RD14381

    Article  CAS  PubMed  Google Scholar 

  5. Jiang W, Lv H, Wang H, Wang D, Sun S, Jia Q, Wang P, Song B, Ni L (2015) Activation of the NLRP3/caspase-1 inflammasome in human dental pulp tissue and human dental pulp fibroblasts. Cell Tissue Res 361(2):541–555. https://doi.org/10.1007/s00441-015-2118-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jorgensen NR, Syberg S, Ellegaard M (2015) The role of P2X receptors in bone biology. Curr Med Chem 22(7):902–914. https://doi.org/10.2174/0929867321666141215094749

    Article  CAS  PubMed  Google Scholar 

  7. Volonte C, Apolloni S, Skaper SD, Burnstock G (2012) P2X7 receptors: channels, pores and more. CNS Neurol Disord Drug Targets 11(6):705–721. https://doi.org/10.2174/187152712803581137

    Article  CAS  PubMed  Google Scholar 

  8. Amstrup J, Novak I (2003) P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx. Biochem J 374(1):51–61. https://doi.org/10.1042/BJ20030585

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yan Z, Li S, Liang Z, Tomić M, Stojilkovic SS (2008) The P2X7 receptor channel pore dilates under physiological ion conditions. J Gen Physiol 132(5):563–573. https://doi.org/10.1085/jgp.200810059

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Smart ML, Panchal RG, Bowser DN, Williams DA, Petrou S (2002) Pore formation is not associated with macroscopic redistribution of P2X7 receptors. Am J Physiol Cell Physiol 283(1):C77–C84. https://doi.org/10.1152/ajpcell.00456.2001

    Article  CAS  PubMed  Google Scholar 

  11. Buell G, Chessell IP, Michel AD, Collo G, Salazzo M, Herren S, Gretener D, Grahames C, Kaur R, Kosco-Vilbois MH, Humphrey PP (1998) Blockade of human P2X7 receptor function with a monoclonal antibody. Blood 92(10):3521–3528

    CAS  PubMed  Google Scholar 

  12. Karasawa A, Michalski K, Mikhelzon P, Kawate T (2017) The P2X7 receptor forms a dye-permeable pore independent of its intracellular domain but dependent on membrane lipid composition. elife 6. https://doi.org/10.7554/eLife.31186

  13. Michel AD, Thompson KM, Simon J, Boyfield I, Fonfria E, Humphrey PPA (2006) Species and response dependent differences in the effects of MAPK inhibitors on P2X(7) receptor function. Br J Pharmacol 149(7):948–957. https://doi.org/10.1038/sj.bjp.0706938

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wickert LE, Blanchette JB, Waldschmidt NV, Bertics PJ, Denu JM, Denlinger LC, Lenertz LY (2013) The C-terminus of human nucleotide receptor P2X7 is critical for receptor oligomerization and N-linked glycosylation. PLoS One 8(5):e63789. https://doi.org/10.1371/journal.pone.0063789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Milius D, Groger-Arndt H, Stanchev D et al (2007) Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells. Toxicology 238(1):60–69. https://doi.org/10.1016/j.tox.2007.05.028

    Article  CAS  PubMed  Google Scholar 

  16. Ferrari D, Pizzirani C, Gulinelli S, Callegari G, Chiozzi P, Idzko M, Panther E, Virgilio F (2007) Modulation of P2X7 receptor functions by polymyxin B: crucial role of the hydrophobic tail of the antibiotic molecule. Br J Pharmacol 150(4):445–454. https://doi.org/10.1038/sj.bjp.0706994

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sun C, Heid ME, Keyel PA, Salter RD (2013) The second transmembrane domain of P2X7 contributes to dilated pore formation. PLoS One 8(4):e61886. https://doi.org/10.1371/journal.pone.0061886

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bradley HJ, Liu X, Collins V, Owide J, Goli GR, Smith M, Surprenant A, White SJ, Jiang LH (2010) Identification of an intracellular microdomain of the P2X7 receptor that is crucial in basolateral membrane targeting in epithelial cells. FEBS Lett 584(23):4740–4744. https://doi.org/10.1016/j.febslet.2010.11.007

    Article  CAS  PubMed  Google Scholar 

  19. Masin M, Young C, Lim K et al (2012) Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: re-evaluation of P2X7 knockouts. Br J Pharmacol 165(4):978–993. https://doi.org/10.1111/j.1476-5381.2011.01624.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Great thanks to Helle Kinggaard Lilja-Fischer for many hours spent on calcium signalling optimization and Susanne Syberg for excellent teaching of YO-PRO-1 dye uptake assay. Also, thanks to Martin Fredensborg Rath for the use of his equipment.

Funding

The study was supported financially by the Lundbeck Foundation and FP7-CIG grant 334443.

Author information

Authors and Affiliations

  1. Department of Clinical Biochemistry, Rigshospitalet, Glostrup, Denmark

    Karin Dreisig, Nikolaj Pagh Kristensen, Maja Wallentin Dommer, Niklas Rye Jørgensen & Birgitte Rahbek Kornum

  2. OPEN, Odense Patient Data Explorative Network Odense University Hospital/Institute of Clinical Research, University of Southern Denmark, Odense, Denmark

    Niklas Rye Jørgensen

  3. Department of Clinical Neurophysiology, Rigshospitalet, Glostrup, Denmark

    Birgitte Rahbek Kornum

Authors
  1. Karin Dreisig
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nikolaj Pagh Kristensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maja Wallentin Dommer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Niklas Rye Jørgensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Birgitte Rahbek Kornum
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Birgitte Rahbek Kornum.

Ethics declarations

Conflict of interest

Karin Dreisig declares that she has no conflict of interest.

Nikolaj Pagh Kristensen declares that he has no conflict of interest.

Maja Wallentin Dommer declares that she has no conflict of interest.

Niklas Rye Jørgensen declares that he has no conflict of interest.

Birgitte Rahbek Kornum declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dreisig, K., Kristensen, N.P., Dommer, M.W. et al. N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake. Purinergic Signalling 14, 83–90 (2018). https://doi.org/10.1007/s11302-017-9598-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11302-017-9598-8

Keywords

Search

Navigation