Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells

Doreen Milius, Helke Gröger-Arndt, Doychin Stanchev, Christine Lange-Dohna, Steffen Rossner, B. Sperlágh, Kerstin Wirkner, Peter Illes

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Recombinant human P2X7 receptors, C-terminally labelled with enhanced green fluorescent protein (P2X7-EGFP), were transiently expressed in HEK293 cells. Activation of these receptors by their preferential agonist 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) induced inward currents and propidium ion uptake indicating the opening of cationic channels and of large pores permeable for dye molecules, respectively. Two mutants of P2X7 receptors (P2X7-EGFP-I568N, -E496A) representing polymorphisms in the P2X7 gene known to interfere with normal receptor-trafficking and with optimal assembly of its subunits, responded with much lower current amplitudes to BzATP than their wild-type counterpart. Similarly, the normal propidium ion uptake induced by BzATP at the wild-type P2X7 receptor was abolished by the two mutants. Confocal laser scanning microscopy indicated that in vitro ischemia of 12 h duration increased the integration of P2X7-EGFP, but not of its two mutants, into the plasma membrane of HEK293 cells. Further, this ischemic stimulus facilitated the current response to BzATP in HEK293 cells permanently transfected with P2X7 receptors. Finally, the fluorescence intensity per cell measured by flow cytometry and P2X7 antibodies directed against an extracellular, but not an intracellular epitope of the receptor, were also increased. In conclusion, P2X7 receptors may alter their trafficking properties during ischemia and thereby contribute to the ATP-induced damage of various cell-types including neurons.

Original languageEnglish
Pages (from-to)60-69
Number of pages10
JournalToxicology
Volume238
Issue number1
DOIs
Publication statusPublished - Aug 16 2007

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Purinergic P2X7 Receptors
HEK293 Cells
Cell membranes
Cell Membrane
Oxygen
Glucose
Propidium
Ischemia
Ions
Flow cytometry
Induced currents
Polymorphism
Confocal Microscopy
Neurons
Epitopes
Flow Cytometry
Microscopic examination
Coloring Agents
Genes
Adenosine Triphosphate

Keywords

  • Apoptosis
  • Cationic channel
  • Ischemia
  • P2X receptor
  • Pore dilation
  • Receptor trafficking

ASJC Scopus subject areas

  • Toxicology

Cite this

Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells. / Milius, Doreen; Gröger-Arndt, Helke; Stanchev, Doychin; Lange-Dohna, Christine; Rossner, Steffen; Sperlágh, B.; Wirkner, Kerstin; Illes, Peter.

In: Toxicology, Vol. 238, No. 1, 16.08.2007, p. 60-69.

Research output: Contribution to journalArticle

Milius, D, Gröger-Arndt, H, Stanchev, D, Lange-Dohna, C, Rossner, S, Sperlágh, B, Wirkner, K & Illes, P 2007, 'Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells', Toxicology, vol. 238, no. 1, pp. 60-69. https://doi.org/10.1016/j.tox.2007.05.028
Milius, Doreen ; Gröger-Arndt, Helke ; Stanchev, Doychin ; Lange-Dohna, Christine ; Rossner, Steffen ; Sperlágh, B. ; Wirkner, Kerstin ; Illes, Peter. / Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells. In: Toxicology. 2007 ; Vol. 238, No. 1. pp. 60-69.
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