Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells.
Recombinant human P2X(7) receptors, C-terminally labelled with enhanced green fluorescent protein (P2X(7)-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 P2X(7) receptors (P2X(7)-EGFP-I568N, -E496A) representing polymorphisms in the P2X(7) 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 P2X(7) receptor was abolished by the two mutants. Confocal laser scanning microscopy indicated that in vitro ischemia of 12h duration increased the integration of P2X(7)-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 P2X(7) receptors. Finally, the fluorescence intensity per cell measured by flow cytometry and P2X(7) antibodies directed against an extracellular, but not an intracellular epitope of the receptor, were also increased. In conclusion, P2X(7) receptors may alter their trafficking properties during ischemia and thereby contribute to the ATP-induced damage of various cell-types including neurons.
Milius
Doreen
D
Rudolf-Boehm-Institute for Pharmacology and Toxicology, University of Leipzig, D-04107 Leipzig, Germany. Doreen.Milius@medizin.uni-leipzig.de
Gröger-Arndt
Helke
H
Stanchev
Doychin
D
Lange-Dohna
Christine
C
Rossner
Steffen
S
Sperlagh
Beata
B
Wirkner
Kerstin
K
Illes
Peter
P
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015687:Cell Hypoxia; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005947:Glucose / Q000172:deficiency / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D009154:Mutation; D010100:Oxygen / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011419:Propidium / Q000378:metabolism / Q000493:pharmacokinetics; D021381:Protein Transport / Q000187:drug effects; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013997:Time Factors; D014162:Transfection
C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011419:Propidium; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D005947:Glucose; D010100:Oxygen