Pore formation is not associated with macroscopic redistribution of P2X7 receptors.
The present study examines whether changes in P2X7 purinergic receptor density precede formation of the cytolytic pore characteristic of this receptor. We fused P2X7 receptors with enhanced green fluorescent protein (EGFP) at the amino or carboxy termini (EGFP-P2X7 and P2X7-EGFP). Electrophysiological characterization in Xenopus oocytes revealed wild-type responses to ATP for GFP-tagged receptors. However, differences in sensitivity to ATP were apparent with the P2X7-EGFP receptor displaying a threefold reduction in ATP sensitivity compared with control. Ethidium ion uptake was used to measure cytolytic pore formation. Comparison of tagged receptors with wild type in HEK-293 and COS-7 cells showed there was no significant difference in ethidium ion uptake, suggesting that fusions with EGFP did not interfere with cytolytic pore formation. Confocal microscopy confirmed that tagged receptors localized to the plasmalemma. Simultaneous monitoring of EGFP and ethidium ion fluorescence revealed that changes in receptor distribution do not precede pore formation. We conclude that it is unlikely that large scale changes in P2X7 receptor density precede pore formation.
Smart
Megan L
ML
The Laboratory of Biophysics and Molecular Physiology, Department of Physiology, The University of Melbourne, Victoria 3010, Australia.
Panchal
Rekha G
RG
Bowser
David N
DN
Williams
David A
DA
Petrou
Steven
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004996:Ethidium / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D007473:Ion Channels / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D009865:Oocytes; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins; D014018:Tissue Distribution; D014981:Xenopus
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007473:Ion Channels; D008164:Luminescent Proteins; C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D004996:Ethidium