title: Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor.
abstract: 1. Extracellular ATP acting on P2X7 receptors opens a channel permeable to small cations, creates an access pathway for the entry of larger molecular weight dyes, and causes cell death. We used whole-cell recording and fluorescence microscopy to measure the time courses of ionic currents, uptake of the propidium dye YO-PRO-1, and membrane disruption, in human embryonic kidney (HEK293) cells expressing the rat P2X7 receptor. 2. The ATP analogue 2', 3'-O-(benzoyl-4-benzoyl)-ATP (30 microM) induced membrane blebbing within 30-40 s of sustained application; this was 5-10 times slower when extracellular sodium was replaced by larger cations. 3. Fluorescence of YO-PRO-1 was detectable within 3 s, and the uptake reached a steady rate within 10-20 s; YO-PRO-1 uptake was greatly enhanced by removing extracellular sodium. 4. Electrophysiological measurements of current reversal potentials with intracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively t'2+LE0i%-i"dilated during 10-20 s to a diameter greater than 1 nm (10 A). With short agonist applications (3-5 s) the pore dilatation and YO-PRO-1 uptake were reversible and repeatable. 5. Polyethylene glycols having molecular weights >= 5000 blocked the increase in cation permeability, YO-PRO-1 uptake and membrane blebbing. 6. We conclude that maximum P2X7 receptor activation causes an exponential dilatation of the ion channel with a time constant of 25 s to a final diameter of 3-5 nm from an initial minimum pore diameter of 0.8 nm.
authors.affiliation: Geneva Biomedical Research Institute, GlaxoWellcome Research and Development, 1228 Plan-les-Ouates, Geneva, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Virginio
authors.forename: A
authors.initials: A
authors.lastname: MacKenzie
authors.forename: R A
authors.initials: RA
authors.lastname: North
authors.forename: A
authors.initials: A
authors.lastname: Surprenant
mesh_terms: D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000818:Animals; D001583:Benzoxazoles; D001711:Biotransformation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000235:genetics / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D004396:Coloring Agents; D004558:Electric Stimulation; D004594:Electrophysiology; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D018408:Patch-Clamp Techniques; D011092:Polyethylene Glycols / Q000494:pharmacology; D011806:Quinolinium Compounds; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D058486:Receptors, Purinergic P2X7
chemical_list: D001583:Benzoxazoles; D004396:Coloring Agents; D005456:Fluorescent Dyes; C546468:P2RX7 protein, human; C546470:P2rx7 protein, rat; D011806:Quinolinium Compounds; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C089813:YO-PRO 1; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate