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Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor
C Virginio1, A MacKenzie, R A North
1Geneva Biomedical Research Institute, GlaxoWellcome Research and Development, 1228 Plan-les-Ouates, Geneva, Switzerland.
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.
Insights
Extracellular ATP binding to P2X7 receptors causes a time-dependent pore dilation, allowing dye entry and cell death. This pore dilation is reversible with short ATP applications and blocked by large molecules.
Area of Science:
- Cell Biology
- Molecular Pharmacology
- Ion Channel Physiology
Background:
- Extracellular ATP (adenosine triphosphate) is a signaling molecule that activates P2X7 receptors.
- P2X7 receptor activation leads to ion channel opening, membrane permeability changes, and cell death.
- The precise kinetics and pore size dynamics of P2X7 receptor-mediated channel opening remain incompletely understood.
Purpose of the Study:
- To investigate the time course of P2X7 receptor channel dilation.
- To characterize the permeability changes associated with P2X7 receptor activation.
- To determine the molecular size limit of the P2X7 receptor pore.
Main Methods:
- Whole-cell patch-clamp electrophysiology to measure ionic currents.
- Fluorescence microscopy using YO-PRO-1 dye uptake to assess pore size.
- Application of ATP analogues and polyethylene glycols of varying molecular weights.
Main Results:
- P2X7 receptor activation by ATP analogues induced membrane blebbing and YO-PRO-1 dye uptake.
- Channel dilation occurred progressively over 10-20 seconds, reaching a diameter >1 nm.
- Extracellular sodium removal enhanced YO-PRO-1 uptake, indicating increased cation permeability.
- Polyethylene glycols >5000 Da blocked dye uptake and membrane blebbing.
- Maximum P2X7 receptor activation resulted in exponential pore dilation to 3-5 nm.
Conclusions:
- P2X7 receptor activation leads to a time-dependent, exponential pore dilation.
- The P2X7 receptor channel can expand to accommodate molecules up to 3-5 nm in diameter.
- This dynamic pore dilation is crucial for ATP-induced cell permeability changes and death.