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Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X₇ receptor

C. Virginio *, A. MacKenzie *¹, R. A. North *¹ and A. Surprenant *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.

The ATP analogue 2',3'-O-(benzoyl-4-benzoyl)-ATP (30 µM) induced membrane blebbing within 30-40 s of sustained application; this was 5-10 times slower when extracellular sodium was replaced by larger cations.

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.

Electrophysiological measurements of current reversal potentials with intracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively t'²⁺LE0%-"dilated during 10-20 s to a diameter greater than 1 nm (10 Å). With short agonist applications (3-5 s) the pore dilatation and YO-PRO-1 uptake were reversible and repeatable.

Polyethylene glycols having molecular weights 5000 blocked the increase in cation permeability, YO-PRO-1 uptake and membrane blebbing.

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.

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