Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X(7) receptor

1. Extracellular ATP acting on P2X(7) receptors opens a channel permeable t o small cations, creates an access pathway for the entry of larger molecula r weight dyes, and causes cell death. We used whole-cell recording and fluo rescence microscopy to measure the time courses of ionic currents, uptake o f the propidium dye YO-PRO-1, and membrane disruption, in human embryonic k idney (HEK293) cells expressing the rat P2X(7) receptor. 2. The ATP analogue 2',3'- 0-(benzoyl-4-benzoyl)-ATP (30 mu M) induced memb rane 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 reach ed a steady rate within 10-20 s; YO-PRO-1 uptake was greatly enhanced by re moving extracellular sodium. 4. Electrophysiological measurements of current reversal potentials with in tracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively dilated during 10-20 s to a diameter great er than 1 nm (10 Angstrom). With short agonist applications (3-5 s) the por e dilatation and YO-PRO-1 uptake were reversible and repeatable. 5. Polyethylene glycols having molecular weights greater than or equal to 5 000 blocked the increase in cation permeability, YO-PRO-1 uptake and membra ne blebbing. 6. We conclude that maximum P2X(7) receptor activation causes an exponentia l dilatation of the ion channel with a time constant of 25s to a final diam eter of 3-5 nm from an initial minimum pore diameter of 0.8 nm.