P2X receptors are ligand-gated ion channels activated by the binding of extracellular adenosine 5'-triphosphate (ATP). Brief (< 1 s) applications of ATP to nodose ganglion neurons or to cells transfected with P2X2 or P2X4 receptor cDNAs induce the opening of a channel selectively permeable to small cations within milliseconds. We now show that, during longer ATP application (10-60 s), the channel also becomes permeable to much larger cations such as N-methyl-D-glucamine and the propidium analog YO-PRO-1. This effect is enhanced in P2X2 receptors carrying point mutations in the second transmembrane segment. Progressive dilation of the ion-conducting pathway during prolonged activation reveals a mechanism by which ionotropic receptors may alter neuronal function.

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 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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A cDNA was isolated from a human monocyte library that encodes the P2X 7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2,3-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X 7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X 7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.Cell surface receptors for ATP can be divided into metabotropic (P2Y/P2U) and ionotropic (P2X) classes. The metabotropic class belong to the superfamily of G protein-coupled receptors with seven transmembrane segments; the ionotropic class are ligand-gated channels, currently thought to be multisubunit proteins with two transmembrane domains per subunit (for review see Ref. 1). P2Z receptors have been distinguished from other P2 receptors in three main ways (2-4). First, activation of this receptor leads not only to an inward ionic current but also to cell permeabilization. Second, 2Ј,3Ј-(4-benzoyl)benzoyl ATP is the most effective agonist, and ATP itself is of rather low potency. Third, responses are strongly inhibited by extracellular magnesium ions, which has been interpreted to indicate that ATP Ϫ4 is the active agonist (for review see Ref. 5).A seventh member of the P2X receptor family was isolated recently from a rat cDNA library that, when expressed in human embryonic kidney (HEK293) cells, exhibits these three properties (6). This receptor (rP2X 7 ) 1 is thus considered to represent a P2Z receptor. The protein is structurally related to other members of the P2X family; there is 35-40% amino acid identity in the region of homology, but the C terminus is 239 amino acids long in the rP2X 7 receptor compared with 27-120 amino acids in the others. The rP2X 7 receptor functions both as a channel permeable to small cations and as a cytolytic pore (6). Brief applications of ATP (1-2 s) transiently open the channel, and this is generally similar in properties to other P2X receptors. Repeated or prolonged applications of agonist cause cell permeabilization; reducing the extracellular magnesium concentration much potentiates this effect. The permeabilization involves the cytoplasmic C terminus of the protein because it does not occur with a P2X 7 receptor lacking the last 177 residues, although this truncation does not affect the function as a small cation channel. The P2Z receptor has been implicated in lysis of antigenpresenting cells by cytotoxic T lymphocytes, in the mi...

1. Complementary DNAs for the ATP-gated ion channel subunits P2X, (from human bladder) and P2X2 (from rat phaeochromocytoma (PC12) cells) were used to express the receptors in human embryonic kidney cells by stable transfection, and in Chinese hamster ovary cells by viral infection. 2. Membrane currents evoked by ATP were recorded by the whole-cell patch clamp method.The reversal potential of the current was measured with various intracellular and extracellular solutions and used to compute the relative permeability of the P2X receptor channels. 3. There was no difference between the two receptors with respect to their permeability to monovalent organic cations. The relative permeabilities (Px/PNa) were 2-3, 1P0, 1 0, 0 95, 0'72, 0 5, 0-29, 0-16, 0 04 and 0 03 for guanidinium, potassium, sodium, methylamine, caesium, dimethylamine, 2-methylethanolamine, tris(hydroxymethyl)-aminomethane, tetraethylammonium and N-methyl-D-glucamine, respectively (values for P2X2 receptor). 4. The calcium permeability of P2X1 receptors was greater than that of P2X2 receptors. Under biionic conditions (112 mm calcium outside, 154 mm sodium inside), Pca/PNa values were 3 9 and 2 2, respectively (corrected for ionic activities). 5. ATP-evoked currents in cells expressing the P2X2 receptor were strongly inhibited when the extracellular calcium concentration was increased (0 3-30 mM); the action of ATP could be restored by increasing the ATP concentration. ATP-evoked currents in cells expressing the P2X1 receptor were not inhibited by such increases in the extracellular calcium concentration.