The Permeabilizing ATP Receptor, P2X7

Abstract

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...

The P2X7 receptor mediates the uptake of organic cations in canine erythrocytes and mononuclear leukocytes: comparison to equivalent human cell types

“…Mg 2+ ions inhibited canine P2X 7 in a concentrationdependent fashion, as previously observed for recombinant human and rat P2X 7 [21,23,24] and similar to that for human erythrocyte P2X 7 . The ability of Mg 2+ to impair P2X 7 is a well-established phenomenon [39], although its mechanism of action remains unknown.…”

“…Consistent with our previous observations [17], the rate of BzATP-and ATP-induced 86 Rb + efflux from canine erythrocytes was approximately 100-fold greater than from human erythrocytes. Moreover, as previously observed for human and rat P2X 7 [23,30], ATP was a partial agonist compared to BzATP. This phenomenon however is not common to all mammalian species.…”

“…Mg 2+ ions are well known to inhibit human and rat P2X 7 [21,23,24], however the effect of these ions on canine P2X 7 is unknown. Therefore, using an approach similar for KN-62, 86 Rb + -loaded erythrocytes in KCl medium were pre-incubated with varying concentrations of Mg 2+ for 5 min and the ATP-induced 86 Rb + effluxes measured.…”

“…Comparison of published sequences of canine and human P2X 7 [23,34] demonstrate that these receptors contain a threonine and asparagine at residue 284 respectively. Although we have not directly sequenced the P2X 7 of the canine donors used in our study, we suggest that the residue difference at position 284 in these two species accounts for the observed differences in EC 50 values for ATP.…”

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

“…The pore is permeable to molecules with molecular masses up to 900 Da, irrespective of the charge [36]. To measure P2X 7 -dependent pore formation, monocytes were suspended in potassium glutamate basic salt solution [37] containing ethidium bromide before ATP and NAD + were added at different concentrations for 15 min at 37°C.…”