Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X(7) receptors on all cells examined from eight of eight subjects, as well as P2X(2) at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of Rb-86(+) (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of similar to95 muM. Other nucleotides also induced an efflux of Rb-86(+) from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP ( BzATP)>ATP>2-methylthio-ATP (2MeSATP)>adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of Rb-86(+) from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X(7) receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an Rb-86(+) influx approximately equal in magnitude to ATP-stimulated Rb-86(+) efflux from the same cells. BzATP also stimulated the influx of Na-22(+) into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of Rb-86(+) and Na-22(+) were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X(7) receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X(7) receptor.