A Glu-496 to Ala Polymorphism Leads to Loss of Function of the Human P2X7 Receptor

The P2X7 receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X7 is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X7. In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A→C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X7 function that was half that of germline P2X7. Results of transfection experiments showed that the mutant P2X7 receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-γ, which up-regulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with germline (8.6versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7receptor.