A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages

Abstract

The P2X7 receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X7 receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5′-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X7 polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C → G), which changes Thr357 to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X7 function was measured by ATP-induced ethidium+ influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X7 in either HEK-293 cells or Xenopus oocytes gave P2X7 function of ∼50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X 7, restored P2X7 function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X7 function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X 7 receptor function. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.