A His-155 to Tyr Polymorphism Confers Gain-of-Function to the Human P2X7 Receptor of Human Leukemic Lymphocytes

Abstract

The P2X7R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X7R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X7R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X7R function was monitored by measuring ATP-induced Ca2+ influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X7 allelic variant. Ca2+ influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca2+ flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X7R confirmed an increased ATP-dependent activation of the P2X7 489T mutant with respect to the wild type receptor, as assessed by both by [Ca2+]i influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X7R.

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

“…1c) [59]. However, no significant increases of expression and pore formation of this variant were observed (Figs.…”

“…This novel variant with dual substitutions we term Variant A. We also isolated full-length cDNA corresponding to previously reported gain-of-function SNP (H155Y) and loss-of-function SNP (E496A) [54,59]. Additional cDNAs encoding nonsynonymous SNPs (H270R and A348T) were also obtained (Fig.…”

“…Five loss-of-function nonsynonymous allelic SNPs in P2X 7 have been described to date, of which three are located in the carboxy-terminal cytoplasmic tail (T357S, E496A, and I568N) and two are in the extracellular loop [54][55][56][57][58]. Cabrini et al have characterized the first gain-of-function polymorphism identified (H155Y) [59]. Another putative gain-of-function polymorphism described recently, the Q460R substitution, appears to enhance pore activity of the P2X 7 receptor [58].…”

P2X7 receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness

“…Importantly, P2X 7 R were not expressed in non-cancerous mammary cells (MCF-10A, 184A1 and Human Mammary Epithelial Cell) whereas they were markedly expressed in highly invasive MDA-MB-435s cells. Many polymorphisms have been identified in the P2RX7 gene, some of them being responsible for important functional alterations and associated to many diseases (Cabrini et al, 2005;Shemon et al, 2006;Roger et al, 2010b). We therefore performed P2X 7 R complementary DNA sequencing in MDA-MB-435s cells.…”

Expression, signaling, and function of P2X7 receptors in bone

“…Polymorphisms that have been identified within the coding region of the human P2X7 receptor have a wide range of outcomes, such as gain-of-function [81], loss-of-function [82,83], impairment of cytokine release [84,85], and altered cell death [86]. The relatively common Glu496Ala polymorphism results in reduced pore-forming ability [82] without altering channel function [87], whereas the Ile568Asn polymorphism prevents normal channel trafficking and function [88].…”