An Ile-568 to Asn Polymorphism Prevents Normal Trafficking and Function of the Human P2X7 Receptor

Abstract

The P2X 7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X 7 receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X 7 function was measured by ATPinduced fluxes of Rb ؉ , Ba 2؉ , and ethidium ؉ into various lymphocyte subsets and was decreased to values of ϳ25% of normal. The expression of the P2X 7 receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X 7 carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-␥ up-regulated P2X 7 function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X 7 receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.The purinergic P2X 7 receptor is a ligand-gated channel, selective for cationic permeants, which has a wide distribution including cells of the immune and hemopoietic system (1, 2). Activation of this receptor by brief exposure to extracellular ATP opens a channel that allows Ca 2ϩ and Na ϩ influx and K ϩ efflux and that initiates a cascade of intracellular downstream events. These include the stimulation of phospholipase D (3, 4), the activation of membrane metalloproteases (5-7), and the stimulation of intracellular caspases, which eventually lead to the apoptotic death of the target cell (8, 9). P2X 7 activation also leads to extensive membrane blebbing (10), which is a typical morphological feature of the apoptotic process. P2X 7 receptors have two transmembrane domains with intracellular amino and carboxyl termini, and the P2X 7 receptor differs from other members of the P2X receptor family in having a long carboxyl terminus of 240 amino acids from the inner membrane face (11). This long carboxyl terminus is necessary for the permeability properties of the P2X 7 receptor because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye Yo-Pro-1 (12). P2X 7 has an oligomeric structure in the membrane based on trimeric or larger complexes of identical subunits (13,14), and there is evidence that P2X 7 interacts with a number of structural and adhesion proteins in a complex at the cell surface (15). Phosphorylation of a tyrosine at amino acid 343 of the P2X 7 primary structure has been proposed as being important for maintaining the full activity of the P2X 7 channel (15). A number of regulatory d...

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

“…Three loss-of function SNPs (T357S, E496A, and I568N) and one gain-of-function SNP (Q460R) are located in the long intracellular domain [54][55][56]58]. These loss-of-function polymorphisms lead not only to reduced P2X 7 pore function but also to impaired ATP-induced mycobacterial killing by macrophages [56,60,61,81,82].…”

“…However, the majority of these have not been validated at the population genetic level and their functional effects are unclear. 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].…”

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]. In this regard, Ohlendorff et al found that Glu496Ala and Ile568Asn single-nucleotide polymorphisms in the human P2X7 receptor are associated with 10-year fracture risk in postmenopausal women [79].…”

Orthodontic mechanotransduction and the role of the P2X7 receptor

“…In addition, one polymorphism was proven to impair ATP-induced IL-1β release from human monocytes and one to affect normal trafficking of this receptor Wiley et al, 2003). If the role of the P2X7 receptor in tooth movement is shown in an experimental model controlling for multiple variables, it could provide a strong base for association studies of these polymorphisms with ERR, and rate of tooth movement.…”