An Arg307 to Gln Polymorphism within the ATP-binding Site Causes Loss of Function of the Human P2X7 Receptor

Abstract

The P2X 7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X 7 receptor have been observed, explained in part by loss-of-function polymorphisms that change Glu 496 to Ala (E496A) and Ile 568 to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg 307 (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X 7 function was measured by ATP-induced fluxes of Rb ؉ , Ba 2؉ , and ethidium ؉ into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X 7 carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X 7 and blocks P2X 7 function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X 7 function in cells heterozygous for the R307Q to a value 10 -40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X 7 , and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X 7 receptor, and since the affected Arg 307 is homologous to those amino acids essential for ATP binding to P2X 1 and P2X 2 , it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X 7 .In cells of the hemopoietic and immune systems, extracellular ATP can induce cytolysis of lymphocytes (1), monocytes/ macrophages (2), and dendritic cells (3). It is generally accepted that these cytolytic effects of ATP are mediated by the P2X 7 receptor, which is a ligand-gated cation channel activated by extracellular ATP and highly expressed on these cell types (4, 5 P2X receptors have an oligomeric structure in the plasma membrane based on trimeric or larger complexes of identical subunits (20,21). Moreover the values of Hill coefficients derived from the sigmoid ATP dose-response curves of the P2X 7 (P2Z) receptor are consistent with multiple ATP-binding sites in each P2X 7 trimer (8,22,23). All seven members of the P2X receptor family have two transmembrane domains with intracellular amino and carboxyl termini. Little is known of the conformation of the extracellular domain containing the ATPbinding site(s). An analysis of the P2X subtype sequence homology has shown that the two transmembrane domains M1 and M2 are separated by an extracellular sequence containing a cysteine-rich region (residues 110 -170) followed by a segment from Phe 188 -Val 321 that may form six antiparallel ␤-pleated sheets homologous with members of the class II aminoacyl-tRNA synthetases (24). The ATP-...

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

“…Net dye uptake indicates the percentage dye uptake induced by BzATP with control (no BzATP treatment) subtracted. The data shown are representative of more than three independent experiments and two-tailed Student's t tests (unpaired) compared to Reference (R) yielded significance levels of <0.05(*), <0.01(**), or <0.001 (***) as shown in b-e of-function (H155Y and A166G) variations are located in the first cysteine-rich domain and only one loss-of-function SNP (R307Q) is at the C-terminus between the second cysteine-rich domain and second membrane-spanning domain (this study and references [57][58][59]). The large extracellular loop is believed to be responsible for ligandbinding and is quite conserved among the P2X receptors, although the fine structure of the ligand-binding pocket is still uncertain.…”

“…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].…”

Orthodontic mechanotransduction and the role of the P2X7 receptor

“…From the genetics perspective, there are two polymorphisms that lead to a null allele of the P2X7 gene, one of them affecting 1-2% of the Caucasian population, has already been reported (Gu et al, 2004;Skarratt et al, 2005). 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).…”

“…P2X7R has also direct clinical relevance because there are many functional human polymorphisms in the P2X7R gene, which can induce different levels of affinity of these receptors to ATP. One of these polymorphisms, a null allele that occurs in 2% of the Caucasian population, is especially important because it results in reduced expression of the ion channel by 50% (Gu et al, 2004). It is hypothesized that variability in the expression of the P2X7R gene is an important factor in the individual variation to applied orthodontic loads.…”

P2X7 receptor polymorphism impairs extracellular adenosine 5′-triphosphate-induced interleukin-18 release from human monocytes

“…4,5 In addition, the K þ ionophore, nigericin, has recently been shown to cause the release of IL-18 from human monocytes. 16 Given the central role of IL-18 in immunity and inflammation, the Glu 496 Ala polymorphism 11 and other, rarer loss-of-function polymorphisms in the P2X 7 receptor 23,24 may have clinical significance. Impaired ATPinduced release of IL-18 may play an important role in diseases where reduced or absent levels of IL-18 are known to increase susceptibility to mycobacteria and certain bacteria, and severity of septic arthritis and endotoxic shock.…”