Residues 155 and 348 Contribute to the Determination of P2X7 Receptor Function via Distinct Mechanisms Revealed by Single-nucleotide Polymorphisms (original) (raw)
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Point mutations confer loss of ATP-induced human P2X7 receptor function
FEBS Letters, 2002
Residues considered essential for ATP binding to the human P2X 7 receptor (hP2X 7 R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild-type or sitedirected mutants of hP2X 7 R constructs and channel/pore activity measured in the presence of ATP or 2P P,3P P-O-(4-benzoylbenzoyl)-ATP (BzATP). Barium uptake and ethidium influx into HEK293 cells were abolished in cells expressing K193A and K311A mutants, and were partially reduced in cells expressing mutant P210A. K193A and K311A mutations also completely abolished responses to ATP and BzATP in Xenopus oocytes as measured by electrophysiology. These results indicate that K193 and K311 are essential residues in ATP binding in the hP2X 7 R. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
Structural and Functional Basis for Understanding the Biological Significance of P2X7 Receptor
International Journal of Molecular Sciences
The P2X7 receptor (P2X7R) possesses a unique structure associated to an as yet not fully understood mechanism of action that facilitates cell permeability to large ionic molecules through the receptor itself and/or nearby membrane proteins. High extracellular adenosine triphosphate (ATP) levels—inexistent in physiological conditions—are required for the receptor to be triggered and contribute to its role in cell damage signaling. The inconsistent data on its activation pathways and the few studies performed in natively expressed human P2X7R have led us to review the structure, activation pathways, and specific cellular location of P2X7R in order to analyze its biological relevance. The ATP-gated P2X7R is a homo-trimeric receptor channel that is occasionally hetero-trimeric and highly polymorphic, with at least nine human splice variants. It is localized predominantly in the cellular membrane and has a characteristic plasticity due to an extended C-termini, which confers it the capac...
Journal of Biological Chemistry, 2004
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 .
Identification of Thr 283 as a key determinant of P2X 7 receptor function
British Journal of Pharmacology, 2006
Background and purpose: The ATP-gated P2X 7 receptor is an unusual ion channel that couples to multiple downstream signalling cascades. We noted differences in mouse cDNA sequences that may indicate polymorphisms; the aim of this study was to compare function and expression of these mouse P2X 7 receptor mutations. Experimental approach: There are three differences in the sequences of P2X 7 cDNA cloned from mouse NTW8 microglial cells or C57 BL/6 mice: [Phe 11 ,Ala 221 ,Met 283 ]P2X 7 in the former and [Leu 11 ,Thr 221 ,Thr 283 ]P2X 7 in the latter. We expressed these receptors and measured membrane currents, ethidium uptake, calcium influx and surface membrane expression. We also carried out these assays on the previously described polymorphism observed between C57 BL/6 and Balb/c mice ([Leu 451 ]P2X 7 vs [Pro 451 ]P2X 7 ).
Significance of P2X7 Receptor Variants to Human Health and Disease
Recent Patents on DNA &# 38; Gene …, 2011
The human P2X7 receptor is a trimeric ligand-gated cation channel coded by the P2RX7 gene located at chromosome position 12q24. P2X7 is expressed in a wide variety of normal and disease-associated cell types. Activation of this receptor by extracellular adenosine 5'-triphosphate results in numerous downstream events including the release of pro-inflammatory mediators, cell proliferation or death, and killing of intracellular pathogens. As a result, P2X7 plays important roles in inflammation, immunity, bone homeostasis, neurological function and neoplasia. The P2RX7 gene encodes a P2X7 subunit 595 amino acids in length, however splice isoforms that can alter receptor expression and function, and modify the signaling properties downstream of receptor activation also exist. Moreover, the relative amount of P2X7 function varies between human individuals due to numerous single nucleotide polymorphisms resulting in either loss-or gain-offunction. Combinations of these polymorphisms give rise to various haplotypes that can also modify P2X7 function. Collectively, P2X7, and its splice and polymorphic variants are attracting considerable interest in relation to human health and disease, including the development and publication of a number of patents.
Mammalian P2X7 receptor pharmacology: comparison of recombinant mouse, rat and human P2X7 receptors
British Journal of Pharmacology, 2009
Background and purpose: Acute activation of P2X7 receptors rapidly opens a non-selective cation channel. Sustained P2X7 receptor activation leads to the formation of cytolytic pores, mediated by downstream recruitment of hemichannels to the cell surface. Species-and single-nucleotide polymorphism-mediated differences in P2X7 receptor activation have been reported that complicate understanding of the physiological role of P2X7 receptors. Studies were conducted to determine pharmacological differences between human, rat and mouse P2X7 receptors. Experimental approach: Receptor-mediated changes in calcium influx and Yo-Pro uptake were compared between recombinant mouse, rat and human P2X7 receptors. For mouse P2X7 receptors, wild-type (BALB/c) and a reported loss of function (C57BL/6) P2X7 receptor were also compared. Key results: BzATP [2,3-O-(4-benzoylbenzoyl)-ATP] was more potent than ATP in stimulating calcium influx and Yo-Pro uptake at rat, human, BALB/c and C57BL/6 mouse P2X7 receptors. Two selective P2X7 receptor antagonists, A-740003 and A-438079, potently blocked P2X7 receptor activation across mammalian species. Several reported P2X1 receptor antagonists [e.g. MRS 2159 (4-[(4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl}-2-pyridinyl)azo]-benzoic acid), PPNDS and NF279] blocked P2X7 receptors. NF279 fully blocked human P2X7 receptors, but only partially blocked BALB/c P2X7 receptors and was inactive at C57BL/6 P2X7 receptors. Conclusions and implications: These data provide new insights into P2X7 receptor antagonist pharmacology across mammalian species. P2X7 receptor pharmacology in a widely used knockout background mouse strain (C57BL/6) was similar to wild-type mouse P2X7 receptors. Several structurally novel, selective and competitive P2X7 receptor antagonists show less species differences compared with earlier non-selective antagonists.
Selective Knock-Down of P2X7 ATP Receptor Function by Dominant-Negative Subunits
Molecular Pharmacology, 2004
Among the family of P2X ATP-gated cation channels, the P2X 7 receptor is a homomeric subtype highly expressed in immune cells of the monocyte-macrophage lineage. We report here that the WC167-168AA mutation in the ectodomain of P2X 7 produced nonfunctional subunits with strong dominant-negative effect on wild-type P2X 7 receptors (77% inhibition with cotransfection of wild-type and mutant DNA at a ratio of 3:1). The C168A single mutant was also very effective in suppressing P2X 7 receptor function (72% reduction at a DNA ratio of 3:1), indicating the major role played by the C168A mutation in this inhibition. The dominant-negative effect is selective; the mutant subunit did not suppress the function of other receptor-channel subtypes. The reduced current responses in cells coexpressing wild-type and dominant-negative subunits display wild-type
Current Medicinal Chemistry
P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-A crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures.