Home LiteratureArticle Details
PMID: 9606184 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.

The EMBO journal ·Vol. 17 ·No. 11 ·1998-06-01 ·Pages 3016-28

Nicke A, Bäumert HG, Rettinger J, Eichele A, Lambrecht G, Mutschler E, Schmalzing G

Abstract

P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. blue native PAGE/cross-linking/P2X receptor/quaternary structure.

MeSH Terms
Animals Cross-Linking Reagents Dimerization Electrophoresis, Polyacrylamide Gel Glucosides/chemistry Glycosylation/drug effects Hexosaminidases/pharmacology Histidine/genetics Ion Channels/chemistry,metabolism Ligands Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase Membrane Proteins/chemistry,physiology Oocytes/metabolism Purinergic P2 Receptor Antagonists Pyridoxal Phosphate/analogs & derivatives,pharmacology Receptors, Cholinergic/biosynthesis,chemistry Receptors, Purinergic P2/biosynthesis,chemistry,metabolism Receptors, Purinergic P2X Receptors, Purinergic P2X3 Xenopus laevis
Chemicals
Cross-Linking Reagents Glucosides Ion Channels Ligands Membrane Proteins Purinergic P2 Receptor Antagonists Receptors, Cholinergic Receptors, Purinergic P2 Receptors, Purinergic P2X Receptors, Purinergic P2X3 pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid octyl-beta-D-glucoside Histidine Pyridoxal Phosphate Hexosaminidases Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Nicke A
Biocenter of the Johann Wolfgang Goethe-University of Frankfurt, Frankfurt, Germany.
Bäumert H G
Rettinger J
Eichele A
Lambrecht G
Mutschler E
Schmalzing G
References (70)
70 references, click to expand
  1. Structure of gap junction intercellular channels.
    Curr Opin Struct Biol. 1996 Apr;6(2):183-92 PMID: 8728651
  2. The emerging three-dimensional structure of a receptor. The nicotinic acetylcholine receptor.
    Eur J Biochem. 1996 Aug 1;239(3):539-57 PMID: 8774696
  3. Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines.
    Neuron. 1996 Aug;17(2):343-52 PMID: 8780657
  4. Families of ion channels with two hydrophobic segments.
    Curr Opin Cell Biol. 1996 Aug;8(4):474-83 PMID: 8791456
  5. Purinergic receptors: their role in nociception and primary afferent neurotransmission.
    Curr Opin Neurobiol. 1996 Aug;6(4):526-32 PMID: 8794102
  6. Protein folds in channel structure.
    Curr Opin Struct Biol. 1996 Aug;6(4):499-510 PMID: 8794169
  7. Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli.
    Biophys J. 1997 Oct;73(4):1925-31 PMID: 9336188
  8. Molecular assembly of the extracellular domain of P2X2, an ATP-gated ion channel.
    Biochem Biophys Res Commun. 1997 Nov 26;240(3):618-22 PMID: 9398614
  9. Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.
    J Biol Chem. 1998 Jan 9;273(2):681-4 PMID: 9422716
  10. The heterotetrameric architecture of the epithelial sodium channel (ENaC).
    EMBO J. 1998 Jan 15;17(2):344-52 PMID: 9430626
  11. A tetrameric subunit stoichiometry for a glutamate receptor-channel complex.
    Neuroreport. 1998 Jan 26;9(2):327-31 PMID: 9507977
  12. Evidence for a tetrameric structure of recombinant NMDA receptors.
    J Neurosci. 1998 Apr 15;18(8):2954-61 PMID: 9526012
  13. The adhesion molecule on glia (AMOG/beta 2) and alpha 1 subunits assemble to functional sodium pumps in Xenopus oocytes.
    J Biol Chem. 1992 Oct 5;267(28):20212-6 PMID: 1383200
  14. PPADS, a novel functionally selective antagonist of P2 purinoceptor-mediated responses.
    Eur J Pharmacol. 1992 Jul 7;217(2-3):217-9 PMID: 1330591
  15. Membrane glycoprotein folding, oligomerization and intracellular transport: effects of dithiothreitol in living cells.
    EMBO J. 1993 May;12(5):2151-7 PMID: 8491203
  16. Disulfide-linked aggregation of thyroglobulin normally occurs during nascent protein folding.
    Am J Physiol. 1993 Sep;265(3 Pt 1):C704-11 PMID: 8214027
  17. Quaternary structure of the native GABAA receptor determined by electron microscopic image analysis.
    J Neurochem. 1994 Feb;62(2):815-8 PMID: 7507518
  18. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.
    Anal Biochem. 1994 Mar;217(2):220-30 PMID: 8203750
  19. Cloned glutamate receptors.
    Annu Rev Neurosci. 1994;17:31-108 PMID: 8210177
  20. A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP.
    Nature. 1994 Oct 6;371(6497):516-9 PMID: 7523951
  21. Acetylcholine receptor channel imaged in the open state.
    Nature. 1995 Jan 5;373(6509):37-43 PMID: 7800037
  22. Epithelial sodium channels.
    Curr Opin Nephrol Hypertens. 1994 Sep;3(5):487-96 PMID: 7804746
  23. Characterization and chromosomal localization of a human P2X receptor from the urinary bladder.
    Receptors Channels. 1995;3(4):283-9 PMID: 8834001
  24. P2X receptors: a third major class of ligand-gated ion channels.
    Ciba Found Symp. 1996;198:91-105; discussion 105-9 PMID: 8879820
  25. Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.
    EMBO J. 1996 Sep 16;15(18):4798-805 PMID: 8890153
  26. P2-purinoceptor antagonists: I. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by small aromatic isothiocyanato-sulphonates.
    Naunyn Schmiedebergs Arch Pharmacol. 1996 Oct;354(4):481-90 PMID: 8897452
  27. Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
    J Physiol. 1996 Oct 1;496 ( Pt 1):165-73 PMID: 8910205
  28. P2X receptors: an emerging channel family.
    Eur J Neurosci. 1996 Oct;8(10):2221-8 PMID: 8921315
  29. Ionic permeability of, and divalent cation effects on, two ATP-gated cation channels (P2X receptors) expressed in mammalian cells.
    J Physiol. 1996 Dec 1;497 ( Pt 2):413-22 PMID: 8961184
  30. Characterization of recombinant human P2X4 receptor reveals pharmacological differences to the rat homologue.
    Mol Pharmacol. 1997 Jan;51(1):109-18 PMID: 9016352
  31. Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates.
    Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1136-41 PMID: 9037019
  32. The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA.
    J Biol Chem. 1997 Feb 28;272(9):5482-6 PMID: 9038151
  33. Development and perspectives of the purinoceptor concept.
    J Auton Pharmacol. 1996 Dec;16(6):295-302 PMID: 9131402
  34. Design and pharmacology of selective P2-purinoceptor antagonists.
    J Auton Pharmacol. 1996 Dec;16(6):341-4 PMID: 9131412
  35. Identification of amino acid residues contributing to the pore of a P2X receptor.
    EMBO J. 1997 Jun 16;16(12):3446-54 PMID: 9218787
  36. Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking.
    Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8202-7 PMID: 9223339
  37. Structural motif and characteristics of the extracellular domain of P2X receptors.
    Biochem Biophys Res Commun. 1997 Jul 30;236(3):670-5 PMID: 9245711
  38. Ultrastructure of the 5-hydroxytryptamine3 receptor.
    J Neurochem. 1995 Mar;64(3):1401-5 PMID: 7861173
  39. Purinoceptors: are there families of P2X and P2Y purinoceptors?
    Pharmacol Ther. 1994;64(3):445-75 PMID: 7724657
  40. The use of a new series of cleavable protein-crosslinkers on the Escherichia coli ribosome.
    FEBS Lett. 1974 Nov 15;48(2):288-92 PMID: 4373293
  41. Effect of pyridoxal phosphate on the DNA binding site of activated hepatic glucocorticoid receptor.
    J Biol Chem. 1978 Jul 25;253(14):4886-91 PMID: 670167
  42. The permeability of the endplate channel to organic cations in frog muscle.
    J Gen Physiol. 1980 May;75(5):469-92 PMID: 6247422
  43. Assembly of influenza hemagglutinin trimers and its role in intracellular transport.
    J Cell Biol. 1986 Oct;103(4):1179-91 PMID: 2429970
  44. Quaternary structure of influenza virus hemagglutinin after acid treatment.
    J Virol. 1986 Dec;60(3):833-9 PMID: 3783818
  45. Selective radiolabeling of cell surface proteins to a high specific activity.
    Biochemistry. 1987 Feb 10;26(3):743-50 PMID: 3552030
  46. Posttranslational oligomerization and cooperative acid activation of mixed influenza hemagglutinin trimers.
    J Cell Biol. 1988 Mar;106(3):629-39 PMID: 3279048
  47. Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer.
    Proc Natl Acad Sci U S A. 1988 Oct;85(19):7394-8 PMID: 2459705
  48. Identification of synaptophysin as a hexameric channel protein of the synaptic vesicle membrane.
    Science. 1988 Nov 18;242(4881):1050-3 PMID: 2461586
  49. Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes.
    Biochem J. 1989 Jun 1;260(2):395-9 PMID: 2548475
  50. Bivalent ligands and the message-address concept in the design of selective opioid receptor antagonists.
    Trends Pharmacol Sci. 1989 Jun;10(6):230-5 PMID: 2549665
  51. The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization.
    J Biol Chem. 1990 May 25;265(15):8869-73 PMID: 2111326
  52. Ligand-gated ion channels in the brain: the amino acid receptor superfamily.
    Neuron. 1990 Oct;5(4):383-92 PMID: 1698394
  53. Primary structure and functional expression of the alpha-, beta-, gamma-, delta- and epsilon-subunits of the acetylcholine receptor from rat muscle.
    Eur J Biochem. 1990 Dec 12;194(2):437-48 PMID: 1702709
  54. Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure.
    J Biol Chem. 1991 Jun 15;266(17):11192-8 PMID: 2040627
  55. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.
    Anal Biochem. 1991 Dec;199(2):223-31 PMID: 1812789
  56. Solubilization and molecular size determination of the P2x purinoceptor from rat vas deferens.
    J Biol Chem. 1992 Sep 5;267(25):17581-7 PMID: 1387637
  57. Evolutionary history of the ligand-gated ion-channel superfamily of receptors.
    Trends Neurosci. 1995 Mar;18(3):121-7 PMID: 7754520
  58. Dimensions of the narrow portion of a recombinant NMDA receptor channel.
    Biophys J. 1995 Mar;68(3):866-75 PMID: 7538803
  59. A vector for the synthesis of cRNAs encoding Myc epitope-tagged proteins in Xenopus laevis oocytes.
    Gene. 1995 Jul 28;160(2):213-7 PMID: 7543868
  60. Quality control in the secretory pathway.
    Curr Opin Cell Biol. 1995 Aug;7(4):523-9 PMID: 7495572
  61. A P2X purinoceptor expressed by a subset of sensory neurons.
    Nature. 1995 Oct 5;377(6548):428-31 PMID: 7566119
  62. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons.
    Nature. 1995 Oct 5;377(6548):432-5 PMID: 7566120
  63. Ion-channel assembly.
    Trends Neurosci. 1995 Jun;18(6):280-7 PMID: 7571003
  64. Structure and function of glutamate and nicotinic acetylcholine receptors.
    Curr Opin Neurobiol. 1995 Jun;5(3):310-7 PMID: 7580153
  65. A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans.
    EMBO J. 1995 Nov 1;14(21):5170-8 PMID: 7489706
  66. Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel.
    Nature. 1995 Dec 14;378(6558):730-3 PMID: 7501021
  67. Determination of the subunit stoichiometry of an inwardly rectifying potassium channel.
    Neuron. 1995 Dec;15(6):1441-7 PMID: 8845166
  68. An antagonist-insensitive P2X receptor expressed in epithelia and brain.
    EMBO J. 1996 Jan 2;15(1):55-62 PMID: 8598206
  69. Pentameric subunit stoichiometry of a neuronal glutamate receptor.
    Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2741-4 PMID: 8610111
  70. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).
    Science. 1996 May 3;272(5262):735-8 PMID: 8614837
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/17.11.3016
Published
1998-06-01
Pages
3016-28
Language
English
Region
England
NLM ID
8208664
PMCID
PMC1170641
Subset
IM
External Links
PubMed source DOI: 10.1093/emboj/17.11.3016 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2