Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 Jun 15;16(12):3446–3454. doi: 10.1093/emboj/16.12.3446

Identification of amino acid residues contributing to the pore of a P2X receptor.

F Rassendren 1, G Buell 1, A Newbolt 1, R A North 1, A Surprenant 1
PMCID: PMC1169970  PMID: 9218787

Abstract

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.

Full Text

The Full Text of this article is available as a PDF (494.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akabas M. H., Karlin A. Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit. Biochemistry. 1995 Oct 3;34(39):12496–12500. doi: 10.1021/bi00039a002. [DOI] [PubMed] [Google Scholar]
  2. Akabas M. H., Kaufmann C., Archdeacon P., Karlin A. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit. Neuron. 1994 Oct;13(4):919–927. doi: 10.1016/0896-6273(94)90257-7. [DOI] [PubMed] [Google Scholar]
  3. Akabas M. H., Stauffer D. A., Xu M., Karlin A. Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science. 1992 Oct 9;258(5080):307–310. doi: 10.1126/science.1384130. [DOI] [PubMed] [Google Scholar]
  4. Brake A. J., Wagenbach M. J., Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature. 1994 Oct 6;371(6497):519–523. doi: 10.1038/371519a0. [DOI] [PubMed] [Google Scholar]
  5. Buell G., Collo G., Rassendren F. P2X receptors: an emerging channel family. Eur J Neurosci. 1996 Oct;8(10):2221–2228. doi: 10.1111/j.1460-9568.1996.tb00745.x. [DOI] [PubMed] [Google Scholar]
  6. Buell G., Lewis C., Collo G., North R. A., Surprenant A. An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J. 1996 Jan 2;15(1):55–62. [PMC free article] [PubMed] [Google Scholar]
  7. Canessa C. M., Horisberger J. D., Rossier B. C. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature. 1993 Feb 4;361(6411):467–470. doi: 10.1038/361467a0. [DOI] [PubMed] [Google Scholar]
  8. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  9. Collo G., North R. A., Kawashima E., Merlo-Pich E., Neidhart S., Surprenant A., Buell G. Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. J Neurosci. 1996 Apr 15;16(8):2495–2507. doi: 10.1523/JNEUROSCI.16-08-02495.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Evans R. J., Lewis C., Buell G., Valera S., North R. A., Surprenant A. Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). Mol Pharmacol. 1995 Aug;48(2):178–183. [PubMed] [Google Scholar]
  11. Evans R. J., Lewis C., Virginio C., Lundstrom K., Buell G., Surprenant A., North R. A. 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):413–422. doi: 10.1113/jphysiol.1996.sp021777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grussenmeyer T., Scheidtmann K. H., Hutchinson M. A., Eckhart W., Walter G. Complexes of polyoma virus medium T antigen and cellular proteins. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7952–7954. doi: 10.1073/pnas.82.23.7952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hollmann M., Maron C., Heinemann S. N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1. Neuron. 1994 Dec;13(6):1331–1343. doi: 10.1016/0896-6273(94)90419-7. [DOI] [PubMed] [Google Scholar]
  14. Holmgren M., Liu Y., Xu Y., Yellen G. On the use of thiol-modifying agents to determine channel topology. Neuropharmacology. 1996;35(7):797–804. doi: 10.1016/0028-3908(96)00129-3. [DOI] [PubMed] [Google Scholar]
  15. Hong K., Driscoll M. A transmembrane domain of the putative channel subunit MEC-4 influences mechanotransduction and neurodegeneration in C. elegans. Nature. 1994 Feb 3;367(6462):470–473. doi: 10.1038/367470a0. [DOI] [PubMed] [Google Scholar]
  16. Huang M., Chalfie M. Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans. Nature. 1994 Feb 3;367(6462):467–470. doi: 10.1038/367467a0. [DOI] [PubMed] [Google Scholar]
  17. Karlin A. Structure of nicotinic acetylcholine receptors. Curr Opin Neurobiol. 1993 Jun;3(3):299–309. doi: 10.1016/0959-4388(93)90121-e. [DOI] [PubMed] [Google Scholar]
  18. Kuner T., Wollmuth L. P., Karlin A., Seeburg P. H., Sakmann B. Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines. Neuron. 1996 Aug;17(2):343–352. doi: 10.1016/s0896-6273(00)80165-8. [DOI] [PubMed] [Google Scholar]
  19. Kürz L. L., Zühlke R. D., Zhang H. J., Joho R. H. Side-chain accessibilities in the pore of a K+ channel probed by sulfhydryl-specific reagents after cysteine-scanning mutagenesis. Biophys J. 1995 Mar;68(3):900–905. doi: 10.1016/S0006-3495(95)80266-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lewis C., Neidhart S., Holy C., North R. A., Buell G., Surprenant A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature. 1995 Oct 5;377(6548):432–435. doi: 10.1038/377432a0. [DOI] [PubMed] [Google Scholar]
  21. Lingueglia E., Champigny G., Lazdunski M., Barbry P. Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel. Nature. 1995 Dec 14;378(6558):730–733. doi: 10.1038/378730a0. [DOI] [PubMed] [Google Scholar]
  22. MacKinnon R. Pore loops: an emerging theme in ion channel structure. Neuron. 1995 May;14(5):889–892. doi: 10.1016/0896-6273(95)90327-5. [DOI] [PubMed] [Google Scholar]
  23. McLaughlin J. T., Hawrot E., Yellen G. Covalent modification of engineered cysteines in the nicotinic acetylcholine receptor agonist-binding domain inhibits receptor activation. Biochem J. 1995 Sep 15;310(Pt 3):765–769. doi: 10.1042/bj3100765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. North R. A. Families of ion channels with two hydrophobic segments. Curr Opin Cell Biol. 1996 Aug;8(4):474–483. doi: 10.1016/s0955-0674(96)80023-8. [DOI] [PubMed] [Google Scholar]
  25. Numa S. A molecular view of neurotransmitter receptors and ionic channels. Harvey Lect. 1987;83:121–165. [PubMed] [Google Scholar]
  26. Rossier B. C., Canessa C. M., Schild L., Horisberger J. D. Epithelial sodium channels. Curr Opin Nephrol Hypertens. 1994 Sep;3(5):487–496. doi: 10.1097/00041552-199409000-00003. [DOI] [PubMed] [Google Scholar]
  27. Stauffer D. A., Karlin A. Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. Biochemistry. 1994 Jun 7;33(22):6840–6849. doi: 10.1021/bi00188a013. [DOI] [PubMed] [Google Scholar]
  28. Surprenant A., Buell G., North R. A. P2X receptors bring new structure to ligand-gated ion channels. Trends Neurosci. 1995 May;18(5):224–229. doi: 10.1016/0166-2236(95)93907-f. [DOI] [PubMed] [Google Scholar]
  29. Waldmann R., Champigny G., Lazdunski M. Functional degenerin-containing chimeras identify residues essential for amiloride-sensitive Na+ channel function. J Biol Chem. 1995 May 19;270(20):11735–11737. doi: 10.1074/jbc.270.20.11735. [DOI] [PubMed] [Google Scholar]
  30. Yang N., Horn R. Evidence for voltage-dependent S4 movement in sodium channels. Neuron. 1995 Jul;15(1):213–218. doi: 10.1016/0896-6273(95)90078-0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES