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

François Rassendren, Gary Buell, Alison Newbolt, R. Alan North, Annmarie Surprenant

Research output: Contribution to journalArticlepeer-review


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'.
Original languageEnglish
Pages (from-to)3446-3454
Number of pages8
JournalEMBO Journal
Issue number12
Publication statusPublished - 16 Jun 1997


  • Cysteine scanning mutagenesis
  • Ligand-gated channel
  • Methanethiosulfonate
  • P2X receptors
  • Substituted cysteine accessibility method


Dive into the research topics of 'Identification of amino acid residues contributing to the pore of a P2X receptor'. Together they form a unique fingerprint.

Cite this