Interaction between cysteines introduced into each transmembrane domain of the rat P2X 2 receptor

Valeria Spelta, Lin Hua Jiang, R. Jayne Bailey, Annmarie Surprenant, R. Alan North

Research output: Contribution to journalArticlepeer-review

Abstract

1. ATP-gated ion channels (P2X receptors) contain two hydrophobic segments that are presumed to span the plasma membrane (TM1 and TM2). Pairs of cysteines were introduced by mutagenesis into the rat P2X 2 receptor, one in TM1 one in TM2, at positions where single substitutions have previously been shown to confer sensitivity to methanethiosulfonates. The receptors were expressed in HEK293 cells; interactions between the cysteines were sought by measuring the effects on ionic currents of dithiothreitol and methanethiosulfonates. 2. Nine pairs gave normally functioning channels: F44C/I328C, F44C/N333C, F44C/L338C, Q37C/I328C, Q37C/N333C, Q37C/T336C, Q37C/L338C, G30C/I328C, G30C/N333C. None formed functionally detectable disulfide bonds. 3. Currents at the F44C/L338C receptor had time course and ATP-sensitivity similar to those for the F44C mutation alone. Methyl-methanethiosulfonate bound to L338C but did not inhibit ionic current. Methyl-methanethiosulfonate inhibited currents at F44C, but not at F44C/L338C. 4. Ethylammonium-methylthiosulfonate inhibited currents at both F44C and L338C, but not at F44C/L338C. It reversed the rapid current deactivation at F44C/L338C. 5. The results suggest that a methanethiosulfonate binding to L338C prevents binding to F44C; this might indicate proximity of these two residues.
Original languageEnglish
Pages (from-to)131-136
Number of pages5
JournalBritish Journal of Pharmacology
Volume138
Issue number1
DOIs
Publication statusPublished - Jan 2003

Keywords

  • Cysteine mutagenesis
  • Electrophysiology
  • Methanethiosulfonates
  • P2X receptors
  • Sulfhydryl group

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