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Interaction between cysteines introduced into each transmembrane domain of the rat P2X 2 receptor
Author(s) -
Spelta Valeria,
Jiang LinHua,
Bailey R Jayne,
Surprenant Annmarie,
North R Alan
Publication year - 2003
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0705018
Subject(s) - dithiothreitol , chemistry , transmembrane domain , receptor , ion channel , mutagenesis , biophysics , hek 293 cells , transmembrane protein , stereochemistry , biochemistry , mutation , biology , enzyme , gene
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. 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. 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. Ethylammonium‐methylthiosulfonate inhibited currents at both F44C and L338C, but not at F44C/L338C. It reversed the rapid current deactivation at F44C/L338C. The results suggest that a methanethiosulfonate binding to L338C prevents binding to F44C; this might indicate proximity of these two residues.British Journal of Pharmacology (2003) 138 , 131–136. doi: 10.1038/sj.bjp.0705018