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Conformational flexibility of the agonist binding jaw of the human P 2 X 3 receptor is a prerequisite for channel opening
Author(s) -
Kowalski M,
Hausmann R,
Dopychai A,
Grohmann M,
Franke H,
Nieber K,
Schmalzing G,
Illes P,
Riedel T
Publication year - 2014
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 - 10.1111/bph.12830
Subject(s) - agonist , cysteine , xenopus , protein subunit , receptor , biophysics , chemistry , hek 293 cells , binding site , gating , ion channel , mutant , microbiology and biotechnology , biochemistry , biology , gene , enzyme
Background and Purpose It is assumed that ATP induces closure of the binding jaw of ligand‐gated P 2 X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP ‐induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. Experimental Approach A molecular model of the pain‐relevant human (h)P 2 X 3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP . Key Results A series of cysteine double mutant hP 2 X 3 receptors, expressed in HEK 293 cells or X enopus laevis oocytes, exhibited depressed current responses to α,β‐methylene ATP (α,β‐ meATP ) due to the formation of spontaneous inter‐subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β‐ meATP transmembrane current. Amino‐reactive fluorescence labelling of the H is‐tagged hP 2 X 3 receptor and its mutants expressed in HEK 293 or X . laevis oocytes demonstrated the formation of inter‐subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. Conclusions and Implications In conclusion, spontaneous tightening of the binding jaw of the hP 2 X 3 receptor by inter‐subunit cross‐linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist‐evoked currents without interfering with binding, subunit assembly or trafficking.