Open AccessRoles of conserved ectodomain cysteines of the rat P2X4 purinoreceptor in agonist binding and channel gating
Author(s) -
MB Rokic,
Vendula Tvrdoňová,
Vojtěch Vávra,
Marie Jindřichová,
Tomáš Obšil,
SS Stojilkovic,
Hana Zemková
Publication year - 2010
Publication title -
physiological research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.647
H-Index - 70
eISSN - 1802-9973
pISSN - 0862-8408
DOI - 10.33549/physiolres.931979
Subject(s) - cysteine , alanine , threonine , chemistry , gating , mutant , transmembrane domain , receptor , biochemistry , serine , arginine , amino acid , allosteric regulation , biophysics , stereochemistry , biology , phosphorylation , enzyme , gene
Mammalian P2X receptors contain 10 conserved cysteine residuesin their ectodomains, which form five disulfide bonds (SS1-5).Here, we analyzed the relevance of these disulfide pairs in ratP2X4 receptor function by replacing one or both cysteines withalanine or threonine, expressing receptors in HEK293 cells andstudying their responsiveness to ATP in the absence andpresence of ivermectin, an allostenic modulator of thesechannels. Response to ATP was not altered when both cysteinesforming the SS3 bond (C132-C159) were replaced withthreonines. Replacement of SS1 (C116-C165), SS2 (C126-C149)and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairswith threonines resulted in decreased sensitivity to ATP andfaster deactivation times. The maximum current amplitude wasreduced in SS2, SS4 and SS5 double mutants and could bepartially rescued by ivermectin in SS2 and SS5 double mutants.This response pattern was also observed in numerous singleresidue mutants, but receptor function was not affected whenthe 217 cysteine was replaced with threonine or arginine or whenthe 261 cysteine was replaced with alanine. These resultssuggest that the SS1, SS2 and SS4 bonds contribute substantiallyto the structure of the ligand binding pocket, while the SS5 bondlocated towards the transmembrane domain contributes toreceptor gating.