Conserved cysteine residues in the extracellular loop of the human P2X(1) receptor form disulfide bonds and are involved in receptor trafficking to the cell surface.
P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteine-alanine mutants in the human P2X(1) receptor. Mutants were expressed in Xenopus laevis oocytes and effects on ATP potency, cell-surface expression, and N-biotinoylaminoethyl methanethiosulfonate (MTSEA-Biotin) labeling of free cysteines were determined. For the majority of single mutants, only a modest decrease (2- to 5-fold) in ATP potency was recorded. For mutants C261A and C270A, the peak current amplitudes were reduced by 93.6 +/- 2.0 and 95.0 +/- 1.0%, respectively; this was a result of low cell-surface expression of these mutant receptors. Wild-type receptors showed no labeling with MTSEA-biotin suggesting that all 10 cysteine residues in the extracellular loop are disulfide-bonded. Mutation of cysteines at positions 126, 132, 149, 159, 217, and 227 resulted in MTSEA-biotinylation of a free cysteine residue created by the disruption of a disulfide bond and provides direct biochemical evidence for at least three disulfide bonds. Based on phenotypic comparisons of single and double cysteine mutants, we propose the following disulfide bond pairs in the human P2X(1) receptor: C117-C165, C126-C149, C132-C159, C217-C227, and C261-C270. None of these bonds are individually essential for channel function. However, trafficking of the receptor to the cell membrane is severely reduced by disruption of the C261-C270 disulfide bond or disruption of C117-C165 together with another bond.
Ennion
Steven J
SJ
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.
Evans
Richard J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D019921:Biotinylation; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004594:Electrophysiology; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes; D017434:Protein Structure, Tertiary; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D019163:Reducing Agents / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D004220:Disulfides; D008565:Membrane Proteins; C419543:N-biotinylaminoethyl methanethiosulfonate; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D019163:Reducing Agents; D008623:Mercaptoethanol; D001710:Biotin; D000255:Adenosine Triphosphate; D003545:Cysteine; D000409:Alanine; D004229:Dithiothreitol