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Vol. 61, Issue 2, 303-311, February 2002
Department of Cell Physiology and Pharmacology, University of
Leicester, Leicester, United Kingdom
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
P2X1 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 P2X1 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.
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