Beschreibung:
<jats:p>Mammalian P2X receptors contain 10 conserved cysteine residues
in their ectodomains, which form five disulfide bonds (SS1-5).
Here, we analyzed the relevance of these disulfide pairs in rat
P2X4 receptor function by replacing one or both cysteines with
alanine or threonine, expressing receptors in HEK293 cells and
studying their responsiveness to ATP in the absence and
presence of ivermectin, an allostenic modulator of these
channels. Response to ATP was not altered when both cysteines
forming the SS3 bond (C132-C159) were replaced with
threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149)
and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs
with threonines resulted in decreased sensitivity to ATP and
faster deactivation times. The maximum current amplitude was
reduced in SS2, SS4 and SS5 double mutants and could be
partially rescued by ivermectin in SS2 and SS5 double mutants.
This response pattern was also observed in numerous single
residue mutants, but receptor function was not affected when
the 217 cysteine was replaced with threonine or arginine or when
the 261 cysteine was replaced with alanine. These results
suggest that the SS1, SS2 and SS4 bonds contribute substantially
to the structure of the ligand binding pocket, while the SS5 bond
located towards the transmembrane domain contributes to
receptor gating.</jats:p>