ABSTRACT
One feature of the amino acid sequence of P2X receptors identified from mammalian species, Xenopus laevis and zebrafish is the conservation of ten cysteines in the extracellular loop. Little information is available about the role of these conserved ectodomain cysteines in the function of P2X receptors. Here, we investigated the possibility that ten conserved cysteine residues in the extracellular loop of the rat P2X4 receptor may regulate zinc potentiation of the receptor using a series of individual cysteine to alanine point mutations and functional characterization of recombinant receptors expressed in Xenopus oocytes. For the C116A, C132A, C159A, C165A, C217A and C227A mutants, 10 µM zinc did not significantly affect the current activated by an EC40 concentration of ATP. By contrast, 5 µM zinc shifted the ATP concentration-response curve to the right in a parallel manner for both the C261A and C270A mutants and the magnitudes of those shifts were similar to that of the wildtype receptor. Interestingly, for the C126A and C149A mutants, 5µM zinc potentiated ATP-activated current, but increased the maximal response to ATP by 90% and 81% respectively, without significantly changing the EC50 value of ATP. Thus, these results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the potentiation of the rat P2X4 receptor by zinc.
Subject(s)
Adenosine Triphosphate/administration & dosage , Cysteine/metabolism , Receptors, Purinergic P2X4/metabolism , Zinc/pharmacology , Amino Acid Sequence , Animals , Conserved Sequence , Cysteine/chemistry , Disulfides/chemistry , Dose-Response Relationship, Drug , Female , Oocytes , Point Mutation , Rats , Receptors, Purinergic P2X4/chemistry , Receptors, Purinergic P2X4/genetics , Species Specificity , Xenopus laevisABSTRACT
Relatively little information is available about the molecular mechanism of ethanol inhibition of P2X receptors. Here, we investigated the possibility that 10 conserved cysteine residues in the extracellular loop of the rat P2X4 receptor may regulate ethanol inhibition of the receptor using a series of individual cysteine to alanine point mutations. Each of the mutated receptors generated robust inward current in response to ATP and the mutations produced less than a sixfold change in the ATP EC50 value. For the C116A, C126A, C149A, and C165A mutants, 100 mM ethanol did not significantly affect the current activated by an EC40 concentration of ATP. By contrast, for the C261A and C270A mutants, ethanol inhibited ATP-activated current in a competitive manner similar to that for the wild-type receptor. Interestingly, for the C132A, C159A, C217A, and C227A mutants, ethanol inhibited ATP-activated current, but decreased the maximal response to ATP by 70-75% without significantly changing the EC50 value of ATP, thus exhibiting a noncompetitive-type inhibition. The results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the inhibition of the rat P2X4 receptor by ethanol.
