title: Conserved extracellular cysteines differentially regulate the potentiation produced by Zn2+ in rat P2X4 receptors.
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.
authors.affiliation: Wuhan Institutes of Biomedical Sciences, Jianghan University, Wuhan 430056, PR China. licy.whibs@yahoo.cn
authors.forename: Chao-Ying
authors.initials: CY
authors.lastname: Li
authors.forename: Ke-Ming
authors.initials: KM
authors.lastname: Xiong
authors.forename: Yu-Xiang
authors.initials: YX
authors.lastname: Wu
authors.forename: Yu-Wei
authors.initials: YW
authors.lastname: Liu
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.forename: Randall R
authors.initials: RR
authors.lastname: Stewart
authors.forename: Robert W
authors.initials: RW
authors.lastname: Peoples
authors.forename: Chu-Li
authors.initials: CL
authors.lastname: Yi
mesh_terms: D000255:Adenosine Triphosphate / Q000008:administration & dosage*; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005260:Female; D009865:Oocytes; D017354:Point Mutation; D051381:Rats; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D014982:Xenopus laevis; D015032:Zinc / Q000494:pharmacology*
chemical_list: D004220:Disulfides; D058848:Receptors, Purinergic P2X4; D000255:Adenosine Triphosphate; D015032:Zinc; D003545:Cysteine