Conserved extracellular cysteines differentially regulate the inhibitory effect of ethanol in rat P2X4 receptors.
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.
Yi
Chu-Li
CL
Wuhan Institute of Neuroscience & Drug Research, Jianghan University, Wuhan 430056, PR China. Chuli_yi@hotmail.com
Liu
Yu-Wei
YW
Xiong
Ke-Ming
KM
Stewart
Randall R
RR
Peoples
Robert W
RW
Tian
Xiang
X
Zhou
Li
L
Ai
Yong-Xun
YX
Li
Zhi-Wang
ZW
Wang
Qin-Weng
QW
Li
Chao-Ying
CY
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D000431:Ethanol / Q000633:toxicity*; D009154:Mutation; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4; D014981:Xenopus
C548228:P2rx4 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D000431:Ethanol; D003545:Cysteine