Conserved extracellular cysteines differentially regulate the inhibitory effect of ethanol in rat P2X 4 receptors Chu-Li Yi a,1 , Yu-Wei Liu a,1 , Ke-Ming Xiong b , Randall R. Stewart b , Robert W. Peoples c , Xiang Tian a , Li Zhou a , Yong-Xun Ai a , Zhi-Wang Li a , Qin-Weng Wang d, * , Chao-Ying Li a, * a Wuhan Institute of Neuroscience & Drug Research, Jianghan University, Wuhan 430056, PR China b Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA c Department of Biomedical Sciences, Marquette University, Milwaukee, WI 53201, USA d Research Center of Behavioral Science, Medical School of Ningbo University, Ningbo 315211, PR China article info Article history: Received 28 January 2009 Available online 11 February 2009 Keywords: P2X P2X 4 receptor Cysteine Disulfide bond Mutation Ethanol Competitive inhibition Noncompetitive inhibition abstract 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 P2X 4 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 EC 50 value. For the C116A, C126A, C149A, and C165A mutants, 100 mM ethanol did not significantly affect the current acti- vated by an EC 40 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 EC 50 value of ATP, thus exhib- iting a noncompetitive-type inhibition. The results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the inhibition of the rat P2X 4 receptor by ethanol. Ó 2009 Elsevier Inc. All rights reserved. P2X receptors are ligand-gated ion channels that are activated by extracellular ATP, and a growing body of evidence suggests that P2X receptors have important functions in the central and periph- eral nervous systems. P2X receptors are widely distributed in the central nervous system, including cerebral cortex, hippocampus, thalamus, hypothalamus, midbrain, cerebellum, and spinal cord, and in sensory and autonomic ganglia in the peripheral nervous system [1–3]. Activation of postsynaptic P2X receptors elicits excitatory postsynaptic potentials in both central and peripheral neurons [2,3], and activation of presynaptic P2X receptors en- hances release of neurotransmitters, including glutamate, c-ami- nobutyric acid, noradrenaline, and acetylcholine [2,3]. To date, seven P2X receptor subunits, designated P2X 1 to P2X 7 , have been cloned and these subunits have been found to be widely distrib- uted in the nervous system [1–3]. Among the seven cloned P2X receptors, the P2X 4 subunit has been found to be the most abun- dant P2X receptor expressed in brain [1–3]. Previous studies have revealed that pharmacological concentra- tions of ethanol can differentially regulate the function of P2X receptors. For instance, ethanol inhibits ATP-activated current in bullfrog dorsal root ganglion neurons [4–6], freshly isolated adult rat hippocampal CA1 neurons [7], and rat recombinant P2X 2 [8] and P2X 4 receptors [8–11], and potentiates ATP-activated current mediated by recombinant rat P2X 3 receptors [10,12]. Only a few recent investigations have begun to explore the molecular basis for the differential regulation of P2X receptor function by ethanol. It has been found that in the rat P2X 4 receptor, mutations of histi- dines 140 and 286 do not significantly alter ethanol inhibition of ATP-activated current, but mutation of 241 alters the mechanism by which ethanol inhibits P2X 4 receptor function: from apparently competitive to noncompetitive [11]. In addition, a recent study using chimeric strategies found that ectodomain segments at TM interfaces played key roles in determining qualitative and quanti- tative responses to ethanol of P2X 2 and P2X 3 receptors [12]. How- ever, more information is required to better understand how the molecular structure of P2X receptors determines the effect of ethanol on these receptor-channels. In the present study, we inves- tigated whether conserved cysteine residues in the extracellular loop of rat P2X 4 receptors are involved in the regulation of ethanol on these receptors. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.02.018 * Corresponding authors. Fax: +86 27 84225827 (C.-Y. Li); +86 574 87608638 (Q.-W. Wang). E-mail addresses: Chuli_yi@hotmail.com (C.-L. Yi), liu-yuwei@163.com (Y.-W. Liu), wangqinwen@nbu.edu.cn (Q.-W. Wang), licwhindr@gmail.com (C.-Y. Li). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications 381 (2009) 102–106 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc