P2X4 receptor (P2X4R) is a member of a family of ligand-gated ion channels that are activated by extracellular ATP. P2X4Rs are widely distributed throughout the central nervous system and are inhibited by ethanol. Building evidence suggests that ethanol inhibition may play a role in mediating and/or modulating some of the cellular and behavioral effects of ethanol. However, there is a paucity of information regarding the sites of ethanol action on P2XRs. Recent work in our laboratory, utilizing a chimeric strategy in P2X2R/P2X3Rs, suggests that regions in P2X3R ectodomain near the transmembrane (TM) domains and the TM1 domain itself may represent important targets for ethanol. My K01 proposal extends this line of investigation to P2X4Rs by testing the hypothesis that the ectodomain region and the TM1 domain of P2X4Rs contain sites of ethanol action. The primary goals of this project are: 1) Provide extensive hands-on training for myself in cellular, molecular and electrophysiological techniques that will be necessary in order to take the next step towards becoming an independent alcohol researcher;2) To identify important sites of ethanol action in P2X4Rs. Results from the proposed studies will set the stage for new directions of independent investigation for myself in alcohol/purinergic research. To accomplish these goals two Specific Aims are proposed.
Aim 1 investigates the role of the ectodomain and TM1 domain of P2X4Rs in ethanol action using advanced electrophysiological techniques and recombinant, cellular expression systems. In these studies I will utilize alanine scanning mutagenesis of the indicated regions of P2X4Rs to identify key amino acids involved in ethanol action.
Aim 2 investigates the effects of ethanol on P2X4Rs and P2X4-like receptors in dissociated neurons. These later studies will utilize P2X4R-transfected neurons as well as neurons from control and P2X4R-knockout mice. Results of proposed studies will identify ethanol targets in endogenous and recombinant P2X4Rs. The long-term goal of the proposed work is to determine the role of P2X4Rs in mediating and/or modulating behavioral effects of ethanol and where warranted to identify novel therapeutic targets for the treatment of alcoholism. My K01 proposal represents advanced experimental training and new directions in my scientific career and provides me with an opportunity to become an independent alcohol researcher working towards a faculty position at USC School of Pharmacy.
|Asatryan, Liana; Khoja, Sheraz; Rodgers, Kathleen E et al. (2015) Chronic ethanol exposure combined with high fat diet up-regulates P2X7 receptors that parallels neuroinflammation and neuronal loss in C57BL/6J mice. J Neuroimmunol 285:169-79|
|Naito, A; Muchhala, K H; Trang, J et al. (2015) Manipulations of extracellular Loop 2 in ?1 GlyR ultra-sensitive ethanol receptors (USERs) enhance receptor sensitivity to isoflurane, ethanol, and lidocaine, but not propofol. Neuroscience 297:68-77|
|Yardley, Megan M; Neely, Michael; Huynh, Nhat et al. (2014) Multiday administration of ivermectin is effective in reducing alcohol intake in mice at doses shown to be safe in humans. Neuroreport 25:1018-23|
|Wyatt, Letisha R; Finn, Deborah A; Khoja, Sheraz et al. (2014) Contribution of P2X4 receptors to ethanol intake in male C57BL/6 mice. Neurochem Res 39:1127-39|
|Naito, Anna; Muchhala, Karan H; Asatryan, Liana et al. (2014) Glycine and GABA(A) ultra-sensitive ethanol receptors as novel tools for alcohol and brain research. Mol Pharmacol 86:635-46|
|Asatryan, Liana; Yardley, Megan M; Khoja, Sheraz et al. (2014) Avermectins differentially affect ethanol intake and receptor function: implications for developing new therapeutics for alcohol use disorders. Int J Neuropsychopharmacol 17:907-16|
|Bortolato, Marco; Yardley, Megan M; Khoja, Sheraz et al. (2013) Pharmacological insights into the role of P2X4 receptors in behavioural regulation: lessons from ivermectin. Int J Neuropsychopharmacol 16:1059-70|
|Popova, Maya; Trudell, James; Li, Kaixun et al. (2013) Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors. Purinergic Signal 9:621-32|
|Yardley, Megan M; Wyatt, Letisha; Khoja, Sheraz et al. (2012) Ivermectin reduces alcohol intake and preference in mice. Neuropharmacology 63:190-201|
|Asatryan, Liana; Nam, Hyung W; Lee, Moonnoh R et al. (2011) Implication of the purinergic system in alcohol use disorders. Alcohol Clin Exp Res 35:584-94|
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