title: A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors.
abstract: ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration-response curves and ethanol (10-200 mM)-induced changes in ATP EC(10)-gated currents were determined using two-electrode voltage clamp (-70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50-61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321-337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I(max), EC(50), or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, USA.
authors.forename: Maya
authors.initials: M
authors.lastname: Popova
authors.forename: Liana
authors.initials: L
authors.lastname: Asatryan
authors.forename: Olga
authors.initials: O
authors.lastname: Ostrovskaya
authors.forename: Letisha R
authors.initials: LR
authors.lastname: Wyatt
authors.forename: Kaixun
authors.initials: K
authors.lastname: Li
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Alkana
authors.forename: Daryl L
authors.initials: DL
authors.lastname: Davies
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005260:Female; D017354:Point Mutation / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000235:genetics*; D058848:Receptors, Purinergic P2X4; D014982:Xenopus laevis
chemical_list: D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D000431:Ethanol; D000255:Adenosine Triphosphate