title: Identification of P2X(4) receptor transmembrane residues contributing to channel gating and interaction with ivermectin.
abstract: Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X(4) receptor-channel function by interacting with residues of transmembrane (TM) helices in the open conformation state. In this paper, we used cysteine-scanning mutagenesis of rat P2X(4)-TMs to identify and map residues of potential importance for channel gating and interaction with IVM. The receptor function was unchanged by mutations in 29 different residues, and among them, the IVM effects were altered in Gln(36), Leu(40), Val(43), Val(47), Trp(50), Asn(338), Gly(342), Leu(346), Ala(349), and Ile(356) mutants. The substitution-sensitive Arg(33) and Cys(353) mutants could also be considered as IVM-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both TMs, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the IVM-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met(31), Tyr(42), Gly(45), Val(49), Gly(340), Leu(343), Ala(344), Gly(347), Thr(350), Asp(354), and Val(357) map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.
authors.affiliation: Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology of the Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20, Prague 4, Czech Republic.
authors.forename: Irena
authors.initials: I
authors.lastname: Jelínkova
authors.forename: Vojtech
authors.initials: V
authors.lastname: Vávra
authors.forename: Marie
authors.initials: M
authors.lastname: Jindrichova
authors.forename: Tomas
authors.initials: T
authors.lastname: Obsil
authors.forename: Hana W
authors.initials: HW
authors.lastname: Zemkova
authors.forename: Hana
authors.initials: H
authors.lastname: Zemkova
authors.forename: Stanko S
authors.initials: SS
authors.lastname: Stojilkovic
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000977:Antiparasitic Agents / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating; D007559:Ivermectin / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4
chemical_list: D000977:Antiparasitic Agents; C548226:P2RX4 protein, human; C548228:P2rx4 protein, rat; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D007559:Ivermectin