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.

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

Zobrazit více v PubMed

J Gen Physiol. 2004 Mar;123(3):281-93 PubMed

J Biol Chem. 2006 Oct 27;281(43):32649-59 PubMed

J Neurosci. 2004 Aug 4;24(31):6968-78 PubMed

Br J Pharmacol. 1998 Nov;125(5):924-47 PubMed

Mol Pharmacol. 1987 Dec;32(6):749-52 PubMed

J Biol Chem. 2003 Mar 21;278(12):10182-8 PubMed

Nihon Yakurigaku Zasshi. 2003 Dec;122(6):527-38 PubMed

Neuron. 2007 Apr 19;54(2):263-74 PubMed

J Neurosci. 1999 Sep 1;19(17):7289-99 PubMed

J Biol Chem. 2005 Mar 18;280(11):10759-65 PubMed

J Biol Chem. 2004 Oct 1;279(40):41650-7 PubMed

Biochem Biophys Res Commun. 2006 Oct 20;349(2):619-25 PubMed

Mol Pharmacol. 1998 Feb;53(2):283-94 PubMed

Vet Hum Toxicol. 2000 Feb;42(1):30-5 PubMed

Electrophoresis. 1997 Dec;18(15):2714-23 PubMed

Eur J Pharmacol. 1994 Jul 1;259(2):121-8 PubMed

J Biol Chem. 2008 Feb 22;283(8):5110-7 PubMed

J Biol Chem. 2001 Apr 20;276(16):12556-64 PubMed

J Neurochem. 2007 Aug;102(4):1139-50 PubMed

Pharmacol Rev. 1998 Sep;50(3):413-92 PubMed

J Gen Physiol. 2005 Apr;125(4):347-59 PubMed

J Biol Chem. 2005 Feb 18;280(7):6118-29 PubMed

Nature. 2006 Aug 3;442(7102):527-32 PubMed

Physiol Rev. 2002 Oct;82(4):1013-67 PubMed

Nature. 1994 Oct 20;371(6499):707-11 PubMed

J Biol Chem. 2001 Aug 17;276(33):30934-41 PubMed

EMBO J. 1997 Oct 1;16(19):5867-79 PubMed

EMBO J. 1997 Jun 16;16(12):3446-54 PubMed

J Biol Chem. 2001 May 4;276(18):14902-8 PubMed

EMBO J. 1998 Jun 1;17(11):3016-28 PubMed

J Neurosci. 1998 Apr 1;18(7):2350-9 PubMed

J Biol Chem. 2004 Feb 20;279(8):6426-33 PubMed

J Neurosci. 2004 Aug 18;24(33):7378-86 PubMed

Yao Xue Xue Bao. 1998 Jun;33(6):449-52 PubMed

J Neurosci. 2001 Aug 15;21(16):5885-92 PubMed

Allosteric regulation of the P2X4 receptor channel pore dilation

Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains

Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors