Abstract
Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X4 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 P2X4-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 Gln36, Leu40, Val43, Val47, Trp50, Asn338, Gly342, Leu346, Ala349, and Ile356 mutants. The substitution-sensitive Arg33 and Cys353 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 Met31, Tyr42, Gly45, Val49, Gly340, Leu343, Ala344, Gly347, Thr350, Asp354, and Val357 map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.
Similar content being viewed by others
References
Burkhart CN (2000) Ivermectin: an assessment of its pharmacology, microbiology and safety. Vet Hum Toxicol 42:30–35
Albers-Schonberg G, Arison BH, Chabala JC, Douglas AW, Eskola P, Fisher MH, Lusi A, Mrozik H, Smith JL, Tolman RL (1981) Avermectins. Structure determination. J Am Chem Soc 103:4216–4221
Springer JP, Arison BH, Hirshfield JM, Hoogsteen K (1981) The absolute stereochemistry and confromation of avermectin B2a. J Am Chem Soc 103:4221–4224
Hu X, Gu J, Chen L, HuangPu Y (1998) [Studies on the crystal structure of ivermectin (H2B1a)]. Yao Xue Xue Bao 33:449–452
Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LH, Schaeffer JM, Arena JP (1994) Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans. Nature 371:707–711
Dent JA, Davis MW, Avery L (1997) avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans. EMBO J 16:5867–5879
Ikeda T (2003) Pharmacological effects of ivermectin, an antiparasitic agent for intestinal strongyloidiasis: its mode of action and clinical efficacy. Nippon Yakurigaku Zasshi 122:527–538
Sigel E, Baur R (1987) Effect of avermectin B1a on chick neuronal γ-aminobutyrate receptor channels expressed in Xenopus oocytes. Mol Pharmacol 32:749–752
Krusek J, Zemková H (1994) Effect of ivermectin on γ-aminobutyric acid-induced chloride currents in mouse hippocampal embryonic neurones. Eur J Pharmacol 259:121–128
Shan Q, Haddrill JL, Lynch JW (2001) Ivermectin, an unconventional agonist of the glycine receptor chloride channel. J Biol Chem 276:12556–12564
Krause RM, Buisson B, Bertrand S, Corringer PJ, Galzi JL, Changeux JP, Bertrand D (1998) Ivermectin: a positive allosteric effector of the α7 neuronal nicotinic acetylcholine receptor. Mol Pharmacol 53:283–294
Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA (1999) Allosteric control of gating and kinetics at P2X4 receptor channels. J Neurosci 19:7289–7299
Priel A, Silberberg SD (2004) Mechanism of ivermectin facilitation of human P2X4 receptor channels. J Gen Physiol 123:281–293
Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492
Nicke A, Baumert HG, Rettinger J, Eichele A, Lambrecht G, Mutschler E, Schmalzing G (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J 17:3016–3028
Barrera NP, Ormond SJ, Henderson RM, Murrell-Lagnado RD, Edwardson JM (2005) Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize. J Biol Chem 280:10759–10765
North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067
Khakh BS, North RA (2006) P2X receptors as cell-surface ATP sensors in health and disease. Nature 442:527–532
Jelinkova I, Yan Z, Liang Z, Moonat S, Teisinger J, Stojilkovic SS, Zemkova H (2006) Identification of P2X4 receptor-specific residues contributing to the ivermectin effects on channel deactivation. Biochem Biophys Res Commun 349:619–625
Zemkova H, Yan Z, Liang Z, Jelinkova I, Tomic M, Stojilkovic SS (2007) Role of aromatic and charged ectodomain residues in the P2X4 receptor functions. J Neurochem 102:1139–1150
He ML, Zemkova H, Stojilkovic SS (2003) Dependence of purinergic P2X receptor activity on ectodomain structure. J Biol Chem 278:10182–10188
Silberberg SD, Li M, Swartz KJ (2007) Ivermectin interaction with transmembrane helices reveals widespread rearrangements during opening of P2X receptor channels. Neuron 54:263–274
Rassendren F, Buell G, Newbolt A, North RA, Surprenant A (1997) Identification of amino acid residues contributing to the pore of a P2X receptor. Embo J 16:3446–3454
Egan TM, Haines WR, Voigt MM (1998) A domain contributing to the ion channel of ATP-gated P2X2 receptors identified by the substituted cysteine accessibility method. J Neurosci 18:2350–2359
Jiang LH, Rassendren F, Spelta V, Surprenant A, North RA (2001) Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X2 receptor. J Biol Chem 276:14902–14908
Li Z, Migita K, Samways DS, Voigt MM, Egan TM (2004) Gain and loss of channel function by alanine substitutions in the transmembrane segments of the rat ATP-gated P2X2 receptor. J Neurosci 24:7378–7386
Khakh BS, Egan TM (2005) Contribution of transmembrane regions to ATP-gated P2X2 channel permeability dynamics. J Biol Chem 280:6118–6129
Silberberg SD, Chang TH, Swartz KJ (2005) Secondary structure and gating rearrangements of transmembrane segments in rat P2X4 receptor channels. J Gen Physiol 125:347–359
Agboh KC, Webb TE, Evans RJ, Ennion SJ (2004) Functional characterization of a P2X receptor from Schistosoma mansoni. J Biol Chem 279:41650–42657
Hassinen T, Perakyla M (2001) New energy terms for reduced protein models implemented in an off-lattice force field. J Comput Chem 22:1229–1242
Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723
Colquhoun D (1998) Binding, gating, affinity and efficacy: the interpretation of structure–activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125:924–947
Zemkova H, He ML, Koshimizu TA, Stojilkovic SS (2004) Identification of ectodomain regions contributing to gating, deactivation, and resensitization of purinergic P2X receptors. J Neurosci 24:6968–6978
Haines WR, Voigt MM, Migita K, Torres GE, Egan TM (2001) On the contribution of the first transmembrane domain to whole-cell current through an ATP-gated ionotropic P2X receptor. J Neurosci 21:5885–5892
Yan Z, Liang Z, Obsil T, Stojilkovic SS (2006) Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate. J Biol Chem 281:32649–32659
Samways DS, Migita K, Li Z, Egan TM (2008) On the role of the first transmembrane domain in cation permeability and flux of the ATP-gated P2X2 receptor. J Biol Chem 283:5110–5117
Rettinger J, Schmalzing G (2004) Desensitization masks nanomolar potency of ATP for the P2X1 receptor. J Biol Chem 279:6426–6433
Migita K, Haines WR, Voigt MM, Egan TM (2001) Polar residues of the second transmembrane domain influence cation permeability of the ATP-gated P2X2 receptor. J Biol Chem 276:30934–30941
Acknowledgments
This study was supported by the Intramural Research Program of the NICHD, NIH, the Grant Agency of the Czech Republic (305/07/0681 and 303/07/0915), the Internal Grant Agency of Academy of Sciences (IAA5011408 and IAA500110702, Research Project AVOZ 50110509), and the Centrum for Neuroscience (Research Project LC554).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplemental Fig. 1
The time required for solution exchange was measured by recording K+ current using standard patch pipette filled with intracellular solution positioned about 500 μm from the common outlet of the application system. Altered KCl concentrations generated a current (gray trace) with the rates of 130 and 220 ms (black lines) for the wash-in and washout periods, respectively. (PDF 78.2 KB)
Rights and permissions
About this article
Cite this article
Jelínkova, I., Vávra, V., Jindrichova, M. et al. Identification of P2X4 receptor transmembrane residues contributing to channel gating and interaction with ivermectin. Pflugers Arch - Eur J Physiol 456, 939–950 (2008). https://doi.org/10.1007/s00424-008-0450-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00424-008-0450-4