Abstract
P2X receptors are trimeric eukaryotic ATP-gated cation channels. Extracellular ATP—their physiological ligand—is released as a neurotransmitter and in conditions of cell damage such as inflammation, and substantial evidence implicates P2X receptors in diseases including neuropathic pain, cancer, and arthritis. In 2009, the first P2X crystal structure, Danio rerio P2X4 in the apo- state, was published, and this was followed in 2012 by the ATP-bound structure. These structures transformed our understanding of the conformational changes induced by ATP binding and the mechanism of ligand specificity, and enabled homology modeling of mammalian P2X receptors for ligand docking and rational design of receptor modulators. P2X receptors are attractive drug targets, and a wide array of potent, subtype-selective modulators (mostly antagonists) have been developed. In 2016, crystal structures of human P2X3 in complex with the competitive antagonists TNP-ATP and A-317491, and Ailuropoda melanoleuca P2X7 in complex with a series of allosteric antagonists were published, giving fascinating insights into the mechanism of channel antagonism. In this article we not only summarize current understanding of small-molecule modulator binding at P2X receptors, but also use this information in combination with previously published structure-function data and molecular docking experiments, to hypothesize a role for the dorsal fin loop region in differential ATP potency, and describe novel, testable binding conformations for both the semi-selective synthetic P2X7 agonist 2′-(3′)-O-(4-benzoyl)benzoyl ATP (BzATP), and the P2X4-selective positive allosteric modulator ivermectin. We find that the distal benzoyl group of BzATP lies in close proximity to Lys-127, a residue previously implicated in BzATP binding to P2X7, potentially explaining the increased potency of BzATP at rat P2X7 receptors. We also present molecular docking of ivermectin to rat P2X4 receptors, illustrating a plausible binding conformation between the first and second transmembrane domains which not only tallies with previous mutagenesis studies, but would also likely have the effect of stabilizing the open channel structure, consistent with the mode of action of this positive allosteric modulator. From our docking simulations and analysis of sequence homology we propose a series of mutations likely to confer ivermectin sensitivity to human P2X1.
Highlights
INTRODUCTIONOur understanding of the relationship between the structure and the function of the ATP-gated P2X receptor family of ion channels has been transformed by a series of crystal structures, from the first structure of a P2X receptor, that of Danio rerio P2X4.1 (zfP2X4) in the apo-state, published in 2009 (Kawate et al, 2009), via structures of zfP2X4 bound to ATP (Hattori and Gouaux, 2012), a Gulf Coast tick (Amblyomma maculatum) P2X structure (Kasuya et al, 2016), human P2X3 in the apo-, ATP- and antagonist-bound states (Mansoor et al, 2016), zfP2X4 bound to the partial agonist CTP (Kasuya et al, 2017a), to the most recently determined structures of giant panda (Ailuropoda melanoleuca) P2X7 (Karasawa and Kawate, 2016) and chicken P2X7 (Kasuya et al, 2017b)
This article demonstrates how the growing collection of P2X receptor crystal structures, from different species and subtypes, in complex with orthosteric agonists and both orthosteric and allosteric antagonists, has transformed our understanding of how small molecule modulators bind to and influence P2X receptor function, and has enabled us to effectively model novel binding conformations for other ligands based on the available molecular evidence
We have been able to compare the binding of ATP at zfP2X4, Gulf Coast tick P2X and human P2X3, and to analyze the molecular basis for competitive and non-competitive antagonism at human P2X3 and panda and chicken P2X7
Summary
Our understanding of the relationship between the structure and the function of the ATP-gated P2X receptor family of ion channels has been transformed by a series of crystal structures, from the first structure of a P2X receptor, that of Danio rerio P2X4.1 (zfP2X4) in the apo-state, published in 2009 (Kawate et al, 2009), via structures of zfP2X4 bound to ATP (Hattori and Gouaux, 2012), a Gulf Coast tick (Amblyomma maculatum) P2X structure (Kasuya et al, 2016), human P2X3 in the apo-, ATP- and antagonist-bound states (Mansoor et al, 2016), zfP2X4 bound to the partial agonist CTP (Kasuya et al, 2017a), to the most recently determined structures of giant panda (Ailuropoda melanoleuca) P2X7 (Karasawa and Kawate, 2016) and chicken P2X7 (Kasuya et al, 2017b) These impressive achievements, along with their enabling of the interpretation of a large body of prior mutagenesis data (reviewed in Chataigneau et al, 2013; Jiang et al, 2013; Alves et al, 2014; Samways et al, 2014; Grimes and Young, 2015; Habermacher et al, 2016; Kawate, 2017), have led to significant breakthroughs in our understanding of channel architecture, ligand binding, and the mechanisms of channel opening, desensitization and both orthosteric and allosteric antagonism. The effects of these truncations and/or mutations on protein function are significant in some cases; the original zfP2X4 apo-state crystal structure was significantly
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