Tightening of the ATP-binding sites induces the opening of P2X receptor channels. EMBO J

Faculté de Pharmacie, Laboratoire de Biophysicochimie des Récepteurs Canaux, UMR 7199 CNRS, Conception et Application de Molécules Bioactives, Université de Strasbourg, Illkirch, France.
The EMBO Journal (Impact Factor: 10.43). 03/2012; 31(9):2134-43. DOI: 10.1038/emboj.2012.75
Source: PubMed


The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

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Available from: Antoine Taly
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    • "These pairs play important roles in ion channel functions, such as ATP binding and ATP induced conformational changes, providing a zinc binding site and stabilizing the conformation of the open channel conformation state [7] [8] [9] [10] [11] [12] [13]. All of these interfaces are exclusively involved in body-to-body, head-to-dorsal-fin and body-to-left-flipper interactions. "
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    ABSTRACT: P2X receptors are trimeric ATP-activated non-selective cation channels. The ATP binding pocket is positioned between two neighboring subunits. Accompanying ligand binding, subunit-subunit contacts are most likely involved in receptor function and drive a conformational change to open the ion permeation pathway. In this way, we sought to determine the function of side chains of the zebrafish P2X4 receptor ectodomain left-flipper-to-dorsal-fin interface residues in ligand binding. By Combining site-directed mutagenesis and electrophysiology methods, we showed that cysteine substitutions of I212, S215, Y216 and L217 resulted in decreased sensitivity to ATP. In addition, the ATP induced current at L217C was completely inhibited by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)), indicating a role for this residue in ATP action. Deletion of residues 285-293 from the zebrafish P2X4 receptor abolished channel function. However, insertion of the same sequence frame into a homologous position of the rat P2X6 receptor did not rescue channel function, suggesting that these residues are necessary but not sufficient for achieving the correct ATP-induced conformation.
    Full-text · Article · Aug 2014 · Neuroscience Letters
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    • "The ATP binding site is ~40 Å from the membrane-spanning segments, which constitute the ionic pore of P2X receptors. The recent ATP-bound crystal structure, and previous studies utilizing normal mode analysis (Du et al., 2012; Jiang et al., 2012a), metal-bridging experiments (Jiang et al., 2012a), electron microscopy (Roberts et al., 2012), and voltage-clamp fluorometry (Lorinczi et al., 2012), have now revealed a plausible activation mechanism that can be dissected into five steps (Figure 4): binding of ATP4− (Li et al., 2013) to a pocket located at the interface between each subunit (first step) leads to the tightening of the head domain relative to the dorsal fin (second step). Because the ribose and adenine base interact hydrophobically with L217 and I232 (chain B), which are part of the dorsal fin, closure of the binding “jaw” induces the upward movement of the dorsal fin. "
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    ABSTRACT: P2X receptors are ATP-gated non-selective cation channels involved in many different physiological processes, such as synaptic transmission, inflammation, and neuropathic pain. They form homo- or heterotrimeric complexes and contain three ATP-binding sites in their extracellular domain. The recent determination of X-ray structures of a P2X receptor solved in two states, a resting closed state and an ATP-bound, open-channel state, has provided unprecedented information not only regarding the three-dimensional shape of the receptor, but also on putative conformational changes that couple ATP binding to channel opening. These data provide a structural template for interpreting the huge amount of functional, mutagenesis, and biochemical data collected during more than fifteen years. In particular, the interfacial location of the ATP binding site and ATP orientation have been successfully confirmed by these structural studies. It appears that ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the opening of the ion channel. These structural data thus represent a firm basis for understanding the activation mechanism of P2X receptors.
    Full-text · Article · Dec 2013 · Frontiers in Cellular Neuroscience
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    • "The structure of the zebrafish P2X4.1R in the open state (Hattori and Gouaux, 2012) and the analysis of cysteine accessibility in the mammalian P2XRs (Kawate et al., 2011; Samways et al., 2011; Jiang et al., 2012; Roberts et al., 2012) provide consistent evidence in support of the notion that the three lateral fenestrations, located just above the plasma membrane and linking to the extracellular vestibule, act as the pathways for ions to enter or exit the aforementioned transmembrane ion-conducting pathway (Figure 3B). The lateral fenestrations contain negatively charged residues, Asp59 and Asp61 in the zebrafish P2X4.1R (corresponding to Arg53 and Glu55 in the human P2X7R; highlighted in blue in Figure 1). "
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    ABSTRACT: The mammalian P2X7 receptors (P2X7Rs), a member of the ionotropic P2X receptor family with distinctive functional properties, play an important part in mediating extracellular ATP signaling in health and disease. A clear delineation of the molecular mechanisms underlying the key receptor properties, such as ATP-binding, ion permeation, and large pore formation of the mammalian P2X7Rs, is still lacking, but such knowledge is crucial for a better understanding of their physiological functions and contributions in diseases and for development of therapeutics. The recent breakthroughs in determining the atomic structures of the zebrafish P2X4.1R in the closed and ATP-bound open states have provided the long-awaited structural information. The human P2RX7 gene is abundant with non-synonymous single nucleotide polymorphisms (NS-SNPs), which generate a repertoire of human P2X7Rs with point mutations. Characterizations of the NS-SNPs identified in patients of various disease conditions and the resulting mutations have informed previously unknown molecular mechanisms determining the mammalian P2X7R functions and diseases. In this review, we will discuss the new insights into such mechanisms provided by structural modeling and recent functional and genetic linkage studies of NS-SNPs.
    Full-text · Article · May 2013 · Frontiers in Pharmacology
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