Molecular determinants for agonist recognition and discrimination in P2X2 receptors

Gasparri, Federica; Wengel, Jesper; Grütter, Thomas; Pless, Stephan A.

doi:10.1085/jgp.201912347

Cited by 13 publications

(22 citation statements)

References 52 publications

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“…Additionally, the approach does not rely on the ribosomal machinery and thus delivers a homogenous protein population by avoiding the potential for non-specific incorporation, which can affect protein manipulation using non-sense suppression approaches [32][33][34][35][36] .…”

Section: Discussionmentioning

confidence: 99%

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Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Khoo

Galleano

Gasparri

et al. 2020

Preprint

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AbstractManipulation of proteins by chemical modification is a powerful way to decipher their function or harness that function for therapeutic purposes. Despite recent progress in ribosome-dependent and semi-synthetic chemical modifications, these techniques sometimes have limitations in the number and type of modifications that can be simultaneously introduced or their application in live eukaryotic cells. Here we present a new approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into soluble and membrane proteins expressed in eukaryotic cells. We insert synthetic peptides into proteins of interest via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating different aspects of GFP, NaV1.5 and P2X2 receptor function. Because the approach can introduce virtually any chemical modification into both intracellular and extracellular regions of target proteins, we anticipate that it will overcome some of the drawbacks of other semi-synthetic or ribosome-dependent methods to engineer proteins.

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Section: Discussionmentioning

confidence: 99%

“…While the location of the ATP-binding site in the extracellular domain is undisputed, the details of how conserved basic side chains coordinate the phosphate tail of ATP remain unclear 33 . However, ribosome-based non-sense suppression approaches, using e.g.…”

Section: Insertion Of Ncaas Into the P2x2 Receptor Extracellular Domainmentioning

confidence: 99%

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Khoo

Galleano

Gasparri

et al. 2020

Preprint

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“…They used molecular dynamics simulations to demonstrate that the loss of an intramolecular hydrogen bond between the 2′ hydroxyl and a γ-phosphate oxygen could destabilize the U-shaped conformation of ATP required for efficient agonist action. In terms of rat P2X2 receptors, Gasparri et al found that for 3′-dATP the EC 50 was increased by a factor of 3, but that 2′-dATP displayed very little agonist activity 33 . Similarly in our study, both 3′-and 2′-dATP displayed reduced activity compared to ATP, with 2′-dATP having the lowest activity, suggesting broad agreement between the agonist profiles of P2X1 and P2X2.…”

Section: Discussionmentioning

confidence: 99%

“…The knowledge of the structure of the P2X binding pocket and its interactions with the primary ligand offers a unique opportunity to understand the mechanistic basis of ligand specificity of the receptor. The ligand specificity of P2X receptors has been explored in previous studies (reviewed in [30][31][32], and most recently in a systematic study of agonist recognition and specificity in rat P2X2 receptors 33 , where a series of ATP analogues with modifications to the base and ribose were examined. In our study we have further expanded the range of molecules tested on rat P2X2 by using a library of 80 adenosine nucleotides.…”

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Drosophila taste neurons as an agonist-screening platform for P2X receptors

Grimes

Griffiths

Pasqualetto

et al. 2020

Sci Rep

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van der Goes van naters 1 ✉ The P2X receptor family of ATP-gated cation channels are attractive drug targets for pain and inflammatory disease, but no subtype-selective agonists, and few partially selective agonists have been described to date. As proof-of-concept for the discovery of novel P2X receptor agonists, here we demonstrate the use of Drosophila taste neurons heterologously expressing rat P2X2 receptors as a screening platform. We demonstrate that wild-type rat P2X2 expressed in Drosophila is fully functional (Atp ec 50 8.7 µM), and that screening of small (2 µl) volumes of a library of 80 adenosine nucleotide analogues is rapid and straightforward. We have determined agonist potency and specificity profiles for rat P2X2 receptors; triphosphate-bearing analogues display broad activity, tolerating a number of substitutions, and diphosphate and monophosphate analogues display very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists AtpγS and AtpαS, we were also able to identify a novel agonist, the synthetic analogue 2-fluoro-ATP, and to confirm its agonist activity on rat P2X2 receptors expressed in human cells. These data validate our Drosophila platform as a useful tool for the analysis of agonist structure-activity relationships, and for the screening and discovery of novel P2X receptor agonists. P2X receptors are ATP-gated ion channels which play diverse roles in physiology and thereby affect a variety of diseases and conditions, including, although not exhaustively: pain sensation 1 , cancer 2 , arthritis 3,4 , osteoporosis 5 , and hypertension 6. Together with the metabotropic P2Y receptors, P2X receptors mediate cellular responses to extracellular ATP 7. ATP functions as an intercellular signaling molecule through release as a (co)transmitter in many synapses, during muscle contraction in skeletal and heart muscle, and by receptor-mediated release in many non-excitable tissues. ATP is also released from cells following damage or stress to the cell membrane by a variety of conductive mechanisms 8 , and is released constitutively at low flux rates 9. Seven P2X genes in mammals (P2RX1-P2RX7), some of which may be alternatively spliced, encode channel subunits that trimerize to form a complex repertoire of homo-and heterotrimeric P2X receptors. Each subunit has two transmembrane domains connected by a large extracellular loop; N-and C-termini are intracellular. P2X genes have also been identified in many other classes of vertebrates and invertebrates, as well as in lower organisms, such as slime mold, where the P2X receptor has an intracellular function in vesicle fusion 10,11. The P2X2 subunit in mammals functions as a homotrimer, or in association with other subunits, in signaling in both the peripheral nervous system (PNS) and central nervous system (CNS). In the PNS, P2X2 receptors of the carotid sinus nerve endings are crucial to carotid body oxygen signal transduction 6. Functions of P2X2 receptors have also been re...

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“…The papers published by the winners attest to the quality of work in JGP . The awards were presented in San Diego by the Society of General Physiology (SGP) President, Crina Nimigean, to Stephan Pless (Cranefield Awardee) for his work on P2X receptors (Gasparri et al, 2019), Sarah Codding (Postdoc Award) for her study of hERG channels (Codding and Trudeau, 2019), and Aaron Bozzi (Student Award) for his work on metal transporters (Bozzi et al, 2019).…”

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confidence: 99%

First steps

Eisner¹

2020

Journal of General Physiology

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Molecular determinants for agonist recognition and discrimination in P2X2 receptors

Abstract: P2X receptors (P2XRs) are ligand-gated cation channels involved in pain and inflammation. Gasparri et al. show that the backbone carbonyl atoms of amino acid residue Thr184 are involved in ligand discrimination, while those of Lys69 contribute mostly to ligand recognition by rat P2X2Rs.

Cited by 13 publications

References 52 publications

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Drosophila taste neurons as an agonist-screening platform for P2X receptors

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