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P2X2 receptor subunit interfaces are missense variant hotspots where mutations tend to increase apparent ATP affinity

View ORCID ProfileFederica Gasparri, View ORCID ProfileDebayan Sarkar, Sarune Bielickaite, View ORCID ProfileMette Homann Poulsen, View ORCID ProfileAlexander Sebastian Hauser, View ORCID ProfileStephan Alexander Pless
doi: https://doi.org/10.1101/2021.03.26.436616
Federica Gasparri
Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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Debayan Sarkar
Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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Sarune Bielickaite
Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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Mette Homann Poulsen
Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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Alexander Sebastian Hauser
Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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  • For correspondence: stephan.pless@sund.ku.dk alexander.hauser@sund.ku.dk
Stephan Alexander Pless
Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
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  • For correspondence: stephan.pless@sund.ku.dk alexander.hauser@sund.ku.dk
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ABSTRACT

Background and Purpose P2X receptors (P2XRs) are trimeric ligand-gated ion channels (LGICs) that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes can assemble as homo- or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2XRs is well established, but little is known about the spectrum and prevalence of human genetic variations and the functional implications in specific domains.

Experimental Approach Here we examine the impact of P2X2 receptor (P2X2R) inter-subunit interface missense variants identified in the human population or through structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches.

Key results We demonstrate that predicted ECD inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity.

Conclusions and Implications We provide the first structural mapping of the mutational burden across the human population in a LGIC and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other LGICs, P2X2Rs have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

Bullet point summary

  • ‘What is already known’:

    • P2X2 receptors are ATP-activated ion channels implicated in hearing and nociceptice pathways

  • ‘What this study adds’:

    • A structural mapping of missense variants observed in the human population

    • We identify the intersubunit-interface as a variant hotspot and decipher functional impact of mutations

  • ‘Clinical significance’:

    • The development of both inhibitors and activators of P2X2 receptor function may be required

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Availability of data: The data that support the findings of this study are available from the corresponding author upon reasonable request.

  • FUNDING INFORMATION: We acknowledge the Lundbeck Foundation (R139-2012-12390; SAP - R278-2018-180; ASH), Carlsberg Foundation (2013-01-0439; SAP), Beckett Foundation (39414/42389; SAP), Hartmann Foundation (R73-A27283; SAP), Hørslev Foundation (203866; SAP).

  • Minor text edits

  • Abbreviations

    ATP
    adenosine 5′-triphosphate
    PDB
    protein data bank
    CADD
    Combined Annotation-Dependent Depletion
    CRC
    concentration-response curve
    ECD
    extracellular domain
    gnomAD
    Genome Aggregation Database
    ICD
    intracellular domain
    LGIC
    ligand-gated ion channels
    MAFFT
    Multiple Alignment using Fast Fourier Transform
    P2XR
    P2X receptors
    PBS
    phosphate-buffered saline
    PISA
    Protein, Interfaces, Structures and Assemblies
    TEVC
    two-electrode voltage clamp
    TMD
    transmembrane domain
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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    Posted January 17, 2022.
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    P2X2 receptor subunit interfaces are missense variant hotspots where mutations tend to increase apparent ATP affinity
    Federica Gasparri, Debayan Sarkar, Sarune Bielickaite, Mette Homann Poulsen, Alexander Sebastian Hauser, Stephan Alexander Pless
    bioRxiv 2021.03.26.436616; doi: https://doi.org/10.1101/2021.03.26.436616
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    P2X2 receptor subunit interfaces are missense variant hotspots where mutations tend to increase apparent ATP affinity
    Federica Gasparri, Debayan Sarkar, Sarune Bielickaite, Mette Homann Poulsen, Alexander Sebastian Hauser, Stephan Alexander Pless
    bioRxiv 2021.03.26.436616; doi: https://doi.org/10.1101/2021.03.26.436616

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