Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Protein semisynthesis underscores role of a conserved lysine in activation and desensitization of acid-sensing ion channels

Debayan Sarkar, Iacopo Galleano, Sofie Yuewei Ou, Gül Refika Uzun, Keith K. Khoo, Gerbrand Jan van der Heden van Noort, View ORCID ProfileStephan Alexander Pless
doi: https://doi.org/10.1101/2023.01.23.525296
Debayan Sarkar
1Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Iacopo Galleano
1Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
2Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sofie Yuewei Ou
1Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gül Refika Uzun
1Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Keith K. Khoo
1Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gerbrand Jan van der Heden van Noort
2Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stephan Alexander Pless
1Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Stephan Alexander Pless
  • For correspondence: stephan.pless@sund.ku.dk
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

SUMMARY

Acid-sensing ion channels (ASICs) are trimeric ligand-gated ion channels that open a cation-conducting pore in response to proton-binding to their extracellular domain (ECD). This activation is typically short-lived, as channel opening is followed by a rapid and spontaneous pore closure due to a process termed fast desensitization. This intrinsic channel closing mechanism is important, because excessive activation of ASICs during periods of prolonged acidosis in conditions such as inflammation and ischemia, has been linked to several pathologies, including pain and stroke. Structural and functional data suggest that a conserved lysine in the ECD (Lys211 in mASIC1a) plays a role in receptor function. However, the precise physico-chemical contributions to activation and fast desensitization are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring side chains invariably changes multiple parameters, such as size, charge, and H-bonding pattern simultaneously. Here, we study the contribution of Lys211 to activation and fast desensitization of mASIC1a using tandem protein-trans splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct extensive optimization efforts to improve apparent splicing yields and subsequently interrogate the function of semisynthetic mASIC1a using two-electrode voltage clamp. Our data show that both charge and side chain length of Lys211 are crucial to activation and fast desensitization of mASIC1a, with the latter being sensitive to even the most subtle mutational disruptions. Our work emphasizes that tPTS can enable atomic-scale interrogations of how side chains contribute to the function of complex membrane proteins.

Competing Interest Statement

The authors have declared no competing interest.

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.
Back to top
PreviousNext
Posted January 24, 2023.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Protein semisynthesis underscores role of a conserved lysine in activation and desensitization of acid-sensing ion channels
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Protein semisynthesis underscores role of a conserved lysine in activation and desensitization of acid-sensing ion channels
Debayan Sarkar, Iacopo Galleano, Sofie Yuewei Ou, Gül Refika Uzun, Keith K. Khoo, Gerbrand Jan van der Heden van Noort, Stephan Alexander Pless
bioRxiv 2023.01.23.525296; doi: https://doi.org/10.1101/2023.01.23.525296
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
Protein semisynthesis underscores role of a conserved lysine in activation and desensitization of acid-sensing ion channels
Debayan Sarkar, Iacopo Galleano, Sofie Yuewei Ou, Gül Refika Uzun, Keith K. Khoo, Gerbrand Jan van der Heden van Noort, Stephan Alexander Pless
bioRxiv 2023.01.23.525296; doi: https://doi.org/10.1101/2023.01.23.525296

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Biophysics
Subject Areas
All Articles
  • Animal Behavior and Cognition (4110)
  • Biochemistry (8813)
  • Bioengineering (6518)
  • Bioinformatics (23456)
  • Biophysics (11788)
  • Cancer Biology (9205)
  • Cell Biology (13318)
  • Clinical Trials (138)
  • Developmental Biology (7433)
  • Ecology (11407)
  • Epidemiology (2066)
  • Evolutionary Biology (15146)
  • Genetics (10433)
  • Genomics (14041)
  • Immunology (9169)
  • Microbiology (22152)
  • Molecular Biology (8808)
  • Neuroscience (47559)
  • Paleontology (350)
  • Pathology (1428)
  • Pharmacology and Toxicology (2491)
  • Physiology (3730)
  • Plant Biology (8079)
  • Scientific Communication and Education (1437)
  • Synthetic Biology (2220)
  • Systems Biology (6037)
  • Zoology (1252)