The ion selectivity filter is not an activation gate in TRPV1-3 channels

  1. Andrés Jara-Oseguera  Is a corresponding author
  2. Katherine E Huffer
  3. Kenton J Swartz  Is a corresponding author
  1. National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Abstract

Activation of TRPV1 channels in sensory neurons results in opening of a cation permeation pathway that triggers the sensation of pain. Opening of TRPV1 has been proposed to involve two gates that appear to prevent ion permeation in the absence of activators: the ion selectivity filter on the external side of the pore and the S6 helices that line the cytosolic half of the pore. Here we measured the access of thiol-reactive ions and compounds across the selectivity filters in rodent TRPV1-3 channels. Although our results are consistent with structural evidence that the selectivity filters in these channels are dynamic, they demonstrate that cations can permeate the ion selectivity filters even when channels are closed. Our results suggest that the selectivity filters in TRPV1-3 channels do not function as activation gates but might contribute to coupling structural rearrangements in the external pore to those in the cytosolic S6 gate.

Data availability

All data generated or analysed during this study are shown in the manuscript and supporting files. The corresponding authors can be contacted if the raw data is required by anyone.

Article and author information

Author details

  1. Andrés Jara-Oseguera

    Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    For correspondence
    andres.jara-oseguera@nih.gov
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5921-9320
  2. Katherine E Huffer

    Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    No competing interests declared.
  3. Kenton J Swartz

    Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    For correspondence
    swartzk@ninds.nih.gov
    Competing interests
    Kenton J Swartz, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3419-0765

Funding

National Institute of Neurological Disorders and Stroke (K99 Pathway to Independence Award)

  • Andrés Jara-Oseguera

National Institute of Neurological Disorders and Stroke (Intramural Research Program NS002945)

  • Kenton J Swartz

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. László Csanády, Semmelweis University, Hungary

Publication history

  1. Received: August 20, 2019
  2. Accepted: November 13, 2019
  3. Accepted Manuscript published: November 14, 2019 (version 1)
  4. Version of Record published: December 2, 2019 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 2,676
    Page views
  • 484
    Downloads
  • 25
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Andrés Jara-Oseguera
  2. Katherine E Huffer
  3. Kenton J Swartz
(2019)
The ion selectivity filter is not an activation gate in TRPV1-3 channels
eLife 8:e51212.
https://doi.org/10.7554/eLife.51212

Further reading

    1. Structural Biology and Molecular Biophysics
    Seoyoon Kim, Daehyo Lee ... Duyoung Min
    Tools and Resources

    Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne (DBCO) cycloaddition and traptavidin binding, enabling the estimation of the folding 'speed limit' of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 h at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane (TM) homodimer for 9 h at 12 pN, and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling (HMM) analysis, respectively. The Kramers rate framework yields a considerably low speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to μs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.

    1. Computational and Systems Biology
    2. Structural Biology and Molecular Biophysics
    Jinyeop Song, Bo-Seong Jeong ... Byung-Ha Oh
    Research Article

    Immunoglobulin G (IgG) antibodies are widely used for diagnosis and therapy. Given the unique dimeric structure of IgG, we hypothesized that, by genetically fusing a homodimeric protein (catenator) to the C-terminus of IgG, reversible catenation of antibody molecules could be induced on a surface where target antigen molecules are abundant, and that it could be an effective way to greatly enhance the antigen-binding avidity. A thermodynamic simulation showed that quite low homodimerization affinity of a catenator, e.g. dissociation constant of 100 μM, can enhance nanomolar antigen-binding avidity to a picomolar level, and that the fold enhancement sharply depends on the density of the antigen. In a proof-of-concept experiment where antigen molecules are immobilized on a biosensor tip, the C-terminal fusion of a pair of weakly homodimerizing proteins to three different antibodies enhanced the antigen-binding avidity by at least 110 or 304 folds from the intrinsic binding avidity. Compared with the mother antibody, Obinutuzumab(Y101L) which targets CD20, the same antibody with fused catenators exhibited significantly enhanced binding to SU-DHL5 cells. Together, the homodimerization-induced antibody catenation would be a new powerful approach to improve antibody applications, including the detection of scarce biomarkers and targeted anticancer therapies.