Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states

  1. Hans Moldenhauer
  2. Ignacio Díaz-Franulic
  3. Horacio Poblete
  4. David Naranjo  Is a corresponding author
  1. Universidad de Valparaíso, Chile
  2. Universidad de Talca, Chile

Abstract

In-silico and in-vitro studies have made progress in understanding protein-protein complexes formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Data availability

Data used for Figures 2 to 7 is available in dryad.org

The following data sets were generated

Article and author information

Author details

  1. Hans Moldenhauer

    Instituto de Neurociencia, Universidad de Valparaíso, Valpararaíso, Chile
    Competing interests
    The authors declare that no competing interests exist.
  2. Ignacio Díaz-Franulic

    Instituto de Neurociencia, Universidad de Valparaíso, Valparaíso, Chile
    Competing interests
    The authors declare that no competing interests exist.
  3. Horacio Poblete

    Center for Bioinformatics and Molecular Simulations, Universidad de Talca, Talca, Chile
    Competing interests
    The authors declare that no competing interests exist.
  4. David Naranjo

    Instituto de Neurociencia, Universidad de Valparaíso, Valparaíso, Chile
    For correspondence
    david.naranjo@uv.cl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3482-5126

Funding

Fondo Nacional de Desarrollo Científico y Tecnológico (3160321)

  • Hans Moldenhauer

Fondo Nacional de Desarrollo Científico y Tecnológico (3170599)

  • Ignacio Díaz-Franulic

Fondo Nacional de Desarrollo Científico y Tecnológico (1171155)

  • Horacio Poblete

Ministerio de Economía, Fomento y Turismo (MiNICAD)

  • Horacio Poblete

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

Reviewing Editor

  1. Leon D Islas, Universidad Nacional Autónoma de México, Mexico

Version history

  1. Received: February 18, 2019
  2. Accepted: July 4, 2019
  3. Accepted Manuscript published: July 4, 2019 (version 1)
  4. Version of Record published: July 26, 2019 (version 2)

Copyright

© 2019, Moldenhauer et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,445
    Page views
  • 176
    Downloads
  • 7
    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. Hans Moldenhauer
  2. Ignacio Díaz-Franulic
  3. Horacio Poblete
  4. David Naranjo
(2019)
Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states
eLife 8:e46170.
https://doi.org/10.7554/eLife.46170

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Riham Ayoubi, Joel Ryan ... Carl Laflamme
    Research Advance

    Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50–75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Rui-Qiu Yang, Yong-Hong Chen ... Cheng-Gang Zou
    Research Article

    An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to maintain fitness remains largely unknown. In Caenorhabditis elegans, Escherichia coli, which is its bacterial diet, proliferates in its intestinal lumen during aging. Here, we demonstrate that progressive intestinal proliferation of E. coli activates the transcription factor DAF-16, which is required for maintenance of longevity and organismal fitness in worms with age. DAF-16 up-regulates two lysozymes lys-7 and lys-8, thus limiting the bacterial accumulation in the gut of worms during aging. During dysbiosis, the levels of indole produced by E. coli are increased in worms. Indole is involved in the activation of DAF-16 by TRPA-1 in neurons of worms. Our finding demonstrates that indole functions as a microbial signal of gut dysbiosis to promote fitness of the host.