Analysis of the Mechanosensor Channel Functionality of TACAN
- Howard Hughes Medical Institute, The Rockefeller University, United States
- Rockefeller University, United States
- The Rockefeller University, United States
Abstract
Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods we found that TACAN, at high protein concentrations, produces heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single particle cryo-EM and observe that it forms a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme-A cofactor, confirmed by mass spectrometry. The TACAN protomers are related in 3-dimensional structure to a fatty acid elongase, ELOVL. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.
Data availability
The B-factor sharpened 3D cryo-EM density map and atomic coordinates of wild-type TACAN have been deposited in Worldwide Protein Data Bank (wwPDB) under accession codes 7N0K and EMD-24107.The B-factor sharpened 3D cryo-EM density map and atomic coordinates of His196Ala, His197Ala mutant TACAN have been deposited in Worldwide Protein Data Bank (wwPDB) under accession codes 7N0L and EMD-24108.
Article and author information
Author details
Roderick MacKinnon
Funding
National Institutes of Health (GM43949)
- Roderick MacKinnon
National Institutes of Health (GM109824)
- Brian Chait
National Institutes of Health (GM103314)
- Brian Chait
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Publication history
- Received: June 11, 2021
- Preprint posted: June 12, 2021 (view preprint)
- Accepted: August 6, 2021
- Accepted Manuscript published: August 10, 2021 (version 1)
- Version of Record published: August 19, 2021 (version 2)
Copyright
© 2021, Niu 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.
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Analysis of the Mechanosensor Channel Functionality of TACAN
eLife 10:e71188.
https://doi.org/10.7554/eLife.71188
Further reading
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- Structural Biology and Molecular Biophysics
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 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 hr 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 homodimer for 9 hr 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 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.
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