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Millisecond dynamics of an unlabeled amino acid transporter
Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538599/ https://www.ncbi.nlm.nih.gov/pubmed/33024106 http://dx.doi.org/10.1038/s41467-020-18811-z |
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author | Matin, Tina R. Heath, George R. Huysmans, Gerard H. M. Boudker, Olga Scheuring, Simon |
author_facet | Matin, Tina R. Heath, George R. Huysmans, Gerard H. M. Boudker, Olga Scheuring, Simon |
author_sort | Matin, Tina R. |
collection | PubMed |
description | Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted Glt(Ph), a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that Glt(Ph) transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution. |
format | Online Article Text |
id | pubmed-7538599 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-75385992020-10-19 Millisecond dynamics of an unlabeled amino acid transporter Matin, Tina R. Heath, George R. Huysmans, Gerard H. M. Boudker, Olga Scheuring, Simon Nat Commun Article Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted Glt(Ph), a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that Glt(Ph) transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution. Nature Publishing Group UK 2020-10-06 /pmc/articles/PMC7538599/ /pubmed/33024106 http://dx.doi.org/10.1038/s41467-020-18811-z Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Matin, Tina R. Heath, George R. Huysmans, Gerard H. M. Boudker, Olga Scheuring, Simon Millisecond dynamics of an unlabeled amino acid transporter |
title | Millisecond dynamics of an unlabeled amino acid transporter |
title_full | Millisecond dynamics of an unlabeled amino acid transporter |
title_fullStr | Millisecond dynamics of an unlabeled amino acid transporter |
title_full_unstemmed | Millisecond dynamics of an unlabeled amino acid transporter |
title_short | Millisecond dynamics of an unlabeled amino acid transporter |
title_sort | millisecond dynamics of an unlabeled amino acid transporter |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538599/ https://www.ncbi.nlm.nih.gov/pubmed/33024106 http://dx.doi.org/10.1038/s41467-020-18811-z |
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