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Molecular architecture of potassium chloride co-transporter KCC2
KCC2 is a neuron specific K(+)-Cl(−) co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological proper...
Autores principales: | , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705597/ https://www.ncbi.nlm.nih.gov/pubmed/29184062 http://dx.doi.org/10.1038/s41598-017-15739-1 |
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author | Agez, Morgane Schultz, Patrick Medina, Igor Baker, David J. Burnham, Matthew P. Cardarelli, Ross A. Conway, Leslie C. Garnier, Kelly Geschwindner, Stefan Gunnarsson, Anders McCall, Eileen J. Frechard, Alexandre Audebert, Stéphane Deeb, Tarek Z. Moss, Stephen J. Brandon, Nicholas J. Wang, Qi Dekker, Niek Jawhari, Anass |
author_facet | Agez, Morgane Schultz, Patrick Medina, Igor Baker, David J. Burnham, Matthew P. Cardarelli, Ross A. Conway, Leslie C. Garnier, Kelly Geschwindner, Stefan Gunnarsson, Anders McCall, Eileen J. Frechard, Alexandre Audebert, Stéphane Deeb, Tarek Z. Moss, Stephen J. Brandon, Nicholas J. Wang, Qi Dekker, Niek Jawhari, Anass |
author_sort | Agez, Morgane |
collection | PubMed |
description | KCC2 is a neuron specific K(+)-Cl(−) co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into “head” and “core” domains connected by a flexible “linker”. Dimers are asymmetrical and display a bent “S-shape” architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2. |
format | Online Article Text |
id | pubmed-5705597 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-57055972017-12-05 Molecular architecture of potassium chloride co-transporter KCC2 Agez, Morgane Schultz, Patrick Medina, Igor Baker, David J. Burnham, Matthew P. Cardarelli, Ross A. Conway, Leslie C. Garnier, Kelly Geschwindner, Stefan Gunnarsson, Anders McCall, Eileen J. Frechard, Alexandre Audebert, Stéphane Deeb, Tarek Z. Moss, Stephen J. Brandon, Nicholas J. Wang, Qi Dekker, Niek Jawhari, Anass Sci Rep Article KCC2 is a neuron specific K(+)-Cl(−) co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into “head” and “core” domains connected by a flexible “linker”. Dimers are asymmetrical and display a bent “S-shape” architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2. Nature Publishing Group UK 2017-11-28 /pmc/articles/PMC5705597/ /pubmed/29184062 http://dx.doi.org/10.1038/s41598-017-15739-1 Text en © The Author(s) 2017 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 Agez, Morgane Schultz, Patrick Medina, Igor Baker, David J. Burnham, Matthew P. Cardarelli, Ross A. Conway, Leslie C. Garnier, Kelly Geschwindner, Stefan Gunnarsson, Anders McCall, Eileen J. Frechard, Alexandre Audebert, Stéphane Deeb, Tarek Z. Moss, Stephen J. Brandon, Nicholas J. Wang, Qi Dekker, Niek Jawhari, Anass Molecular architecture of potassium chloride co-transporter KCC2 |
title | Molecular architecture of potassium chloride co-transporter KCC2 |
title_full | Molecular architecture of potassium chloride co-transporter KCC2 |
title_fullStr | Molecular architecture of potassium chloride co-transporter KCC2 |
title_full_unstemmed | Molecular architecture of potassium chloride co-transporter KCC2 |
title_short | Molecular architecture of potassium chloride co-transporter KCC2 |
title_sort | molecular architecture of potassium chloride co-transporter kcc2 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705597/ https://www.ncbi.nlm.nih.gov/pubmed/29184062 http://dx.doi.org/10.1038/s41598-017-15739-1 |
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