Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Dokumenter
- Fulltext
Forlagets udgivne version, 3,79 MB, PDF-dokument
The sodium–potassium–chloride transporter NKCC1 of the SLC12 family performs Na+-dependent Cl−- and K+-ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+, K+, and 2 Cl−) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl− binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl−-ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl−-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure–function relationship of NKCC1 with broader implications for other SLC12 family members.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | e110169 |
| Tidsskrift | EMBO Journal |
| Vol/bind | 41 |
| Udgave nummer | 23 |
| Antal sider | 15 |
| ISSN | 0261-4189 |
| DOI | |
| Status | Udgivet - 2022 |
Bibliografisk note
Funding Information:
The authors are grateful for technical assistance to Tina Drejer, Tetyana Klymchuk, Anna Marie Nielsen, Bente Andersen and Anne Lillevang. We want to thank Thomas Boesen, Andreas Bøggild, and Taner Drace from the cryo‐EM facility at Aarhus University for assistance with grid screening and numerous overnight data collections. We are thankful to Milena Timcenko, Jeppe Achton Nielsen, Søren Kirk Amstrup, and Jonathan Juhl for help with data processing and fruitful discussions about cryo‐EM. We want to acknowledge Qi Wu for performing MS on the purified hNKCC1 sample in order to assure that sample of the expressed and purified hNKCC1 construct was correct. We acknowledge Diamond Light Source for access and support of the cryo‐EM facilities at the UK's National Electron Bio‐Imaging Centre (eBIC) under proposal AP27 (BI21404) funded by the Wellcome Trust, MRC, and BBRSC. Work on the project was supported by a PhD fellowship from the Lundbeck Foundation to CN (2015‐3225); the Leducq Foundation (17CVD05), the Novo Nordisk Foundation (NNF21OC0067647, NNF17OC0029724, and NNF19OC0058439), and the Independent Research Fund Denmark to RF; from the Brainstruc center (R155‐2015‐2666 and R328‐2019‐546) and a professorship grant (R310‐2018‐3713) funded by the Lundbeck Foundation and research infrastructure grants from the Carlsberg Foundation (CF15‐0821) and the Danish Ministry for Research and Higher Education (EMBION – 5072‐00025B) to PN.
Publisher Copyright:
©2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.
ID: 323969093