Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

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Abstract

Cation-chloride-cotransporters (CCCs) catalyze transport of Cl^- with K⁺ and/or Na⁺ across cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. CCCs are targets of clinically important drugs including loop diuretics and their disruption has been implicated in pathophysiology including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the structure of a CCC, the Mus musculus K⁺-Cl^- cotransporter (KCC) KCC4, in lipid nanodiscs determined by cryo-EM. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We identify binding sites for substrate K⁺ and Cl^- ions, demonstrate the importance of key coordinating residues for transporter activity, and provide a structural explanation for varied substrate specificity and ion transport ratio among CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.

Data availability

The final map of KCC4 in MSP1D1 nanodiscs has been deposited to the Electron Microscopy Data Bank under accession code EMD-20807. Atomic coordinates have been deposited in the PDB under ID 6UKN. Original KCC4 in MSP1D1 nanodiscs micrograph movies have been deposited to EMPIAR.

The following data sets were generated

(2019) Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs
EMDB, EMD-20807.

http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20807
(2019) Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs
PDB, 6UKN.

http://www.rcsb.org/structure/6UKN

Article and author information

Author details

Michelle S Reid

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
The authors declare that no competing interests exist.
David M Kern

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
The authors declare that no competing interests exist.
Stephen Graf Brohawn

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

For correspondence
brohawn@berkeley.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-6768-3406

Funding

New York Stem Cell Foundation (NYSCF-R-N145)

Stephen Graf Brohawn

National Institute of General Medical Sciences (DP2GM123496)

Stephen Graf Brohawn

McKnight Endowment Fund for Neuroscience

Stephen Graf Brohawn

Klingenstein Third Generation Foundation

Stephen Graf Brohawn

National Institute of General Medical Sciences (F32GM128263)

David M Kern

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

Copyright

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.