2. Academic Validation
  3. Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters

Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters

  • EMBO J. 2021 Jul 15;40(14):e107294. doi: 10.15252/embj.2020107294.
Gamma Chi 1 2,  Rebecca Ebenhoch 1 2,  Henry Man 1 2,  Haiping Tang 3,  Laurence E Tremblay 4,  Gabriella Reggiano 5,  Xingyu Qiu 3,  Tina Bohstedt 1 2,  Idlir Liko 6,  Fernando G Almeida 6,  Alexandre P Garneau 4 7,  Dong Wang 1 2,  Gavin McKinley 1 2,  Christophe P Moreau 1,  Kiran D Bountra 6,  Patrizia Abrusci 1 2,  Shubhashish M M Mukhopadhyay 1 2,  Alejandra Fernandez-Cid 1 2,  Samira Slimani 4,  Julie L Lavoie 7,  Nicola A Burgess-Brown 1 2,  Ben Tehan 6,  Frank DiMaio 5,  Ali Jazayeri 6,  Paul Isenring 4,  Carol V Robinson 3,  Katharina L Dürr 1 2 6
Affiliations

Affiliations

  • 1 Nuffield Department of Medicine, Centre of Medicines Discovery, University of Oxford, Oxford, UK.
  • 2 Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
  • 3 Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, UK.
  • 4 Department of Medicine, Nephrology Research Group, Faculty of Medicine, Laval University, Quebec City, QC, Canada.
  • 5 Department of Biochemistry, University of Washington, Seattle, WA, USA.
  • 6 OMass Therapeutics, Ltd., Oxford, UK.
  • 7 Cardiometabolic Axis, School of Kinesiology and Physical Activity Sciences, University of Montréal, Montréal, QC, Canada.
PMID: 34031912 DOI: 10.15252/embj.2020107294
Abstract

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.

Keywords

HDX-MS; nucleotide binding; phospho-regulation; potassium-chloride co-transport; solute carrier.

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