Structural basis for the ion selectivity of potassium-chloride cotransporter KCC4 revealed by cryo-EM titration
image: High-resolution structures of KCC4 in different ion conditions. A 3D reconstruction of KCC4KNO3 colored by local resolution. B Sample of EM maps at transmembrane helices 1 (TM1) and 6 (TM6) of KCC4 in different ion conditions at the contour level of 4 σ. C Cartoon representation of one KCC4KCl subunit (left) and the zoom-in view of three potential ion binding sites S1–S3 (right). S1–S3 are equivalent to the three sites (SK, SCl1, SCl2) that we initially assigned in the KCC16KKR structure. The ions are shown as cyan spheres
Credit: HIGHER EDUCATION PRESS
Potassium-chloride cotransporters KCCs mediate the coupled, electroneutral cotransport of K+ and Cl– across the membrane and are involved in important physiological processes such as cell volume regulation and γ-aminobutyric acid (GABA) and glycine-mediated inhibitory neurotransmission. Although structures of KCCs have been reported, the identification of ions bound in KCCs awaits experimental studies. Here using the cryo-electron microscopy (cryo-EM) titration methods, we present six structures of human KCC4 in different ion conditions at 2.38–2.58 Å resolutions. These structures, along with molecular dynamic simulations, allow us to assign one K+ and two Cl− ions in the substrate-binding pocket. The K+ at S1 and Cl− at the S2 site are tightly coupled in the binding and dissociation, suggesting that the Cl− at S2 but not at S3 is the cotransported one. The S1 site provides coordination that largely matches the K+ dehydration radius and therefore displays higher selectivity to K+ over Na+. This study establishes the structural basis for the K+ selectivity of KCCs by the cryo-EM titration.
The work entitled “Structural basis for the ion selectivity of potassium-chloride cotransporter KCC4 revealed by cryo-EM titration” was published in Biophysics Reports (published in Issue 3, Volume 12, June 2026).