Structural basis for the ion selectivity of potassium-chloride cotransporter KCC4 revealed by cryo-EM titration

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.