Both chloride-binding sites are required for KCC2-mediated transport.

The K⁺-Cl^- cotransporter 2 (KCC2) plays an important role in inhibitory neurotransmission, and its impairment is associated with neurological and psychiatric disorders, including epilepsy, schizophrenia, and autism. Although KCCs transport K⁺ and Cl^- in a 1:1 stoichiometry, two Cl^- coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl^- in Cl₁ and Cl₂. Individual mutations of residues in Cl₁ and Cl₂ reduce or abolish KCC2^WT function, indicating a crucial role of both Cl^- coordination sites for KCC2 function. Structural changes in the extracellular loop 2 by inserting a 3xHA tag switches the K⁺ coordination site to another position. To investigate, whether the extension of the extracellular loop 2 with the 3xHA tag also affects the coordination of the two Cl^- coordination sites, we carried out the analogous experiments for both Cl^- coordinating sites in the KCC2^HA construct. These analyses showed that most of the individual mutation of residues in Cl₁ and Cl₂ in the KCC2^HA construct reduces or abolishes KCC2 function, indicating that the coordination of Cl^- remains at the same position. However, the coupling of K⁺ and Cl^- in Cl₁ is still apparent in the KCC2^HA construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr⁵⁶⁹ in Cl₂ shifted in KCC2^HA. Thus, conformational changes in the extracellular domain affect K⁺ and Cl^--binding sites. However, the effect on the Cl^--binding sites is subtler.