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 Cl and Cl. Individual mutations of residues in Cl and Cl reduce or abolish KCC2 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 construct. These analyses showed that most of the individual mutation of residues in Cl and Cl in the KCC2 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 Cl is still apparent in the KCC2 construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr in Cl shifted in KCC2. Thus, conformational changes in the extracellular domain affect K and Cl-binding sites. However, the effect on the Cl-binding sites is subtler.