Role of cystein residues in the normal operation and assembly of K+‐Cl− cotransporter isoform 2 (KCC2)

K+‐Cl− cotransporters (KCCs) regulate intracellular K+ and Cl− concentration as well as cell volume. Their transport activity is sensitive to N‐ethylmaleimide (NEM), a thiol reagent that alkylates available sulfhydryl groups, and their C‐terminus can interact with other CCCs to form oligomers. These features suggest that a number of cystein residues within the KCCs play an important role in carrier activity and/or assembly. To determine whether this is the case, we have exploited a mutagenic approach using rat KCC2 as a model. Substitutions (C8G, C742G, C854G and C982G) were generated by site‐directed mutagenesis and expressed in X. laevis oocytes. Substitution C854G increased while C982G decreased Rb+ transport without altering cell surface protein expression. They responded as did wild type (wt) carrier to the inhibitory effect of 250 μM furosemide but were more sensitive to the inhibitory effect of 1 mM NEM. Coimmunoprecipitation studies showed that the quantity of carriers precipitated with wt KCC2 was lower for C854G than for wt KCC2 or C982G. Lastly, FRAP studies in KCC2‐transfected HEK‐293 cells showed that NEM increased post‐bleach fluorescence recovery time in a concentration‐dependent manner. Collectively, the data suggest that C854 and C982 play an important role in KCC2 function, perhaps by supporting homo‐ and/or heterooligomeric structure formation. This work was funded by CIHR and CFI.