A dominant negative mutant of the KCC1K-Cl cotransporter - Both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunc t strategy for the treatment of sickle cell anemia. However, structure-func tion relationships among the polypeptide products of the four K-Cl cotransp orter (KCC) genes are little understood. We have investigated the importanc e of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl co transport function expressed in Xenopus oocytes. Truncation of as few as ei ght C-terminal amino acids (aa) abolished function despite continued polype ptide accumulation and surface expression. These C-terminal loss-of-functio n mutants lacked a dominant negative phenotype. Truncation of the N-termina l 46 aa diminished function. Removal of 89 or 117 aa (Delta (N)117) abolish ed function despite continued polypeptide accumulation and surface expressi on and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta (N)117 was co-immunoprecipitated with wild type KCC1 polypepti de, and its co-expression did not reduce wild type KCC1 at the oocyte surfa ce. Delta (N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.