K+-Cl- cotransporter-3 has two major amino terminal variants, KCC3a and KCC3b. In LLC-PK1 cells, exogenously expressed KCC3a co-immunoprecipitated with endogenous Na+,K+-ATPase alpha 1-subunit (alpha 1NaK), accompanying significant increases of the Na+,K+-ATPase activity. Exogenously expressed KCC3b did not co-immunoprecipitate with endogenous alpha 1NaK inducing no change of the Na+,K+-ATPase activity. A KCC inhibitor attenuated the Na+,K+-ATPase activity in rat gastric mucosa in which KCC3a is predominantly expressed, while it had no effects on the Na+,K+-ATPase activity in rat kidney in which KCC3b is predominantly expressed. In these tissue samples, KCC3a co-immunoprecipitated with alpha 1NaK, while KCC3b did not. Our results suggest that the NH2-terminus of KCC3a is a key region for association with alpha 1NaK, and that KCC3a but not KCC3b can regulate the Ne+,K+-ATPase activity. (C) 2010 Elsevier Inc. All rights reserved.