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 alpha1-subunit (alpha1NaK), accompanying significant increases of the Na(+),K(+)-ATPase activity. Exogenously expressed KCC3b did not co-immunoprecipitate with endogenous alpha1NaK 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 alpha1NaK, while KCC3b did not. Our results suggest that the NH(2)-terminus of KCC3a is a key region for association with alpha1NaK, and that KCC3a but not KCC3b can regulate the Na(+),K(+)-ATPase activity.
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