K+-Cl- cotransporter-3a (KCC3a) is associated with Na+,K+-ATPase alpha 1-subunit (alpha 1NaK) in lipid rafts of gastric acid-secreting cells and positively regulates Na+,K+-ATPase activity. Here, effects of cholesterol on association of KCC3a with alpha 1NaK in lipid rafts were studied in LLC-PK1 cells stably expressing KCC3a. In the cells, lipid rafts destructed by methyl-beta-cyclodextrin (M beta CD) could be reconstructed by exogenous addition of cholesterol accompanying a shift of both KCC3a and alpha 1NaK from non-rafts to rafts. The KCC3a-increased Na+,K+-ATPase activity was abolished by M beta CD, and recovered by repletion of cholesterol without changing expression levels of KCC3a and alpha 1NaK in the cells. KCC3a was co-immunoprecipitated with alpha 1NaK even after destruction of lipid rafts by M beta CD, indicating that molecular association of KCC3a with alpha 1NaK still retains in the non-raft environment. Our results suggest that cholesterol is essential for eliciting up-regulation of Na+,K+-ATPase activity by KCC3a in the KCC3a-alpha 1NaK complex. (C) 2012 Elsevier Inc. All rights reserved.