Role of cholesterol in functional association between K(+)-Cl(-) cotransporter-3a and Na+,K(+)-ATPase.
K(+)-Cl(-) cotransporter-3a (KCC3a) is associated with Na(+),K(+)-ATPase α1-subunit (α1NaK) in lipid rafts of gastric acid-secreting cells and positively regulates Na(+),K(+)-ATPase activity. Here, effects of cholesterol on association of KCC3a with α1NaK in lipid rafts were studied in LLC-PK1 cells stably expressing KCC3a. In the cells, lipid rafts destructed by methyl-β-cyclodextrin (MβCD) could be reconstructed by exogenous addition of cholesterol accompanying a shift of both KCC3a and α1NaK from non-rafts to rafts. The KCC3a-increased Na(+),K(+)-ATPase activity was abolished by MβCD, and recovered by repletion of cholesterol without changing expression levels of KCC3a and α1NaK in the cells. KCC3a was co-immunoprecipitated with α1NaK even after destruction of lipid rafts by MβCD, indicating that molecular association of KCC3a with α1NaK 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-α1NaK complex.
Department of Pharmaceutical Physiology, University of Toyama, Toyama 930-0194, Japan. d1061211@ems.u-toyama.ac.jp
Kyosuke
K
Fujita
Takuto
T
Fujii
Takahiro
T
Shimizu
Noriaki
N
Takeguchi
Hideki
H
Sakai
D000818:Animals; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism* / Q000494:pharmacology; D021962:Membrane Microdomains / Q000378:metabolism*; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology; D000096922:K Cl- Cotransporters
D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase; D027981:Symporters; D047392:beta-Cyclodextrins; C492281:Slc12a6 protein, rat; D000096922:K Cl- Cotransporters; C108732:methyl-beta-cyclodextrin