Regulation Of Na/K-Atpase Expression By Cholesterol: Isoform Specificity And The Molecular Mechanism

We have reported that the reduction in plasma membrane cholesterol could decrease cellular Na/K-ATPase alpha 1 expression through a Src-dependent pathway. However, it is unclear whether cholesterol could regulate other Na/K-ATPase alpha-isoforms and the molecular mechanisms of this regulation are not fully understood. Here we used cells expressing different Na/K-ATPase a isoforms and found that membrane cholesterol reduction by U18666A decreased expression of the alpha 1-isoform but not the alpha 2-or alpha 3-iso-form. Imaging analyses showed the cellular redistribution of alpha 1 and alpha 3 but not alpha 2. Moreover, U18666A led to redistribution of alpha 1 to late endosomes/lysosomes, while the proteasome inhibitor blocked alpha 1-reduction by U18666A. These results suggest that the regulation of the Na/K-ATPase alpha-subunit by cholesterol is isoform specific and alpha 1 is unique in this regulation through the endocytosis-proteasome pathway. Mechanistically, loss-of-Src binding mutation of A425P in alpha 1 lost its capacity for regulation by cholesterol. Meanwhile, gain-of-Src binding mutations in alpha 2 partially restored the regulation. Furthermore, through studies in caveolin-1 knockdown cells, as well as subcellular distribution studies in cell lines with different alpha-iso-forms, we found that Na/K-ATPase, Src, and caveolin-1 worked together for the cholesterol regulation. Taken together, these new findings reveal that the putative Src-binding domain and the intact Na/K-ATPase/Src/caveolin-1 complex are indispensable for the isoform-specific regulation of Na/K-ATPase by cholesterol.