β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

Clifford RJ, Kaplan JH. beta-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells. Am J Physiol Renal Physiol 295: F1314-F1323, 2008. First published August 13, 2008; doi:10.1152/ajprenal.90406.2008.-In eukaryotic cells, the apparent maintenance of 1: 1 stoicheometry between the Na-K-ATPase alpha- and beta-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase beta(1)-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged beta(1)-subunits (beta(1)flag) or Myc-tagged beta(1)-subunits (beta(1)myc) under the control of a tetracycline-dependent promoter. The induction of beta(1)flag subunit synthesis in MDCK cells, which increases beta(1)-subunit expression at the plasma membrane by more than twofold, while maintaining stable alpha(1) expression levels, revealed that all mature alpha(1)-subunits associate with alpha(1)-subunits, and no evidence of free beta(1)-subunits was obtained. Consequently, the ratio of assembled beta(1)-to beta(1)-subunits is significantly increased when extra beta-subunits are expressed. An increased beta(1)/alpha(1) stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured beta(1)myc-expressing and beta(1)flag-expressing MDCK cells show colocalization of beta(1)myc and beta(1)flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the beta-subunits. Immunoprecipitation using MDCK cells constitutively expressing beta(1)myc and tetracycline-regulated beta(1)flag subunits confirmed beta-beta-subunit interactions. These results demonstrate that the equimolar ratio of assembled beta(1)/alpha(1)-subunits of the Na-K-ATPase in kidney cells is not fixed by the inherent properties of the interacting subunits. It is likely that cellular mechanisms are present that regulate the individual Na-K-ATPase subunit abundance.