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

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois

Submitted 10 July 2008 ; accepted in final form 12 August 2008

In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase - and β-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 β₁-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged β₁-subunits (β₁flag) or Myc-tagged β₁-subunits (β₁myc) under the control of a tetracycline-dependent promoter. The induction of β₁flag subunit synthesis in MDCK cells, which increases β₁-subunit expression at the plasma membrane by more than twofold, while maintaining stable ₁ expression levels, revealed that all mature β₁-subunits associate with ₁-subunits, and no evidence of "free" β₁-subunits was obtained. Consequently, the ratio of assembled β₁- to ₁-subunits is significantly increased when "extra" β-subunits are expressed. An increased β₁/₁ 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 β₁myc-expressing and β₁flag-expressing MDCK cells show colocalization of β₁myc and β₁flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the β-subunits. Immunoprecipitation using MDCK cells constitutively expressing β₁myc and tetracycline-regulated β₁flag subunits confirmed β-β-subunit interactions. These results demonstrate that the equimolar ratio of assembled β₁/₁-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.

sodium pump; beta; expression; epithelial cell; alpha; ratio

This article has been cited by other articles:

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				T. A. Pressley The stoichiometry of the Na-K pump: one plus one doesn't equal one Am J Physiol Renal Physiol, November 1, 2008; 295(5): F1313 - F1313. [Full Text] [PDF]