Oligomerization of the Na,K-ATPase in cell membranes

Abstract

The higher order oligomeric state of the Na,K-ATPase αβ heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with α-subunits bearing either His6 or FLAG epitopes at the carbozyl terminus. Each of these constructs produced functional Na,K-ATPase αβ heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding α-His/β and α-FLAG/β Na,K-ATPases. Co-immunoprecipitation of the His-tagged α-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,K-ATPase is present in cell membranes in an oligomeric state of at least (αβ)2 composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the α-subunit results in the deletion α-4,5-loop-less (α-4,5LL), which associates with β but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive α-4,5LL/β heterodimer is co-expressed with wild-type αβ, oligomers of wild-type αβ and α-4,5LL/β form in the ER, but the α-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric αβ heterodimer in native cell membranes.