Localization of cytoplasmic and extracellular domains of Na,K-ATPase by epitope tag insertion

We have used epitope tag addition to analyze the transmembrane topology of the Na,K-ATPase catalytic (α) subunit. An antigenic peptide derived from the hemagglutinin (HA) of influenza virus was inserted at 15 different positions within the rat Na,K-ATPase α1 subunit isoform. The functional integrity of the tagged proteins was tested by their capacity to confer ouabain resistance upon human HEK 293 cells. Constructs with the tag at aa positions 119, 173, 318, 815, 881, 953, 987, and 1023 conferred ouabain resistance, and the mutant proteins were detectable in the plasma membrane of transfected cells. In contrast, α1 subunits with insertions at aa positions 338, 797, 805, 868, 895, 910, and 921 were unable to confer drug resistance. Immunofluorescence analysis of permeabilized and intact cells using a monoclonal antibody specific for the HA epitope showed that double tags at positions 119 and 318 were located extracellularly, whereas single or double tags at positions 173, 815, 881, 987, and 1023 were cytoplasmically disposed. These results are consistent with an eight transmembrane domain arrangement for the α subunit. Epitope insertion within TM4, and the region linking transmembrane segments TM6-TM7, caused the loss of α subunit function, suggesting that the integrity of these domains is essential for the proper biosynthesis and/or maturation of the α subunit.