Studies on the membrane topology of the (Na,K)-ATPase

Abstract

The topology of the alpha 1 and beta 1 subunits of the rat (Na,K)-ATPase has been studied by insertion of epitope(s): at the NH2 terminus and COOH terminus and between Glu-117 and Glu-118, Lys-828 and Arg-829, Gln-900 and Trp-901, and Val-939 and Phe-940 of the alpha subunit; and at the NH2 terminus and COOH terminus and between Glu-228 and Tyr-229 of the beta subunit. The epitope-tagged alpha 1 constructs were expressed in HeLa cells to select for stable cell lines expressing a functional (Na,K)-ATPase. The epitope-tagged beta constructs were transiently expressed in Cos-7 cells. The membrane arrangement of the epitopes was revealed by indirect immunofluorescence with cells expressing the (Na,K)-ATPase chains. The results indicate that the alpha subunit has 4 transmembrane segments in the COOH-terminal membrane-bound domain between residues 760 and 938, and that both the NH2 terminus and the COOH terminus are in the cytosol; it was not determined whether there are more transmembrane segments between residue 938 and the COOH terminus. The beta subunit has only one transmembrane-spanning region with the NH2 terminus in the cytosol and the COOH terminus on the extracytoplasmic surface of the plasma membrane.