Residue leu973 of the rat alpha1 subunit of the Na,K-ATPase is located on the cytoplasmic side of the plasma membrane.

In a previous study (Yoon, K. L., and Guidotti, G., 1994 J. Biol. Chem. 269, 28249-28258), we indicated that the alpha subunit of the Na,K-ATPase has 4 transmembrane segments in the COOH terminal domain between residues Lys769 and Val939, and that both the NH2-terminus and the COOH-terminus are in the cytosol. However, there was insufficient information to determine whether there are more transmembrane segments between residues Val939 and the COOH-terminal Tyr1018. To investigate this question, we inserted the influenza virus hemagglutinin (HA)-epitope between Leu973 and Arg974 of the alpha1 chain, expressed the construct in COS-7 and HeLa cells and determined the membrane arrangement by indirect immunofluorescence. The results indicate that Leu973 is not on the extracellular surface of the plasma membrane. Thus, the alpha1 subunit is likely to possess only four complete transmembrane segments in the COOH terminal domain between residues Lys769 and Tyr1018.