Analysis of amino acid residues in the H5-H6 transmembrane and extracellular domains of Na,K-ATPase alpha subunit identifies threonine 797 as a determinant of ouabain sensitivity

Abstract

Several amino acid residues of the alpha subunit of the Na,K-ATPase have been identified which alter ouabain sensitivity. These residues are located in the N-terminal half of the alpha 1 subunit suggesting that this portion of the molecule may represent the binding site for cardiac glycosides. However, not all extracellular and transmembrane regions have been investigated, including the H5-H6 membrane-spanning region. To determine if this region of the alpha subunit contributes to ouabain sensitivity, amino acids which have the potential to form hydrogen bonds were substituted with alanine, a non-hydrogen-bonding amino acid. cDNAs encoding enzyme containing these individual amino acid replacements were expressed in ouabain-sensitive HeLa cells, and the ability of the altered enzymes to confer ouabain resistance was examined. Nineteen amino acid substitutions were investigated. T797A (Thr 797 to Ala) was the only substitution which conferred ouabain resistance to sensitive HeLa cells. Three additional substitutions at this position (T797V, T797S, and T797D) were generated in order to examine the effects of the replacements of Thr 797 on ouabain inhibition of Na,K-ATPase activity. The T797V substitution conferred ouabain resistance, but T797S and T797D substitutions did not. The ouabain-resistant cell lines expressing the T797A and T797V substitutions exhibited Na,K-ATPase activity that was 60 and 70 times more resistant to ouabain than the endogenous HeLa or sheep enzymes. The absence of a hydroxyl group at amino acid 797 may be responsible for the reduced sensitivity of the enzyme with substitutions at this position.(ABSTRACT TRUNCATED AT 250 WORDS)