Contribution to Tl+, K+, and Na+ binding of Asn776, Ser775, Thr774, Thr772, and Tyr771 in cytoplasmic part of fifth transmembrane segment in α-subunit of renal Na,K-ATPase

The sequence (YTLTSNIPEITP)-T-771-P-781 in the fifth transmembrane segment of the alpha-subunit of Na,K-ATPase is unique among cation pump proteins. Here, in search of the molecular basis for Na,K specificity, alanine and conservative substitutions were directed to six oxygen-carrying residues in this segment. The contribution of the residues to cation binding was estimated from direct binding of T1(+) [Nielsen, et al, (1998) Biochemistry 37, 1961-1968], K+ displacement of ATP binding at equilibrium, and Na+-dependent phosphorylation from ATP in the presence of oligomycin. As an intrinsic control, substitution of Thr(781) had no effect on T1(+)(K+) or Nat binding. There are several novel observations from this work. First, the carboxamide group of Asn(776) is equally important for binding T1(+)(K+) or Na+ whereas a shift of the position of the carboxamide of Asn(776) (Asn(776)Gln) causes a large depression of Naf binding without affecting the binding of T1(+)(K+). Second, Thr(774) is important for Nat selectivity because removal of the hydroxyl group reduces the binding of Naf with no effect on binding of T1(+)(K+). Removal of the methyl groups of Thr(774) Or Thr(772) reduces binding of both T1(+)(K+) and Na+, whereas the hydroxyl group of Thr(772) does not contribute to cation binding, Furthermore, the hydroxyl groups of Ser(775) and Tyr(771) are important for binding both T1(+)(K+) and Na+. The data suggest that rotating or tilting of the cytoplasmic part of the fifth transmembrane segment may adapt distances between coordinating groups and contribute to the distinctive Na+/K+ selectivity of the pump.