Contribution to Tl+, K+, and Na+ binding of Asn(776), Ser(775), Thr(774), Thr(772), and Tyr(771) in cytoplasmic part of fifth transmembrane segment in alpha-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. H ere, in search of the molecular basis for Na,K specificity, alanine and con servative substitutions were directed to six oxygen-carrying residues in th is segment. The contribution of the residues to cation binding was estimate d from direct binding of T1(+) [Nielsen, et al, (1998) Biochemistry 37, 196 1-1968], K+ displacement of ATP binding at equilibrium, and Na+-dependent p hosphorylation from ATP in the presence of oligomycin. As an intrinsic cont rol, substitution of Thr(781) had no effect on T1(+)(K+) or Nat binding. Th ere are several novel observations from this work. First, the carboxamide g roup 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 th e 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 bindi ng of both T1(+)(K+) and Na+, whereas the hydroxyl group of Thr(772) does n ot contribute to cation binding, Furthermore, the hydroxyl groups of Ser(77 5) 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 trans membrane segment may adapt distances between coordinating groups and contri bute to the distinctive Na+/K+ selectivity of the pump.