Functional role of oxygen-containing residues in the fifth transmembrane segment of the Na,K-ATPase α subunit

The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, ATPase a subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover, Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity tin the absence of K+). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na+ and K+ affinities (K-1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Naf acting at the extracellular space (Arguello et al. J. Biol. Chem. 271, 24610-24616, 1996), Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme-Na+ interaction was greatly changed in these enzymes, while their activation by K+ did not appear affected Although the Nat It, for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala, n(Hill) = 0.75; Tyr771Ser, n(Hill) = 0.92; Control, n(Hill) = 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na+ dependence (n(Hill) = 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP, Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 <----> E2 equilibrium alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na+ binding and occlusion without participating in K+-enzyme interactions. (C) 1999 Academic Press.