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, K-ATPase α 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 (in the absence of K+). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na+and K+affinities (K1/2for 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 Na+acting at the extracellular space (Argüelloet 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 Na+K1/2for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala,nHill= 0.75; Tyr771Ser,nHill= 0.92; Control,nHill= 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na+dependence (nHill= 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.