Search for: All records

Na+,K+-ATPase actively extrudes three cytoplasmic Na+ ions in exchange for two extracellular K+ ions for each ATP hydrolyzed. The atomic structure with bound Na+ identifies three Na+ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na+ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na+ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na+ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na+ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na+ affinity. This indicates that Na+ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E1-like configuration, allowing Na+ but not K+ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.

Three Na⁺sites are defined in the Na⁺-bound crystal structure of Na⁺, K⁺-ATPase. Sites I and II overlap with two K⁺sites in the K⁺-bound structure, whereas site III is unique and Na⁺specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na⁺at site III, but does not contribute to K⁺coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na⁺, K⁺-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na⁺and K⁺binding properties. Remarkably, Q925 substitutions had minor effects on Na⁺binding from the intracellular side of the membrane – in fact, mutations Q925A and Q925G increased the apparent Na⁺affinity – but caused dramatic reductions of the binding of K⁺as well as Na⁺from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na⁺-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na⁺at the extracellular side.