Distinct effects of Q925 mutation on intracellular and extracellular Na(+) and K(+) binding to the Na(+), K(+)-ATPase

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.