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.