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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.