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

Abstract

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.