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

Hang N Nielsen,Rikke Holm,Bente Vilsen,Jens Peter Andersen,Pablo Artigas,Anja P Einholm,Kerri Spontarelli

doi:10.1038/s41598-019-50009-2

Hang N Nielsen, Rikke Holm + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-50009-2

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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.

Highlights

We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties
The static crystal structure lacks information on which residues are critical to ion binding during the dynamic process where the Na+ sites switch between intracellular- and extracellular-facing orientations in relation to the ion translocation process
The findings show that Q925 contributes little to Na+ binding from the intracellular side – mutations Q925A and Q925G were compatible with Na+ and K+ transport and ATPase activity and increased the apparent Na+ affinity, despite removing an oxygen presumed to participate in Na+-coordination

Summary

Introduction

We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties. Q925 substitutions had minor effects on Na+ binding from the intracellular side of the membrane – 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. We have addressed the specific functional role of Q925 in the various conformational states of the Na+, K+-ATPase by examining the mutants Q925A/G/E/N/L/I/Y We have characterized these mutants enzymatically, with respect to their sided interaction with Na+ and K+. Q925 plays a crucial role in the binding of Na+ and K+ from the extracellular side of the membrane

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