Displacement of the Na+/K+ pump’s transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases

Cellular survival requires the ion gradients built by the Na⁺/K⁺ pump, an ATPase that alternates between two major conformations (E1 and E2). Here we use state-specific engineered-disulfide cross-linking to demonstrate that transmembrane segment 2 (M2) of the pump’s α-subunit moves in directions that are inconsistent with distances observed in existing crystal structures of the Na⁺/K⁺ pump in E1 and E2. We characterize this movement with voltage-clamp fluorometry in single-cysteine mutants. Most mutants in the M1–M2 loop produced state-dependent fluorescence changes upon labeling with tetramethylrhodamine-6-maleimide (TMRM), which were due to quenching by multiple endogenous tryptophans. To avoid complications arising from multiple potential quenchers, we analyzed quenching of TMRM conjugated to R977C (in the static M9–M10 loop) by tryptophans introduced, one at a time, in M1–M2. This approach showed that tryptophans introduced in M2 quench TMRM only in E2, with D126W and L130W on the same helix producing the largest fluorescence changes. These observations indicate that M2 moves outward as Na⁺ is deoccluded from the E1 conformation, a mechanism consistent with cross-linking results and with proposals for other P-type 2 ATPases.

P-type ATPases are enzymes that catalyze formation of ion or lipid gradients across membranes in all phyla. These membrane proteins use homologous E1–E2 mechanisms (Fig. 1A) in which uphill transport of a subtype-specific substrate is powered by ATP hydrolysis (1). In nearly all animal cells the Na⁺/K⁺ pump, a P-type 2C ATPase, exports three Na⁺ and imports two K⁺ into the cell, against their electrochemical gradients, thereby building the ion gradients that energize other essential membrane processes, including electric signaling and secondary-active transport. This ion pump is formed by a catalytic α-subunit, homologous to the catalytic subunits of all other P-type ATPases, and an auxiliary β-subunit. The α-subunit contains the ion-transport sites within its 10 transmembrane α-helices (M1–M10) and the nucleotide (N), phosphorylation (P), and actuator (A) domains formed by its intracellular loops, while the β-subunit, with a single transmembrane segment and a large extracellular globular domain, is necessary for ion occlusion (2) and membrane stability (3). In each catalytic cycle, the pump transits through a set of partial reactions while occupying two major conformations E1 and E2, in what is also known as the Post-Albers mechanism (Fig. 1A).Open in a separate windowFig. 1.(A) Post-Albers kinetic scheme of the Na⁺/K⁺ pump (clockwise forward direction). The pump alternates between two major conformations, E1 and E2, which can be phosphorylated (P) or dephosphorylated. Parentheses indicate ions occluded within the protein. Transitions within the red box E1P(3Na⁺) ↔ E2P + 3 Na⁺_o produce transient charge movement. (B and C) Overall change in structure between Na⁺/K⁺ pump structures E1(3Na⁺) (PDB ID 3WGV; cyan) and E2P-ouabain (PDB ID 4XE5; pink) shown from two angles after aligning through the C-terminal end of the Na⁺/K⁺ pump α-subunit, (B) A lateral view, approximately parallel to the membrane plane (indicated by gray dotted lines), and (C) a view from the extracellular side, approximately perpendicular to the membrane (rotated 90° from the view in B). Residues and distances addressed in the discussion are labeled.Utilizing a combination of generic P-type ATPase as well as pump-specific inhibitors, all intermediate states of the sarcoplasmic/endoplasmic reticulum ATPase (SERCA, another P-type 2 ATPase) have been determined by X-ray crystallography (4–14), but only a handful of states have been resolved for the Na⁺/K⁺ pump (15–20). Considering the high sequence similarity and conserved catalytic cycle, it was expected that the main conformational changes should also be conserved. However, comparison of the available crystal structures in equivalent states reveals significant differences, particularly in the movement of the M1–M2 stalk. In SERCA, transitions between E1P(2Ca²⁺) and E2P states involve kinking as well as movement of the luminal part of M1–M2 perpendicular to the membrane plane (9), a movement that seems largely absent in the available Na⁺/K⁺ pump crystal structures (20) (SI Appendix, Fig. S1). Fig. 1B compares two states of such Na/K pump structures: the aluminum fluoride-inhibited E1 with 3 Na⁺ bound (ref. 20; cyan carbons) and the ouabain- and Mg²⁺-bound beryllium fluoride-inhibited structure (ref. 21; pink carbons). The closest physiologic states to these inhibitor-locked structures are enclosed with a dotted box in the catalytic cycle (Fig. 1A). Displacements in the membrane plane are absent in the side view (Fig. 1B and SI Appendix, Fig. S1), despite large movements in other directions, which are obvious in the external view perpendicular to the membrane (Fig. 1C).The dislodgment of M1–M2 is thought to guide opening of the SERCA ion-binding sites as Ca²⁺ is deoccluded and released toward the luminal side prior to occlusion of the countertransported H⁺. Because a similar mechanism has been proposed for the gastric H⁺/K⁺ pump (22), the absence of such conserved conformational change in the closely related Na⁺/K⁺ pump is perplexing, and this movement has never been evaluated in a functional Na⁺/K⁺ pump in the membrane environment. Here we address the movement of the M1–M2 region in functional Na⁺/K⁺ pumps utilizing cross-linking of engineered disulfides under voltage clamp, as well as voltage-clamp fluorometry (VCF), under conditions in which we can control the occupancy of phosphorylated states in the pump cycle. Our results indicate that the movement of the externalmost part of the α-subunit’s M1–M2 is required for Na⁺/K⁺ pump to function and, thus, conserved through the P-type 2 ATPase family. Furthermore, we illustrate a slight modification to the usual VCF approach, where TMRM is attached to a static residue while relative distance changes are evaluated by scanning with quenching tryptophans in the moving region, allowing one to study both the movement and the structural characteristics of transmembrane segments and their loops in a functional Na⁺/K⁺ pump, a powerful modification that can be exploited to investigate other transport proteins as well.