Flemming Cornelius

The Na⁺,K⁺-ATPase is an integral membrane protein present in all animal plasma membranes. It uses the energy of ATP hydrolysis to pump 3 Na⁺ ions per ATP hydrolysed from the cell cytoplasm into the extracellular fluid in exchange for 2 K⁺ ions. The Na⁺ electrochemical potential gradient it produces is used as an energy source to drive all animal secondary transporters, for example, in nutrient reabsorption in the kidney. In this synthesis of new and previously published results, it is shown that the rate of the mammalian enzyme’s rate-determining E2 → E1 conformational change, during which K⁺ ions are released to the cytoplasm, is strongly dependent on an electrostatic interaction in the E2 state. This electrostatic interaction must be broken to allow the enzyme to convert to the E1 state and allow Na⁺ pumping. The strength of the interaction depends on ionic strength and, even more strongly, on the concentration of divalent metal ions, i.e., Ca²⁺ or Mg²⁺. A likely candidate for the interaction is between the protein’s positively-charged lysine-rich N-terminus and the negatively-charged membrane cytoplasmic surface. Comparison of the Ca²⁺ and Mg²⁺ dissociation constants measured with the physiological levels of the two ions suggests that Mg²⁺, but not Ca²⁺, could play a regulatory role for the Na⁺,K⁺-ATPase. Graphical Abstract The alternative text for this image may have been generated using AI.

Palytoxin (PTX), a potent marine toxin, has long been known to transform Na ⁺ ,K ⁺ -ATPase (NKA), an indispensable ion pump, into a nonselective cation channel. It has been postulated that PTX takes control of the two gates on either side of a channel-like pore. These gates normally open and close alternately, synchronized with chemical events, never opening simultaneously. A critical question is whether palytoxin takes over the control of the two gates or creates a new pathway. Here, we present structures of NKA with bound palytoxin in three different states. PTX binds to NKA in E2P, occupying the physiological Na ⁺ exit pathway, similar to istaroxime, a new-generation cardiotonic steroid. Adding Na ⁺ and ATP/ADP to the NKA·PTX complex induces an open channel traversing the entire membrane alongside the physiological ion pathway. As AlF _x , a stable transition state analog of phosphate replaces phosphate in the NKA·PTX complex preformed in E2P, the complex appears to undergo the normal reaction cycle from E2P to E1· n Na ⁺ . PTX occupies the space between the transmembrane helices M4 and M6, thereby preventing the closure of the extracellular half of the ion pathway. These structures demonstrate that the architecture of NKA is fundamentally different from “a pore with two gates.” Each half of the ion pathway comprises three segments, including a movable component that plays a pivotal role in translocating the bound cations by connecting the constant part to an appropriate inlet. The ion pathway of NKA transforms dynamically, ensuring that the two halves never exist simultaneously.

Na⁺,K⁺‐ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na⁺ and K⁺ across the cell membrane by alternating between the E1 (showing high affinity for Na⁺ and low affinity for K⁺) and E2 (low affinity to Na⁺ and high affinity to K⁺) forms. Presented here are two crystal structures of NKA in E1·Mg²⁺ and E1·3Na⁺ states at 2.9 and 2.8 Å resolution, respectively. These two E1 structures fill a gap in our description of the NKA reaction cycle based on the atomic structures. We describe how NKA converts the K⁺‐bound E2·2K⁺ form to an E1 (E1·Mg²⁺) form, which allows high‐affinity Na⁺ binding, eventually closing the cytoplasmic gate (in E1 ~ P·ADP·3Na⁺) after binding three Na⁺, while keeping the extracellular ion pathway sealed. We now understand previously unknown functional roles for several parts of NKA and that NKA uses even the lipid bilayer for gating the ion pathway.

Na⁺,K⁺‐ATPase (NKA) is one of the most important members of the P‐type ion‐translocating ATPases and plays a pivotal role in establishing electrochemical gradients for Na⁺ and K⁺ across the cell membrane. Presented here is a 3.3 Å resolution structure of NKA in the E2·2K⁺ state solved by cryo‐electron microscopy. It is a stable state with two occluded K⁺ after transferring three Na⁺ into the extracellular medium and releasing inorganic phosphate bound to the cytoplasmic P domain. We describe how the extracellular ion pathway wide open in the E2P state becomes closed and locked in E2·2K⁺, linked to events at the phosphorylation site more than 50 Å away. We also show, although at low resolution, how ATP binding to NKA in E2·2K⁺ relaxes the gating machinery and thereby accelerates the transition into the next step, that is, the release of K⁺ into the cytoplasm, more than 100 times.

Significance: The E2P state of Na ⁺ ,K ⁺ -ATPase, in which the ATPase is phosphorylated and of low affinity to Na ⁺ with the extracellular gate opened, shows different biochemical properties depending on whether the phosphate is transferred from ATP in the forward reaction or from inorganic phosphate (P _i ) in the backward reaction. We present here cryoelectron microscopy structures of Na ⁺ ,K ⁺ -ATPase in the two E2P states, explaining their different biochemical properties established a half century ago. The new electron microscopy maps show previously unseen structural features, including unexpected binding modes of cardiotonic steroids, specific and medically important inhibitors of the ATPase, and stabilization by ATP of the E2P state.

Significance: Cardiotonic steroids (CTSs) are specific inhibitors of Na ⁺ ,K ⁺ -ATPase and have been studied over a long period of time because of their potential therapeutic use in heart failure, hypertension, and cancers. Their affinities and binding kinetics are vastly different and seemingly rather inconsistent in the literature. Recent development of nonconventional CTSs with unexpected behaviors has revived interest in CTSs as potential drugs. In this report, we carried out a systematic study on various CTSs, including rostafuroxin and istaroxime, using kinetics and X-ray crystallography of their complexes with Na ⁺ ,K ⁺ -ATPase, and explain how CTSs block the reaction cycle and why such large differences in inhibitory behaviors arise. The analysis suggests how isoform specificity may be achieved in order to improve their clinical usability.

Circular dichroism of an H⁺, K⁺-ATPase N-terminal peptide at varying trifluoroethanol concentrations is investigated, indicating that its secondary structure is environmentally sensitive.

There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs). Freestanding GUVs are transferred onto a mica support, and after treatment with magnesium chloride, they collapse to form planar lipid bilayer (PLB) patches. Rapid GUV collapse onto the mica preserves the lateral organization of freestanding membranes and thus makes it possible to image 'snapshots' of GUVs up to nanometer resolution by high-resolution microscopy. The method has been applied to classical lipid raft mixtures in which suboptical domain fluctuations have been imaged in both the liquid-ordered and liquid-disordered membrane phases. High-resolution scanning by atomic force microscopy (AFM) of membranes composed of binary and ternary lipid mixtures reconstituted with Na+/K+-ATPase (NKA) has revealed the spatial distribution and orientations of individual proteins, as well as details of membrane lateral structure. Immunolabeling followed by confocal microscopy can also provide information about the spatial distribution of proteins. The protocol opens up a new avenue for quantitative biophysical studies of suboptical dynamic structures in biomembranes, which are local and short-lived. Preparation of GUVs, PLB patches and their imaging takes <24 h.