A Tour of the sodium-potassium pump

Please be patient while the structures in the left frame load. In order to display all of the structures in the tour properly, press 'View' buttons below in order (from 1 to the end).

Pumps are the active transporters: they require energy to catalyze the transport of cations through the cell membrane. The energy required for the pump function can come from light (for example, photosynthetic reaction centers and proton pumping), from a redox process (complexes I to III in mitochondrial membrane) or from hydrolysis of ATP (ATPase pumps). If the mechanism of ATPases involves a phosphorylated enzyme intermediate, then the ATPase belongs to a P-type ATPase family.

The sodium-potassium (Na +-K+) pump is an example of P-type ATPase pump that moves three Na+ ions out and two K+ ions into the cell for each ATP hydrolyzed. The action of Na +-K+ pump maintains a resting membrane potential of -30 mV to -70 mV in mammalian cells. (The potential is negative on the inside of the membrane.)

During the pumping cycle, the pump alternates between two major conformations E1 and E2 (E stands for enzyme). In the E1 conformation, the metal binding sites have high affinity for the metal cations and are open to the cytoplasm. The E2 conformation opens the same metal binding sites to the extracellular environment and changes the metal binding affinity to low. The metal ions are not transported through the membrane but are held at fixed positions within the protein structure while the protein exposes the binding site alternatively to the extracellular and intracellular sides of the membrane. The pump adopts several different states (also known as cycle intermediates or pump forms) in each conformation that differ based on phosphorylation and cations bound. To see these different states and a proposed mechanism, click on thumbnail below.

The display in the left frame shows a ball-and-stick model of the structure of Na +-K+ pump in its E2.2K+.Pi state isolated from shark rectal glands. The protein consists of three different subunits making it an αβγ heterotrimer.

The α subunit contains about 1100 amino acids and is the largest one. The upper half of this subunit is embedded inside the membrane while the bottom half is located in the cytoplasm.

The β subunit has about 100 amino acid residues. Only one helix passes through the membrane while the rest of the subunit is exposed to the extracellular space (a red globule at the top of the structure).

The γ subunit is the smallest one with about 50 amino acids in the primary structure (30 of which form a transmembrane helix). This subunit is also known as the regulatory FXYD protein after a highly conserved FXYD sequence (see below).

Note that the secondary structure of all subunits is almost exclusively composed of α helices. This helix-rich secondary structure provides the protein with flexibility necessary for achieving two distinct conformations.

The α - Subunit

The α-subunit of this Na +-K+ pump consist of four distinct domains.

The nucleotide binding domain (or N-domain) is found in the cytoplasm. It is in charge of binding the ATP and of phosphorylation of P-domain.

The actuator domain (or A-domain) is the protein phosphatase. It is connected to the upper parts of the α subunit through several very flexible hinges (upper part of the domain). This connection allows the A-domain to move relatively freely relative to the rest of the subunit.

These two domains are connected through a salt bridge formed between Arg551 on N-domain and Glu223 located on A-domain. The mutation experiments suggest that this salt bridge is the location of ATP binding. Once ATP binds, the salt bridge is broken and the N- and A-domains are pushed away from each other. This movement exposes the P-domain for phosphorylation.

The phosphorylation site is located in the phosphorylation domain (or P-domain). This domain is highly conserved among all P-type ATP-ases.

Asp376 is the residue that gets phosphorylated.

[MgF4]2- is found in close proximity to Asp376. This anion is frequently used as a mimic for free inorganic phosphate (Pi) in protein crystallography.

One potassium cation is located on the protein surface, on the upper part of the P-domain. Its role appears to be primarily structural (it is not transported across the membrane) and some evidence suggest that it assists during the phosphorylation process.

It is a five-coordinate cationic center with all O-donor ligands. Four donor atoms are neutral with three coming from C=O bonds in the protein backbone (Ala728, Leu725 and Lys726). The fourth is oxygen atom from a loosely bound water molecule. The only negatively charged residue is carboxylate from Asp747.

The last domain is the transport domain (or T-domain). It is a highly flexible bundle consisting of 10 α- helices. Note the flexible hinges that connect T- and A- domains on the left hand side of the display.

The Na+/K+ binding site is located approximately in the middle of T-domain.

The potassium cations are coordinated to the protein by oxygen atoms (red spheres). (The red wireframe structure in the background is a transmembrane segment of the β- subunit.)

The K+ cation closer to the surface of the protein is coordinated by three mainchain carbonyls (Ala330, Val332 & Val329) and three side chain oxygens (Asn783, Glu786 & Asp811). The geometry at this K+ is distorted octahedral.

The second potassium cation is buried deeper inside the T-domain and is coordinated by one main-chain oxygen (Thr779), three side-chain oxygens (Ser782, Asn783 & Asp811) and a water molecule (HOH). The geometry at this K+ center is distorted square pyramidal. Note that oxygens from Asn783 and Asp811 carboxylate groups serve as bridging ligands between two potassium sites.

Three sodium cations bind in the same pocket, but the exact locations and coordinating residues are unknown due to the lack of crystallographic data on sodium-bound Na+-K+ pump.

The β - Subunit

The β-subunit is a 45 kDa protein containing about 170 amino acid residues. It secondary structure is predominantly composed of α-helices. The top part is exposed to the extracellular space. There is only one transmembrane helix, positioned diagonally with respect to the T-domain of the α-subunit. It is has been show that this domain influences K+ affinity: after a complete or partial removal of this domain the affinity for the two cations drops although the pump still performs its function properly. The exact mechanism of the affinity control remains unknown.

The N-terminal of β-subunit contains a highly conserved FYXXFY (Phe-Tyr-X-X-Phe-Tyr) motif, where X residues are hydrophobic (in this case Ile and Leu). The β-subunit interacts with the α-subunit through two Tyr residues of this conserved sequence. This interaction is probably important for the aforementioned affinity control.

The γ - Subunit

The γ-subunit is a small α-protein consisting of about 35 residues. It belongs to a larger family of FXYD regulatory proteins (named after their FXYD characteristic sequence). These regulatory proteins associate with Na+/K+ and some other pumps and regulate their activity in a tissue as well as isoform specific way.

The four residues comprising the conserved sequence are shown here. The X residue in this structure is Thr13. Together with Tyr16 (next residue in the sequence; not shown) these anchor the γ-subunit to the other two pump subunits.

Click on the thumbnail below to see a visual summary of the Na+-K+-ATPase pump structure:

This is the full structure of Na+-K+ pump: feel free to play with this and any other display in this tour.

The structure of Na+-K+ pump was reported by Shinoda, T.; Ogawa, H.; Cornelius, F. and Toyoshima, C. in Nature 2009, 459, 446 (PDB ID 2ZXE).

Other useful sources:

Morth, J. P. et al. (2011). A Structural Overview of the Plasma Membrane Na+-K+-ATPase and H+-ATPase Ion Pumps. Nature Reviews, 12, 60-69. (A very useful current review.)

Morth, J. P. et al. (2007). Crystal Structure of the Sodium-Potassium Pump. Nature, 450, 1043-1050. (An earlier, lower resolution structure of pig renal Na-K pump; PDB ID 3B8E. The text is more informative and detailed than the one cited as a source for this tour.)

Palmgren, M. G., and Nissen, P. (2011). P-Type ATPases. Annu. Rev. Biophys., 40, 243-266. (An excellent current review addressing major P-type ATPases, including Na+-K+-ATPase.)

Tour Copyright: Robert Morris and Alen Hadzovic, 2012.