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Sodium-Potassium Pump

Monica Kriete '11 and Mary Clare Higgins-Luthman '11


I. Introduction

The transmembrane sodium-potassium ATPase is of key importance in living systems. Three sodium ions are pumped out in exchange for the uptake of two potassium ions by utilizing the Gibbs free energy of ATP hydrolysis. This enables cells to establish and maintain an electrochemical gradient used to maintain cell volume and pH. This also provides the energy for secondary active transport of other ions and nutrients, such as protons.

The sodium-potassium ATPase belongs to the P-type ATPase family, so named for the reversible autophosphorylation of an aspartate residue as part of ATP hydrolysis (Brampkamp et. al. 2007). P-type ATPases transport cations, including heavy metals (transported by P1-ATPases) and non-heavy metals, such as sodium, potassium, and calcium (transported by P2-ATPases) (Lutsenko and Kaplan, 1993).

Although most P-type ATPases are single-subunit proteins (Geering), the Na+ ,K+ -ATPase is comprised of three subunits, named alpha, beta, and gamma, all of which are necessary for correct functioning (Morth et. al.). The α-subunit is homologous to single-subunit P-type ATPases, such as the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). The β-subunit is necessary for routing of the alpha-subunit to the plasma membrane, and also plays a role in ion occlusion during exchange. The γ-subunit regulates pumping activity tissue-specifically.

The Na+ /K+ pump is shown crystallized in the E2 state (see below) bound to MgF42-, a phosphate analogue , and two Rb+ ions, which are K+ analogues .


II. Mechanism

The P-type ATPases contain three highly-conserved domains: the actuator (A) domain ; nucleotide-binding (N) domain ; the and the phosphorylation (P) domain . These three domains effect conformational changes in response to ligand binding that result in ion exchange across the cell membrane. In the Na+ /K+ pump, all three domains are contained in the alpha subunit, along with the Na+ and K+ binding sites.  Two sites are present between helices 4, 5 and 6 that serve to bind two K+ or two of the three Na+ ions, which in this model are shown bound to two Rb+ ions. The third Na+ binding site is found on the carboxy-terminal.

In the Na+/K+ pump reaction cycle,three Na+ ions bind to the enzyme in the E1 state, open to the lumen of the cell, with ATP already bound to the N domain. Sodium-ion binding causes movement of the A domain, which results in the occlusion of two of the three sodium ions and the subsequent hydrolysis of ATP at the P domain. The resulting ADP and carboxy-terminal Na+ ion are released to the exterior of the cell, and the enzyme adopts the E2 conformation, open to the exterior of the cell. The remaining two Na+ ions are exchanged for two K+ ions. The enzyme occludes the K+ ions with inorganic phosphate still bound. The phosphate is coordinated by loop of Thr 212, Gly 213, Glu 214, and Ser 215.  Although one of the carboxyl oxygens in Glu 213 is at a hydrogen bond distance from a  fluorine in  MgF42-(the phosphate analog), the high  electronegativity of fluorine makes this conformation unstable. Therefore, Glu 213 may be forced into this configuration by surrounding residues (Toyoshima).  A water molecule replaces the inorganic phosphate, after which ATP-binding prompts conversion back to the E1 state. The K+ ions are released to the cell lumen and the process begins again.  A clearer depiction of the conformational changes between states is given for another P-type ATPase, SERCA.


III. Alpha-Subunit: Potassium Binding

In addition to the A, N and P domains discussed earlier, the α-subunit also contains a transmembrane domain consisting of ten alpha-helices designated αM1-10 αM4 and αM6 are partially unwound to make room for the K+ ions, as in SERCA.

The binding sites for K+ are contained within the transmembrane domain, between helices 
αM4αM5, and αM6, and are designated 1 and 2 . Several residues are involved in ion binding and stabilization. Asp 804 donates a side-chain oxygen ligand to each site .Glu 327 donates a side-chain oxygen to site 2 . The side chains of residues Ser 775, Asn 776, and Glu 779 could all potentially donate ligands for binding, either directly or through an intervening water molecule, and have been assigned roles in liganding by mutagenesis. Asp 808 and Gln 923 may also be involved . Identical or closely-related residues are found at corresponding positions in SERCA. Therefore, cation selectivity must be determined by subtle differences in the side chains of the cation-binding residues between SERCA and the Na+/K+ pump.

IV. Alpha-Subunit: Sodium Binding

Two of the Na+ ions involved in exchange bind to the same sites as K+; the third is proposed to bind to a site comprised of the C-terminal end of the alpha-subunit and several nearby residues. The C-terminal end of the alpha-subunit is extended eight residues relative to SERCA. The first six residues of this extension form an α-helix which is accommodated between the transmembrane helix of the beta-subunitαM7, and αM10 . The final two residues of the extension, Tyr 1015 and Tyr 1016   are recognized by a binding pocket between αM5αM7 and αM8 . Tyr 1016 seems to interact with Lys 766 and Arg 933 in the loop connecting αM8 and αM9. Tyr 771, Thr 807, and Glu 954 are the other residues proposed to be involved this binding site, based on mutagenesis studies. Due to their distance from the C-terminal end, it is likely that these residues stabilize interactions between the helices rather than coordinating the ligands.
The region surrounding the C-terminal extension is highly electropositive, containing six arginine residues . In other types of ion channels, clusters of arginine residues act as voltage sensors that move in response to membrane depolarization; thus, Morth et. al propose that this cluster could function similarly to control the voltage-sensitivity of the Na+/K+ pump.

V. Beta and Gamma Subunits

The Beta Subunit:

The β subunit directs the α subunit to the plasma membrane. Only the transmembrane segment of the β subunit, an α helix, was able to be crystallized. This helix makes direct contact only with αM7and αM10 helix of the alpha subunit. Three amino acid residues, Tyr 39, Phe 42, and Tyr 43, interact with αM7 around Gly 848. The extracellular domain of the β subunit contains a large glycosylated region and is thought to cover the alpha subunit, preventing K+ escape.

The Gamma subunit:

The transmembrane segment of the ɣ subunit is also an alpha helix that interacts minimally with the alpha subunit. Gly 41 of the ɣ subunit interacts with Glu 953 of αM9 of the alpha subunit. Several other residues on αM9, Phe 949, Leu 957, and Phe 960, are thought to interact with the ɣ subunit based on a mutagenesis study . The extracellular domain of the ɣ subunit contains a conserved FXYD motif and moves between the alpha and beta subunits to regulate enzyme activity.

VI. References

Bramkamp, Marc, Karlheinz Altendorf, and Jorg-Christian Greie, 2007. "Common patterns and unique features of P-type ATPases: a comparative view on the KdpFABC complex from Escherichia coli (Review)." Molecular Membrane Biology 24(5-6): 375-386.

Geering, Kathi, 2001. "The Functional Role of Beta-Subunits in Oligomeric P-type ATPases." Journal of Bioenergetics and Biomembranes 33: 425-438.

Lutsenko, Svetlana and Jack H. Kaplan, 1995."Organization of P-Type ATPases: Significance of Structural Diversity" Biochemistry 34, No. 48: 15607-15613.

Morth, J. Preben, et al, 2007. "Crystal structure of the sodium-potassium pump." Nature 450: 1043-1049.

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