Sodium-Potassium Pump Bound with Potassium and Ouabain
Created by Angelica Verdan
The sodium-potassium pump (3A3Y) from Squalus acanthias is an ATPase crucial to maintaining the concentration gradient and action potential across the plasma membrane. This protein pump catalyzes the exchange of three sodium ions for two potassium ions against their concentration gradients by hydrolyzing ATP. The generated action potential across the membrane is then used to energize other transport processes across the plasma membrane. Na+/K+-ATPase in particular is targeted when treating congestive heart failure. Ouabain is a cardiac glycoside, a drug which inhibits the sodium-potassium pump. The drug’s administration increases the contractile force of the heart muscle, slows the rate of beating, and can restore normal function to the heart (Garrett, 2). The molecular weight of the sodium potassium pump is 113,191.11Da and the isoelectric point (pI) of the protein is 5.37, provided by the Expasy Database (Expasy, 1).
Na+/K+-ATPase physiologically exists as a heterotrimer. There are three subunits and each has a single chain. The alpha subunit is responsible for most of the protein’s functions. The alpha subunit is 1023 residues long. It is 45% helical (48 total helices, ten of which are transmembrane alpha helices) and 14% beta sheet consisting of thirty-four strands (3A3Y, 8). The alpha subunit also contains three cytoplasmic domains: the actuator domain (A), phosphorylation domain (P), and nucleotide-binding domain (N). These are involved with binding sodium, potassium, and ATP. The beta subunit is 305 residues long. It is 16% helical (seven total helices) and 16% beta sheet (14 strands) (3A3Y, 8). The beta subunit has a critical role in binding potassium to the ATPase. Only one of its alpha helices is transmembrane and primarily lies on the extracellular side of the membrane. The beta subunit largely coordinates the occlusion or release of the sodium and postassium ions and directs the alpha subunit to the plasma membrane. The gamma subunit, also called the phospholemman-like protein, only consists of 74 residues, but contains a conserved FXDY motif. It is 35% helical with two helices and no beta sheets (3A3Y, 8). The gamma subunit regulates the protein’s activity. Because Na+/K+-ATPase is an integral membrane protein, hydrophobic residues interact with the phospholipid bilayer, while the extraceullar and intracellular regions include hydrophilic residues.
The main function of Na+/K+-ATPase is to maintain the concentration gradients of sodium and potassium ions across the plasma membrane. Three sodium ions are pumped out of the cell and two potassium ions are pumped into the cell when ATP is hydrolyzed. The Na+/K+-ATPase essentially cycles between two main conformations, E1 and E2, for the reaction to occur (Garrett, 2). In the E1 conformation, the Na+/K+-ATPase has a high affinity for sodium and ATP. Once sodium and ATP are bound, Asp-369 in the P domain is phosphorylated, releasing ADP and changing into the E1-P conformation, occluding the three sodium ions. These three sodium ions are at site I, II, and the carboxy-terminal of the protein. Site I is made up of five oxygen atoms from Thr-779, Ser-782, Asn-783, Asp-811, and one water molecule. Site II is coordinated by three main-chain carbonyls: Val-329, Ala-330, and Val-332 (Shinoda, 10). The release of the sodium ion at the carboxy-terminal results in the conformation change into E2-P. The two remaining sodium ions are released and two potassium ions are then bound at site I and II. The sodium-potassium pump dephosphorylates the P domain which occludes the potassium ions. The release of potassium ions returns the Na+/K+-ATPase to its E1 conformation (Garrett, 2).
Ouabain binds to Na+/K+-ATPase when it is in the E2-P conformation. Ouabain binds and stabilizes the occlusion of the potassium ions. As a cardiotonic steroid, it includes a lactone ring, steroid core, and a carbohydrate moiety. Ouabain has a 5-membered lactone and has rhamnose as its carbohydrate (Ogawa, 8). Ouabain is then wedged deeply between the transmembrane M5 and M6 helices, partially unwinding the M4 helix. The extraceullar half of the M4 helix moves away from the M6 helix, resulting in a larger cavity surrounded by the M1-M2 and M4E-M6 helices. The lactone ring of ouabain pushes Gly-326 away and breaks the hydrogen bonds betweenVal-329 and Ala-330 (Ogawa, 8). These two residues form new hydrogen bonds with the carbonyl conjugated to the lactone ring. This unwinds the M4E helix by one turn. The coordination of the sodium ions is partially destroyed because Val-329, which provides the carbonyl oxygen to sodium site II, is displaced. The proximity of the lactone ring to this site thus explains why bound potassium cannot dissociate (Ogawa, 8). Phe-323, Phe-790, Phe-793 are all critical hydrophobic residues that bind the steroid core of ouabain. Thr-804 is another crucial residue for binding the steroid core. The rhamnose in ouabain hydrogen bonds with Arg-887 and Glu-319. The presence of rhamnose increases ouabain’s affinity in comparison to other cardiac glycosides without a sugar moiety (Ogawa, 8).
Other ligands are also essential to Na+/K+-ATPase. Cholesterol interacts with Tyr-40 and Tyr-44 and shields the unwound section of the M7 helix from the bulk lipid. The water molecules interact with the potassium ions. N-acetyl-d-glucosamine was used to stabilize the Na+/K+-ATPase. Magnesium ions interact with and stabilize ATP. Tetrafluoromagnesate(2-) (MgF42-) was used as a phosphate analogue in the phosphorylation site where ATP normally binds (Shinoda, 10).
Because Na+/K+-ATPase is crucial to many animal cells, the P-type ATPase is highly conserved. The PSI-BLAST search, which compares proteins’ primary structure, provides an E value comparing the similarity between many proteins. The lower the E score, the more similar proteins are. A PSI-BLAST search revealed only results with E scores of 0.0, relaying the high conservation of Na+/K+-ATPase and other P-type ATPases (BLAST, 9). The Dali server compares the tertiary structure of a protein by comparing differences in intramolecular differences and provides a Z-score. The higher the Z-score, the more similar the folds are. The Dali server search provided high Z-scores, some upwards of 70 (Dali, 3). This again relays the high conservation of Na+/K+-ATPase across organisms.
The Na+/K+-ATPase with bound ouabain (3N23) in Sus scrofa had a Z-score of 55.0 and an E value of 0.0. Though both are sodium-potassium pumps bound to ouabain, there were still a few differences. The Na+/K+-ATPase in Sus scrofa has six chains for three subunits. The alpha subunit has chains A and C. The beta subunit has chains B and D. The gamma subunit has chains E and G. The total amount of residues is shorter as well (1300 versus 1407). The Na+/K+-ATPase also has an aspartyl phosphate, modified from aspartic acid (PDB, Yatine, 11).
The sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA) in Oryctolagus cuniculus (3FGO) had a Z-score of 49.0. Instead of pumping sodium and potassium across their concentration gradients, SERCA pumps calcium ions against its concentration gradient. This is essential for muscle movement. It is another P-type ATPase. Cyclopiazonic acid (CPA) is used as an inhibitor of SERCA instead (Laursen, 4). SERCA is much smaller in comparison to the Na+/K+-ATPase as it only has one subunit with an A and B chain. It has a total of only 991 residues (PDB, Laursen, 5). The alpha subunits are structurally similar as they contain similar percentages of helices and sheets, though their ligands are different. The CPA binding pocket lies between transmembrane segments M1, M2, M3, and M4 of SERCA which is close, but not the same as the binding of ouabain. The hydrophobic indole group of CPA sits in-between M3 and M4, whereas the tetramic acid moiety has polar interactions with M1 and M2 (Laursen, 4). CPA effectively stabilizes the E2-P state and occludes the calcium ions from escaping.