Protein of the Year!

Okay, I’ll keep this short.

The sodium potassium pump is more important than we realize. It is involved in a wide range of cellular and physiological functions. Here's a quick list of just a few things:

1. Insulin secretion from the pancreas
2. Neuron action potentials (70% of ATP in neurons is for my protein)
3. Heart contraction
4. Import of glucose and amino acids (via facilitated transport)
5. Absorption of water by the kidneys

Bound Potassium Ions

Depending on cell type, there are between 800,000 and 30 million pumps on the surface of cells (1). The sodium potassium pump (Na⁺/K⁺ ATPase) exports 3 Na⁺ ions and then imports 2 K⁺ ion and this ability is very important for establishing an electrochemical gradient across the cell membrane. This gradient is part of most cellular functions dealing with the transport of small molecules or ions across the cell membrane. The electrochemical gradient is so central that 25% of the total energy consumption of a resting human is used for this end. This protein is also a drug target for people with congestive heart failure because by inhibiting this pump, ion concentrations are disrupted and results in more forceful heart contractions. While just about every protein is important for an organism’s survival, few are involved in as many different essential functions as the sodium potassium pump.

 
The sodium potassium pump is also structurally impressive. It is a relatively large protein which spans the cell membrane. The central alpha helices form a tunnel (purple) for sodium and potassium ions and can selectively bind Na⁺ or K⁺ depending upon its state (phosphorylated or dephosphorylated). The army green region is where the ATPase function is located. The yellow spheres represent phosporylation. The discovery of this protein resulted in a Nobel Prize in 1997. Thus, it goes without saying, this protein deserves to be in the protein hall of fame.



For a brief function summary, see the figures below.

Overview of ion pumping cycle

Gradient Quantified

And let's wrap up with some quick pictures.

 

Ouabain - an inhibitor is bound

1. http://www.vivo.colostate.edu/hbooks/molecules/sodium_pump.html

Information about the sodium-potassium pump.

Here are some recent publications (with crystal structures) concerning Na⁺, K⁺-ATPase.

The Na⁺,K⁺-ATPase generates electrochemical gradients across cellular membranes by transporting 3 Na⁺ out and bringing 2 K⁺ into the cell. This enzymatic pump binds ATP and becomes phosporylated which causes a conformational change which releases Na⁺.  Two K⁺ ions bind and cause hydrolysis of P_i which releases K⁺ into the cell. The two conformations of the protein have been termed E1 and E2. The E1 conformation selectively binds sodium while the E2 conformation selectively binds potassium. The sodium-potassium pump is often compared to Ca²⁺-AtPases because they have very similar in functions/mechansims. Note: MgF₄^2- is an analogue of P_i and Rb⁺ is often used as an analogue of K⁺. The claw like structure is on the cytosolic side of the cell.

Deletion of the five most C-terminal residues of  Na⁺, K⁺-ATPase resulted in a 26-fold loss in Na⁺ binding. The authors suggest that the terminal residues interact with an adjacent region which constitutes the Na⁺ binding pocket so its presences helps optimize the sodium binding locations. Asp-369 is a putative site of phophorylation. 

[Morth et al. Nature 2007, 450, 1043-1049.]

The potassium binding is implicated in the dephosphorylation of the enzyme. There are two binding sites for K⁺ ions in the intermembrane section of the protein. One site is formed by essentially five oxygen atoms: one main chain (Thr 779), three side chains (Ser 782, Asn 783 and Asp 811) and one water molecule. The second K⁺ binding site is only 1.3Å away. It is created by three main chain carbonyls (Val 329, Ala 330 and Val 332), three or four side-chain oxygen atoms (Asn 783,Glu 786, Asp 811 and possibly Glu 334) and no water molecules.

The cholesterol molecule appears to shield part of the protein from the hydrophobic lipid core. Na⁺, K⁺-ATPase activity is dependent on cholesterol so this protection from the lipid core is important. Another important chain is the FXYD unit which is believed to be an accessory regulatory protein. In the pictures below, the blue color is the alpha subunit and the green colored portion is the beta subunit. The pink colored region is the FXYD accessory protein which is believed to have a regulatory affect on the enzyme.

[Shinoda, T.; Ogawa, H.; Cornelius, F.; Toyoshima, C. Science 2009, 459, 446.]

Ouabain is an inhibitor of Na⁺, K⁺-ATPase. Many different therapies for congestive heart failure target the sodium pump because blocking the enzyme increases intracellular [Na⁺]. This build up of Na⁺ stops a certain Na⁺/Ca²⁺ exchanger which increases intracellular [Ca²⁺], thereby increasing the strength of heart contractions.

This crystal structure shows Ouabain bound in a low affinity state, but it is very similar to the high affinity state. The bound potassium ions have likely prevented the protein from folding closed into a high affinity state (for Ouabain). Ouabain binds deeply within the transmembrane α-helical region of the protein. The binding of Ouabain disrupts the binding pocket for K⁺ and can also block potassium ions from dissociating from the core of the protein. Ouabain does not bind as strongly in the presence of K⁺ because they compete for some of the same residues, but obviously with Ouabain’s size, the protein cannot function in the presence of Oubain.  Ouabain’s lactone ring and the interactions it has with the protein are very important for binding and there are many mutations which remarkably lower Ouabain binding affinity.

[Ogawa, H.; Shinoda, T.; Cornelius, F.; Toyoshima, C. Proc. Natl. Acad. Sci USA 2009, 106, 13742-13747.]

Na/K ATPase