29 Pumps and ATPases

Prof. M. N. Gupta

1. Objectives

•  To learn how early studies on Na+-K+ pump contributed to our understanding of pumps or transporters.

•  To learn the differences between uniporters, symporters, and antiporters

•  To learn about multidrug transporter and cardiac glycosides

2. Concept Map

3. Description

We now come to the function of the membranes which was historically the first one to be recognised. How do membranes allow transport of the molecules in a regulated fashion?

We look at the transports in the membranes which carry out this function. In the next module we will look at the membrane channels.

A well known example of a pump is the Na+/-K+ Eukaryotic cells maintain a Na+/K+ gradient across its plasma membrane. This also ensures a membrane electrical potential.

Operation of Na+/K+ pump is one kind of ATP-dependent transporter of molecules. The cysteic fibrosis trans-membrane conductance regulator is the second type of transporter which requires energy from ATP hydrolysis.

Ion channels open up/allow ions to pass through when a specific ligand binds the ion linked channel receptors. A well known example of such a specific ligand is acetyl choline at the nerve-muscle junction.

Majority of animal cells have a high [K+/Na+] concentration inside the cell. This is maintained by the Na+-K+ pumps. The movement of these ions require energy in the form of ATP. The enormity of the importance of Na+-K+ pump can be judged by the fact that about 33% of ATP hydrolysed by a resting animal happens for operating this pump.

The Na+-K+ gradient is responsible for many phenomena. It ensures that cell volume remains within a narrow range at all times. It is also responsible for nerve cells and muscle cells being responsive to the electrical stimuli.

The Na+-K+ pump is also responsible for ensuring the transport of sugars and amino acids. It is due to this, that term pump is increasingly replaced by transporter in the recent literature.

Jens Skon, way back in 1957 found that an enzyme, an ATPase which functions only if Na+, K+ and Mg++ were present. The requirement of Mg++ was not a surprise as all ATPases are known to require Mg++.

The requirement of Na+ and K+ was an unusual feature so the enzyme came to be known as Na+-K+ ATPase. Hence, the pump is nothing but an enzyme. The other term transporter also should not cause confusion. What we have is an enzyme which is a hydrolase!

It turns out that all transport proteins or transporters are asymmetric integral membrane proteins. These are capable of existing in two conformers which can interconvert. The transition between two conformational states of the transporter is analogues to the interconversion of allosteric proteins as per the two step model.

In fact, many other features of allosteric proteins are common to tranporters. Such transporters include some which are involved in transport of more than one specie.

Hence, the transporters are classified into uniports, symports and antiports. This classification is based upon the feature of their being involved in transport of a single or more than one specie at the same time and whether the movement is in the same or a different direction.

Uniport catalyses the transport of only one specie at any time. A well known example of this is glucose transporter in red blood cells.

Symport transports two species at the same time. Even the direction of the transport is same for both species.

Some authors prefer a general term of co-transport systems to include both symport and antiports.

Antiports are transporters that involve simultaneous movement of two different species in opposite directions. For this, a well known example is available in the form of chloride-Bicarbonate exchanger.

It may be added that this classification does not worry about whether the transport is active or passive. Only active transport which involves ATP hydrolysis can be viewed as an enzymatic process.

It may be useful at this stage to introduce another classification from the perspective of ATP hydrolysis. Evolution has resulted in design of various kinds of ATPases which differ in structure, mechanistic details and localization within the cells.

The first category is that of P-type ATPases. The key feature of these is reversible phosphorylation of an Asp residue and inhibition by a phosphate analog Vanadate. The structural similarity between the two ions makes the vanadate inhibition understandable.

P-type ATPase have wide distribution. All animal tissues have Na+-K+  ATPase (an anti-porter), Ca++.

ATPase which is a uniporter for Ca++.

These help to maintain ionic gradient between cytosol and extracellular milieu. As an example of their physiological significance, it is a P-type ATPase in cells lining the stomach which ensures acidity in the stomach.

In higher plants, an ATPase creates a difference of 2 pH units across the plasma membrane that creates the electrochemical gradient, Bacteria also operate a P-type ATPase pump to get rid of the metal ions such as Ca2+, Hg2+ from their cells.

The uptake of substances and ionic species by neurospora also is carried out by a pump which drive out H+.

P-type ATPases are characterized by generally having two types of subunit structures. It is the α-unit which has the critical Asp which is involved in phosphorylation.

Not only asp, but sequence around this critical residue is also highly conserved among P-type ATPases. For example, sheep kidney Na+-K+ ATPases, rabbit cardiac Ca2+-ATPase, rat gastric H+-K+ ATPase and E. coli K+-ATPase, which are all P-Type ATPases have considerable homology in the sequence ground the asp residue.

The P-type pumps or transporters or sometime called P-type ion-motive ATPases have a mass of 100 KD and are reversibly interconverted between phosphorylated and dephosphorylated forms.

Their existence is very old as shown by the example of E coli K+-ATPase. The similarity of sequence around the Asp in both bacterial and animal pumps shows that evolution did not tinker much with the design of the P-type ATPases.

While P-type ATPases are more versatile as they transport variety of species, V-types transporters only H+.

They are called V- type as these occur in membranes of vacuoles in yeast and fungi and in plants.

V type ATPase are bigger in size (>400 KD). In these, different subunits are involved in steps like ATP hydrolysis and proton movement. Understandably, these processes are coupled conformationally. This implies that these conformational transitions of the molecule cannot be analyzed by a simple 2 step model.

While these were named V-type after vacuoles, these are not restricted to just yeast, fungi and plants. In animals V-type ATPase are part of the membranes of lysosomes, endosome and secretory vesicles.

V-type ATPase also play an important role in clatherin coated vesicles and golgi apparatus. Thus, the proton movement is coupled to protein targeting.

In fact, acidifying intracellular compartments is their general role. The vacuoles of fungi and plants are able to maintain pH in the range of 3-6, (lower then cytosolic pH) due to the action of V-type ATPases.

V-type ATPase do not undergo reversible phosphorylation cycles. Thus, absence of inhibition by vanedate ion is a good way to distinguish between P-type and V-type transporters.

The low pH in some of these compartments activates proteases and other hydrolytic enzymes. Then trans-membrane domain Vo is a H+ channel. The peripharal domain V1 has ATPase activity.

The V-type ATPases are closer to F-type ATPases. The oxidative phosphorylation by mitochondria coupled to the respiratory chain is the well known example of F-type ATPase.

Hence F-type ATPase involves H+ gradient, however their physiological function is to form ATP rather than hydrolysing it.

While F-type ATPases occur in inner mitochondrial membrane in eukaryotes, higher plants have these in thylakoid membranes. In prokaryotes, these ATP synthesizing enzymes are part of the plasma membranes. Structurally, they have Fc domain which spans membranes and F1 catalytic unit. Sometime F-type ATPases are called ATP synthases. The latter F refers to factors, in early days of work on oxidative phosphorylation, many new proteins which were discovered were simply called factors.

Many eukaryotic cells contain transports of all the above three types. Prokaryotes do not have V type ATPases. Eukaryotes carry out exocytosis and endocytosis. Hence, from evolutionary point of view, V type ATPases, are more recent.

The F1 unit when isolated as a soluble factor acts as an ATPase rather then ATP synthase. So, it is the protein gradient as a result of redox activity of respiratory chain which drives ATPase towards synthesis function.

In mid-1980s, it was found that many tumors were found to be resistant to antitumor compounds. This led to the discovery of fourth type of ATPases which are called multi-drug transporters.

It was found that these transporters present in the plasma membrane of these tumor cells could expel many diverse drugs so, the concentration of the antitumor drugs never reached an effective concentration.

The multidrug transporter is a 170 KD size integral protein having 12 trans-membrane segments and 2 ATP binding sites. The transporter also functions like an ion channel. That function is ATP-independent.

The multidrug transporter is effective in removing both natural and synthetic drugs from the cytosol. The list induces vinablastine, doxorubicin, actinomycin, mitomycin, taxol, colchicine, and paromycin. The common feature is the hydrophobicity of the drugs.

Na+-K+ pump or (Na+-K+)-ATPase is among the most extensively studied active transport system as far as eukaryotic plasma membranes are concerned. As we mentioned earlier, Jens Skon was the person who initiated work on this pump.

The ATPase is a transmembrane protein with two types of subunits. The two α-subunits are nonglycosylated and are of 110KD each and are the sites of binding of ions and catalysis. The 55KD β-subunits (again two in number) do not appear to have any known function except perhaps aid in formation of α2β2 hetrotetramer.

The hydrolysis of one ATP molecule is accompanied by phosphorylation of the asp residue in the protein. The resulting conformational change leads to 3 Na+ ions pumped out and 2 K+ ions pumped in across the plasma membrane.

Both ions move against the concentration gradient. It is active transport and requires the energy in the form of ATP hydrolysis.

The ATP hydrolysis and the pumping of ions in opposite directions is coupled system. Either phenomenon does not occure in isolation.

The ATPase clearly is an antiport and generates a charge separation across the membrane. Expulsion of 3 Na+ is necessary for animal cells to control the osmotic pressure of the cytoplasm. Animal cells do not have supporting cell wall. In the absence of the pump, water would come in, swell the pump, and would lead to its bursting.

The phosphorylation of the Asp residue requires the presence of Na+. Similarly, its dephosphorylation cannot takes place in the absence of K+.

The α-subunit has 8 trans-membrane helices with 2 cytoplasmic domains. It is the cytoplasmic domain at which ATP binds. β-subunit has only one α-helical transmembrane segment but bigger extracellular domain. It is there that the β-subunit is glycosylated.

The pump is present in all cells wherein active transport of Na+ and K+ takes place. Some cells which require this transport more have higher activity. For example, nerve cells are richer in it as compared to RBCs.

When Skon observed the transport and the ATPase activity, it took some brilliant work to establish that the pump and ATPase activities are due to same structure.

For example, it was also found that Na+-K+ concentration gradients affects the pumping action and ATPase activity same way.

There are two cardiac or cardiotonic steroids which inhibit both pumping action and ATPase activity. The Ki of inhibition for both activities by either of steroid derivatives is identical.

The action of these cardiac glycosides helped in gaining insight into Na+-K+ pump. The first one is from digitalis. Degitalis is an extract of purple foxglove leaves and is a mixture of several glycosides. The prominent among those is digitoxin. Digitalis has been around for centuries as a natural product used to treat congestive heart failure.

In fact, it has been shown in the steroid part in this compound called Digitoxigenin unsaturated lactone ring at C-17 is necessary for the physiological effect. Not only that –OH group at C-14 and a cis-fusion of C and D rings are also necessary. Thus the carbohydrate component in digitoxin is not required, only steroid Digitoxigenin has the physiological function.

Another cardiac glycoside Ouabain is derived from lost African ouabio tree also works similarly. While digitalis has been around as a drug, Oubain also has been known for a long time as arrow tips were coated with ouabain by tribals. Even in ouabain, the carbohydrate part is not necessary to inhibit the pump.

Both cardiac steroids bind to the extracellular part the α-subunits. Ouabain, it so turns out, is just not a natural products from an exotic tree. It is actually an animal hormone which regulates cellular Na+ concentration and water balance. It has been hence again also mentioned while discussing steroid hormones!

In order to further understand the action of cardiac glycosides, we should first discuss what we know about the various step in the way the Na+-K+ pump operates. We have already mentioned that the enzyme occurs in two conformational states.

The enzyme (let us say in E1, state) binds intracellular Na+ followed by ATP binding. The complex E1-ATP-3 Na+ is formed. ATP phosphorylates the key asp residue and ADP leaves. Now the complex is E1P-3Na+ is formed.

The high energy bond in aspartyl phosphate drives the conformational change in the enzyme and the complex now is E2-P 3Na+. It now releases 3Na+ which move outside the cell.

2K+ enter and complex E2-P.2K+ is formed. The aspartyl phosphate in the E2-P part is hydrolysed and gives E2-2K+. The E2 conformation reverting to E1 releases 2K+ inside the cell. E1 is ready for another transporter cycle.

Both ATP hydrolysis and ion transport are vectorial processes, so the cycle is unidirectional. Cardiac glycosides stop the hydrolysis of phosphate in the E2-P.2K. The result is increased Na+ concentration inside the cell. This in itself does not explain the action of digitoxin. For understanding that, we also will have to learn about Na+-Ca++ antiport system. However, before we discuss that transporter, let us look at Na+-K+ pump in a little more detail.

The orientation of the ATPase was learnt from studies using model system of erythrocyte ghost cells. Erythrocytes placed in a hypotonic solution swell and develop holes in the membranes. Haemoglobin goes out leaving the pale ghost cells.

Such erythrocytes ghost cells, when placed in a isotonic solution restores the membrane. This allows control on specie which can be resealed inside. Such systems revealed interesting behaviour of the pump.

ATPase functions if Na+ is inside and K+ is outside. ATP has to available inside the Ghost for the pump to function. Cardiac steroids inhibit the pump but only when present outside the cells.

On the other hand, vanodate, a phosphate analog inhibits the pump but only when present on the inside.

These results formed the basis for the mechanistic picture presented earlier.

In the entire one cycle of operation, four forms of the enzyme can be identical: E1, E1-P, E2-P, E2. The turn over number of ATPase is about 100 S-1.

Vanadate inhibits at nanomolar concentrations as it is able to replace Pi in the two forms of the enzymes. In fact vanadate by being able to also form pentacovalent bipyramidal transition state just like Pi, it acts as the inhibitor in most of the phosphoryl transfer reactions.

The pump is said to be electrogenic as it generates the electrical current across the membrane. ATP hydrolysis and the electron/ion movement are coupled. This is in line with two familiar phenomenons. In oxidative phosphorylation blocking ATP formation can stop flow of electrons across the respiratory chain. In muscle contraction, also the similar coupling is witnessed.

If red cells are incubated in hr. concentration of Na+ and lower K+ concentration, the steep ionic gradient reverses the pump activity and ATP is synthesized.

The credit for explaining how ATPase phosphorylation and dephosphorylation causes ions to move belongs to Oleg Jardetzky (1966). According to him, the binding site in E1 and E2 face in different directions.

E1 and E2 have high affinity for Na+ and K+ respectively. E2 is stable in phosphorylated form whereas E1 is stable on dephosphorylated form.

In the beginning of the cycle, binding of Na+ is followed by its phosphorylation. Stability consideration cause E1P→E2P and now the ion binding site faces outside.

K+ binding follows dephosphorylation, another evasion and release of K+ inside. Na+ and K+ do not have drastic difference in their ionic radii. Hence phosphorylation and dephosphorylation can easily cause changes in cavity size by few Aº to change in the affinity of the cavity for Na+-K+.

Ca2+-ATPase (Ca2+ pump

Skeletal muscle has membrane bound tubules and vesicles and is called Sarcoplasmic reticulum. Ca2+ concentration regulates muscle contraction. The (Ca2+) in the cytosol is  0.1 μm as compared to  1500 μM (Ca2+) in the extracellular spaces. This ionic gradient is maintained by Ca2+-ATPase.

With many features of its operation similar to Na+-K+ pumps, Ca2+ pump expels Ca2+ ions (from cytosol) coupled with ATP hydrolysis. The pump also operates across the plasma membrane as (Ca2+) is associated with numerous biochemical processes.

In muscles at rest, (Ca2+) is pumped into the sarcoplasmic reticulum. Excitation of sarcoplasmic reticulum by a nerve impulse results in release of Ca2+ which triggers muscle contraction by troponin and tropomysin. The Ca2+ pump (110 KD) has similar sequence to α-subunit of Na+-K+ pump. Here also, phosphorylation of the asp residue is involved.

Controlling ion concentrations especially Na+, K+ and Ca++ inside the cell turns out to be extremely critical. We will see in the next module that concentration of anions is also similarly regulated.

ATPase in the mitochondrial membranes is linked to respiratory chain and catalyses ATP synthesis by oxidative phosphorylation. In that context, emphasising its hydrolytic activity in its name may have looked odd.

We saw that hydrolytic activity of ATPase is also very important.

We will continue with membrane transport in the next module as well.

Summary

• Na+-K+ pump.
• Classification of transporters
• Classification of ATPase which are transporters
• Cardiac glycosides as inhibitors of transport