An Interview with Manuel and Ann Varela: Who was Jens Christian Skou, and what did he have to do with the Sodium-Potassium Pump?

Jul 2, 2020 by

Jens Christian Skou

Michael F. Shaughnessy –

1) This chapter looks at a scientist from Denmark—who was luckily born into a wealthy family. What do we know about his birth and family?

Jens Christian Skou was a 1997 chemistry Nobel Laureate who discovered the famous Na+ K+ ATPase transporter, colloquially known as the sodium-potassium pump. Skou was born to an affluent family on October 8, 1918, in Lemvig, located in the western region of Denmark on a fjord. Skou had two brothers and one sister. As young children, the brothers spent much of their time playing in the lumberyard with their father and uncle. His father, Magnus Martinus Skou, and his uncle, Peter Skou, were timber and coal wholesalers. His mother, Ane-Margrethe Skou, continued the partnership with Peter, albeit in a minor role, upon the death of her husband. Skou was a young lad of twelve when his father died of pneumonia.

2) Denmark is a small country—but it has some great universities—and schools. Where did Jens go to school, when did he graduate, and what did he study?

Skou began his boarding school education in Haslev, Zealand, at the age of 15, together with 50-60 other boarders. An additional 400 day-pupils attended the school. The school had sprawling park-like grounds with plenty of areas designated for playing fields for sports like football, along with tennis courts on the grounds, and a gymnasium for gymnastics and handball. A scouting program was offered as well. Skou was fortunate enough to spend the major holidays, such as Christmas and Easter, and extended summer and fall break with his family in Lemvig. Skou was considerably interested in the science curriculum; however, mathematics sparked his interest the most.

Skou completed a seven-year program, concentrating on medicine, and graduated with his M.D. in 1944 at the University of Copenhagen, Denmark.

Skou’s medical courses included three years for chemistry, physics, biochemistry, anatomy, and physiology, with four additional years for the clinical training, along with forensic medicine, pharmacology, pathology, and public health.

Between 1944 and 1947, he took his clinical education and training at Hjørring Hospital and Orthopedic Hospital at Aarhus. Skou was a surgical intern and often removed a diseased appendix. The senior surgeon would often have to leave the operating theatre to take part in collecting weapons that had been dropped for the resistance movement. A couple of sources state that in 1954, Skou earned a Ph.D. doctorate in medical science with an emphasis in physiology at Aarhus University, Denmark.

3) Now, the sodium-potassium pump—what exactly is this, and why is it important?

Dr. Jens Skou discovered the sodium-potassium pump. The famous protein is an ATP-energized transporter of sodium (Na+) and potassium (K+) ions across the membrane. The ions of sodium and potassium atoms are electrically charged and are denoted with a plus sign of one, i.e., Na+ and K+. The sodium-potassium pump is membrane-bound, which means that it is embedded within the biological membrane and is continuous with both sides of that membrane. This active ion transporter is known by several names, such as the Na+/ K+ pump, or the Na+-K+ ATPase pump.

Because the pump is energized, it belongs in the class of so-called active transporters. Further, because the pump transports two substrates, Na+ and K+, the protein is known as a co-transporter. Because the pump hydrolyzes ATP, it is known as an ATPase, which cleaves ATP into ADP and inorganic phosphate, Pi. This ATP cleavage provides the energy used to keep the transporter functioning.

https://upload.wikimedia.org/wikipedia/commons/8/89/0308_Sodium_Potassium_Pump.jpg

https://upload.wikimedia.org/wikipedia/commons/8/89/0308_Sodium_Potassium_Pump.jpg

Figure The sodium-potassium ATPase pump

The energy-driven ion transporter serves to maintain a consistent ion concentration across the membrane. The sodium-potassium pump transports Na+ and K+ in opposite directions across the membrane while using the energy from ATP hydrolysis. As one molecule of ATP is used up by its cleavage, three Na+ atoms are pumped outward, while two K+ atoms are pumped inward.

Thus, inside healthy cells, the Na+ concentration is kept low, and the concentration of K+ is maintained at high levels. In biochemical terms, inside the animal cell, the sodium concentration, [Na+], is kept at only 12 mM (millimolar) while the potassium concentration, i.e., [K+], is relatively higher at 140 mM. Conversely, in the spaces outside the animal cell, and blood plasma, the ion concentrations are held to high sodium, [Na+] = 145 mM, and low potassium, [K+] = 4 mM. The sodium-potassium pump functions to preserve these relative concentrations in and out of cells by pumping three Na+ ions out for every two K+ ions into the cell.

https://upload.wikimedia.org/wikipedia/commons/8/83/Blausen_0818_Sodium-PotassiumPump.png

https://upload.wikimedia.org/wikipedia/commons/8/83/Blausen_0818_Sodium-PotassiumPump.png

Figure The sodium-potassium ATPase pump

The ion concentration differences across the cell membrane create a so-called electrical potential, another form of biological energy. The pump, therefore, sets up a so-called membrane potential difference in electrical charge at -50 to -70 mV (millivolts). The electrical membrane potential energy is then used for essential cell functions, like nerve impulse firing, or to drive secondary transport of solutes.

The sodium-potassium pump is one of the most thoroughly investigated proteins in the world. It is known to its atomic level, as its crystal structure was determined to high resolution by co-Nobel Laureate John E. Walker. In the figure, the sodium-potassium pump structure has ribbon-like features, in which the alpha-helices appear as twisted cylinders, and beta-strands are denoted as flat arrows. The thinner lines indicate structures that have undefined turns or bends, which connect the more defined alpha and beta structures.

https://upload.wikimedia.org/wikipedia/commons/thumb/5/52/3b8e.png/451px-3b8e.png

https://upload.wikimedia.org/wikipedia/commons/thumb/5/52/3b8e.png/451px-3b8e.png

Figure Molecular structure of the sodium-potassium ATPase pump

In the figure showing the three-dimensional molecular structure of the sodium-potassium pump, one can see how the pump traverses the membrane, which is depicted as horizontal lines. The sodium-potassium pump structure has several domains.

The membrane domain (M) is the section of the pump that resides in the biological membrane. The membrane itself may consist of, for instance, the cell or plasma membrane, or perhaps a bacterial membrane, or a mitochondrial membrane. The M domain of the pump has about ten alpha-helices, and it provides the tunnel pathway for the ions across the membrane. Six of the ten helices in the M domain are called the T-domain (for transport), and the other four helices are called S-domain (for support). Though not shown in the figures, the T-domain forms the channel through which the ions pass. The S-domain, also not shown, provides a physical support structure for the T-domain.

The phosphorylation domain (P) harbors an aspartate amino acid, which gets a phosphate attached to it or detached from it. The nucleotide-binding domain (NBD) binds ATP and magnesium and performs the phosphate attachment to the P domain. The actuator domain (A) has protein phosphatase activity and serves to remove phosphates from the aspartate of the P domain during the ion transport cycle.

The membrane-bound proteins that use ATP to energize transport are generally called primary active transporters. Secondary active transporters use ion concentration gradients, known as electrochemical potential energy or ion-motive forces, to drive transport. Both primary and active transport systems use energy to drive molecule transport across the membrane.

There are several types of primary active transporters. These types of ATPases are differentiated by specific properties associated with them. For instance, the sodium-potassium pump is a member of the large group of transporters called the P-type ATPases. The “P” denotes the fact that the transporters undergo Phosphorylation during transport.

The F-type ATPases are associated with energy-coupling Factors. One key member is the FoF1 ATPase. The “o” subscript in the Fo term refers to the fact that the antibiotic called oligomycin inhibits its transport function. The “1” in F1 denotes the fact that the protein was the number one factor to be discovered that uses energy to mediate proton transport across the membrane actively.

Another group of ATPases is called the V-type because they reside in Vacuolar compartments. The V-type ATPases are also referred to as ATP synthases. They can also be called VoV1 ATPases. The Vo portion remains in the membrane to mediate transport while the V1 domain harbors the ATPase enzyme, which performs the ATP hydrolysis during that transport.

The A-type ATPases are specific to the transport of Anions across the membrane. Anions are molecules with a negative charge. Thus, the A-type ATPases permit hydrophilic substances to overcome the hydrophobic nature of the membrane barrier and permit transport across the membrane.

The ABC ATPases are transporters with two ATP-binding domains called cassettes. Therefore, the ABC term refers to the ATP-Binding Cassette transporters. The ABC transporters likely constitute the largest family of primary active transporters, and as a group can specify an extensive array of substrates, ranging from ions to building blocks, and nucleotides, to proteins and fatty acids.

All of these types of primary active transporters are ubiquitous in all forms of life, from bacteria to humans. The secondary active transporters are similarly universal. Thus, within organisms, the immense numbers of transporter types are staggering.

4) Anesthesia—many surgeons could not perform surgery without it—what did Jens Skou contribute in this realm?

After receiving his M.D. in 1944, Dr. Skou became an intern in the hospital at Hjørring and the Orthopedic Hospital in Aarhus. During Skou’s first year of internship training, he formed an interest in surgery and began the second year of training in the field. In the surgical wards, no anesthetists were available, and the surgical interns had a severe problem with so-called ether narcosis, a potentially lethal complication from extended respiratory use of the noxious ether during surgery.

Thus, to avoid killing their patients, Skou and his fellow interns used, whenever they could, alternatively applied local or spinal anesthetics on their surgical patients. To do this, Skou invoked a principle of anesthesiology, called the Overton-Meyer theory, which predicted a correlation between lipid solubility of anesthetics and their potency. Skou and colleagues knew those general anesthetics were lipid-soluble, while local anesthetics contained a mixture of charged versus uncharged components in their structures. Thus, he became interested in whether the effectiveness of these anesthetics relied upon their charged (polar, lipid insoluble) or uncharged (non-polar, lipid-soluble) components in their anesthetic nerve-blocking mechanisms. Skou’s interest in understanding the mode of action of anesthetics became the basis for a Ph.D. thesis, even though he already had an M.D. The project would start Skou on the path to the Nobel.

Graduate student and surgical intern Jens Skou first harvested intact sciatic nerves from frog legs, removed their outer sheath coverings, exposed the raw nerves, and examined the ability of various anesthetics to block the muscle contraction activities. While he found that the various general and local anesthetics blocked the nerves-muscle twitching with varying degrees of potency, he also observed that the drugs failed to follow the Overton-Meyer rule, especially for the local anesthetics. That is, he did not see a correlation between lipid solubility and anesthesia potency.

Therefore, Skou paid attention to the lipid environment. He used a new technique, called the Langmuir trough, examine the anesthetics. The Langmuir trough permitted Skou to examine single layers of lipid substances to determine to what extent the anesthetics entered the lipid monolayers. Here, he observed a correlation between lipid entry (solubility) of the anesthetics and their nerve-blocking potencies.

Skou considered the question of sodium permeability across nerve membranes during nerve impulse activity. He reasoned, if sodium permeability increased, a process called depolarization, during regular nerve impulses, then, the anesthetics somehow blocked the sodium transport across the membrane. He further deduced that the anesthetics thus blocked the proteins within nerve membranes from undergoing a conformational change to affect the sodium permeability.

Then, Skou speculated that perhaps the anesthetics’ ability to enter the lipid layers in the Langmuir trough device was somehow tied to enzyme activity. As he would later find, he was correct.

5) Cholinesterase—what exactly is it, and why is it important?

Skou’s inadvertent interest in the neurotransmitter degrading enzyme acetylcholinesterase led to an interest in ATPase—and the Nobel. Skou’s new interest in the ATPase of neurons led to his discovery of the sodium-potassium pump, a finding for which he would garner the Nobel specifically.

During graduate school in the Ph.D. program, while also a surgical intern at Aarhus, Skou had deduced from his studies on frog muscle that anesthetics were related to lipids by way of membrane-bound enzymes. He deduced that the insertion of anesthetics into lipid parts of the biological membrane affected the activity of an enzyme. Thus, Skou searched for a membrane-associated enzyme with high activity that he could readily measure. The first enzyme candidate to come to his attention was acetylcholinesterase. The enzyme had been discovered by Dr. David Nachmansohn, who was a faculty member at Columbia University.

The enzyme acetylcholinesterase catalyzes the conversion of acetylcholine into choline and acetic acid. Choline can be used to make phospholipids. The acetic acid quickly loses a proton (H+) to become acetate. Acetylcholine is a neurotransmitter that is released by motor neurons and diffuses to muscles. There, the molecule binds to its dedicated acetylcholine receptor. The binding of acetylcholine to its receptor causes ion channels to open. The channel opening then allows membrane depolarization to occur in which positively charged ions (called cations) move into muscle cells to trigger muscle contraction.

Acetylcholine also operates during the conduction of nerve impulses along the length of neuronal cells. During neuron firings, depolarization causes sodium entry into presynaptic nerves. The intracellular sodium, in turn, causes packets of acetylcholine to be released into synaptic connections between pre- and post-synaptic neurons. The acetylcholine moves across the synapse and binds to its receptor on the post-synaptic neuron. The acetylcholine-receptor complex then triggers the opening of so-called ligand-gated ion channels. The opening of these channel gates allows cation entry into the post-synaptic neurons, i.e., depolarization, which continues the process of neuronal firing along the nerve.

Skou’s interest in regulating acetylcholinesterase led to his moving to Woods Hole, MA, to work at the Marine Biological Laboratory with Nachmansohn. While in Woods Hole, Skou was exposed to an entirely new style and attitude towards conducting scientific research. The MBL was (and is) a hotbed of scientific activity. The new research approach took the form of daily seminars from investigators whom Skou had read about in textbooks, daily lab meetings for planning daily and nightly research activities, and conducting experiments virtually seven-days and nights per week.

Between these new research endeavors at Woods Hole, he came across a book edited by Nachmansohn describing the work of B. Libet, who discovered a calcium-activated ATPase enzyme in the squid axon and had speculated that the ATPase enzyme was somehow involved in neuronal firings.

After the summer session was over at Woods Hole, Skou returned to Columbia and prepared the acetylcholinesterase enzyme from the electric eel. With his new enzyme in hand, he returned to Aarhus to complete his thesis. In short, he found that the enzyme readily inserted into his lipid monolayers on the Langmuir troughs. Further, in erythrocytes, the local anesthetics potently inhibited the acetylcholinesterase enzymes.

Eventually, Skou would finish his Ph.D. thesis in 1957, publishing six papers in 1954 and a book devoted to the topic of local anesthetics and acetylcholinesterase enzyme action. He would then go on to explore ATPase and nerve action, and the new studies would form the basis for the nomination to the Nobel.

6) Jens Skou won the Nobel Prize, sharing it with two others—and perhaps celebrated with some Aquavit and a “Skoal” toast—But what exactly was the research and why is it important?

Dr. Jens C. Skou took the 1997 Nobel in chemistry for his 1957 discovery of the sodium-potassium pump. He shared the prize with Paul D. Boyer and John E. Walker. In 1974, Boyer (see the chapter on Boyer in this book) discovered the elegant mechanism by which ATP synthase functions. Walker (see the chapter on Walker in this book) took the Nobel for his 1994 determination of the F1 structure of the ATP synthase enzyme.

Here, we shall focus on Skou’s Nobel research discovery—the sodium-potassium pump. For his work on ATPase and ion transport, Skou turned his attention to the crab.

Skou had arranged for European green crabs of the species Carcinus maenas from Aarhus to be sent to him at his lab. There, he dissected out the sciatic neurons from the crab legs, homogenized the separated crab nerves, and purified fragments of their nerve membranes using a centrifuge. Next, Skou took the crab nerve membrane fragments harboring ATPase enzymes and conducted what is considered by many scientists and historians of science as one of the most influential contributions ever to biochemistry, cell physiology, and bioenergetics.

He measured ATPase activity in the presence of increasing concentrations of potassium while adding increasing concentrations of sodium. At little or no ion amounts of sodium or potassium, Skou found little ATPase activity in the crab leg membrane fragments.

However, as Skou increased the amounts of potassium and sodium, the ATPase of the crab-leg neuronal membranes showed a massive spike in its enzyme activity! Furthermore, Skou observed that maximum ATPase activity was obtained when the sodium and potassium mixtures were in equal amounts, about 40 mM each, and which included a low amount of magnesium, about six mM. Strikingly, Skou noted that the sodium and potassium ions worked on ATPase activity in a synergistic fashion, that is, the two ions worked better when together than separately to enhance the crab leg nerve enzyme. This experiment was the first in scientific history to demonstrate ion transport that was driven by ATP!

Skou published the astonishing work as a single author in the prestigious journal Biochimica et Biophysica Acta, in 1957. In the paper, Skou concluded that, with magnesium, ATPase was activated by the combination of sodium and potassium. He also noted that calcium ion inhibited this ATPase activation by the two ions. Skou further speculated that the ATPase mediated the active extrusion of sodium from nerve cells. He did, however, elect not to mention the fact that he had discovered a pump, for fear of generating criticism. But he had indeed discovered the sodium-potassium ATPase-driven pump.

Soon after publication, many others repeated the work, confirming it using a variety of inhibitors and conditions. Investigators from many laboratories extended the results by Skou to show that when sodium was pumped out, potassium was pumped in, even providing a stochiometric basis: three Na+ are pumped out while two K+ are pumped in for every ATPase cleavage of ATP. Skou’s discovery of the famous energy-driven ion pump paved the path for the transporter to become one of the best-understood proteins in the world.

7) Interestingly even after his retirement, he seemed to keep busy—reading journal articles—and when exactly did he die and what is memorable about it? What have I neglected to ask about this remarkable scientist?

In 1988 Skou retired from his post at Aarhus University, and he became Professor Emeritus but maintained a research laboratory. His last publication was in 2015. Upon his retirement, Skou enjoyed his leisure time with fly-fishing and spending time with his grandchildren.

Skou met his wife, Ellen Margrethe Nielsen, while he was in Hjørring Hospital as a patient. He had fallen ill and needed to recuperate in the hospital. She was a nursing student and visited him, and they listened to the English radio together. The couple married in 1948 and later had three daughters. Tragically, their first-born suffered from a genetic disease and died after one and a half years.

During World War II, the Germans invaded and subjugated Denmark in April of 1940 with the hope of gaining a steady food supply source. Thus, the Danish army and fleet remained in operation. Most of the Danish opposed the Germans, but were limited to methods of resistance, due to inadequate access to armaments and nowhere to go into hiding. Sabotage against factories and railways in league with the Germans began to take hold in time with the aid of the English air taskforce. Luckily, for Skou, there was no disruption with his study of medicine. During May and June of 1944, Skou and his classmates were able to take their medical exams and sign the Hippocratic Oath, individually in a secret locale.

At the age of 99 years, just a few months shy of 100, Dr. Jens Christian Skoudied on May 28, 2018, in Risskiv, Aarhus, Denmark.

For additional information regarding this extraordinary investigator, visit these links:

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