Chapter 3: The Emergence of the Steady-State Membrane Pump Concept
Do action potentials necessitate pumps?
As always, this is not medical advice, and reading this does not form a client relationship with me - your health is your responsibility.
Today’s Substack will continue with Chapter 3, The Emergence of the Steady-State Membrane Pump Concept, from Dr. Gilbert Ling’s “In Search of the Physical Basis of Life.”
Please feel free to skip to the parts you wish to read.
Starting with an overall summary of the chapter:
Cells were thought to be impermeable to Na+. Radioactive tracer research in the 1930s and ‘40s showed cells are permeable to Na+ and are constantly exchanging it and K+ with the external cellular environment. The concentration of Na+ is greater extracellularly. As such, a Na+ outward pump was theorized to maintain the theorized cell membrane. ATP became known as the molecule that provided energy for biological work with its “high-energy phosphate bond.” Many doubted the cell produced enough ATP to run the Na+ pump along with everything else due to theoretical calculations. Also, many pointed out major discrepancies would be seen in cells that are at a different temperature, pH, metabolic state, etc. (more on this later). Action potentials seen during nerve and muscle action were driven by a change in cellular K+ and Na+ permeability which leads to electrical currents. The intracellular Na+ is returned to its resting concentration, which gave further support for the pump theory. However, the Na+ could also be excluded as the cytoplasm structure returned. This would point to something similar to a phase change occurring (more to discuss later).
3.1 Major Developments Providing the Background for the Acceptance of the Membrane Pump Theory
3.1.1. The Disproof of the Original Equilibrium Membrane Theory
“General theory for the asymmetrical distribution of living cells [:] Small ions like K+ and Cl- distribute across the cell membrane... large ions like Na+ are permanently and completely barred from entering or leaving the cell.”
Radioactive tracer techniques showed red blood cells, and other cells are permeable to Na+ - radiolabeling is when a compound has certain atoms replaced with radioisotopes with characteristic decay properties. These decays can be “seen” with imaging systems, e.g., positron emission tomography (PET), Geiger and scintillation counters, etc.
“Muscles of rats fed a K+-deficient diet lost a large amount of K+ and at the same time underwent a twofold increase in Na+ concentration... [that] K+ deprivation and gain of cell Na+ did not involve cell damage [is] shown by the complete reversibility of the process: When K+-deprived rats were fed a normal-K+ diet, the muscles readily regained their usual concentrations of K+ and Na+.” - this will become an essential point as the story continues to unfold. This is a reason why many who are in a low bioenergetic state crave salt. Also, this is why consuming at least the RDA from food for potassium (all of the micronutrients) is needed. However, to caution, potassium needs to become intracellular - Ling explains this in future chapters. Those who are not producing adequate ATP and consuming sufficient amino acids, etc., will struggle with this. Again, low and slow unless being actively monitored.
“The experiments... left no doubt that the long-held belief that the cell membranes are in general impermeable to Na+ is wrong, thus undermining the membrane theory of selective ionic distribution, of volume control, and of cellular electrical potentials.”
“[A]lthough all cells studied are permeable to Na+, the rate of exchange, as measured by the half-time of isotopic exchange, varied from second to many hours. This great divergence in ionic permeability is not readily compatible with a belief that all living cells share a very similar membrane - an assumption implicit in Overton’s lipoidal membrane theory.”
“[S]elective ionic accumulation probably has little to do with cell membranes but more to do with cytoplasm.”
“In 1946, Conway too presented calculations suggesting that muscle cells do not have enough energy to operate a Na+ pump.” - confirmed by Ling in 1952.
Conway theorized a pump in 1955 without explaining why he changed course.
3.1.2. The Concept That the Constituents of Living Beings Are in a State of Dynamic Equilibrium
“The availability of both radioactive and nonradioactive isotopes brought new dimension to biochemical methodology… [E]lemental compositions of body parts are not stable but are in a state of constant flux of dynamic equilibrium.” - refer back to my Substack on supplements. This would be the case of oscillation about a local stable equilibrium.
3.1.3. The Hill-Embden Controversy and “A-lactic Acid” Muscle Contraction
Into the 1920s, lactic acid was believed to initiate muscle contraction.
Experiments showing lactic acid followed muscle contraction and muscles could contract without the production of lactic acid led to “the recognition of the key role of the final metabolic product, adenosine triphosphate.”
3.1.4. The High-Energy Phosphate Bond as the Immediate Source of Energy for Biological Work Performance, Including Ionic Pumping
Creatine phosphate was discovered in muscle in the late 1920s. - “[A]fter an intensive burst of contraction the level of phosphocreatine in muscle dropped to a low level. In the presence of oxygen, the normal level of phosphocreatine is soon restored by oxidative metabolism... can [also] be regenerated in the absence of oxygen by anaerobic glycolysis, a process which (instead of converting glucose or glycogen to CO2 and H2O, as in aerobic metabolism) produces lactic acid.” - 40% of total body methyl groups go to synthesizing creatine. This alone is why many need to consider methylation support (considerations for low homocysteine are often first required). Meat provides creatine as well - many supplement creatine monohydrate. I have found some to have a lowered affect when doing so. Others feel good taking it. As always, experiment for yourself. Creatine is considered safe and is currently the most studied supplement. Many will argue for a loading phase where you take a higher dose for a few days and then drop down to a daily maintenance dose. However, I have not seen this needed. Brain creatine changes seem to take years (some research points to 4+y) from supplementation. However, I still think supplementation can be beneficial as it will take the "pressure" off body-wide methylation.
Under anoxia conditions, e.g., using pure nitrogen vs. oxygen, “showed that muscle could still undergo perfectly normal contractions. But with each contraction a certain amount of the limited store of creatine phosphate is used up, until its level finally approaches zero. As this state is approached, the muscle becomes less and less able to go back to the normal relaxed state after each contraction, until finally it enters a permanent state of shortening or rigor mortis.”
“Mechanical work performed by the muscle during its reversible contractions must ultimately derive its energy from the food materials the animal consumes.”
“The contraction of muscle under anoxia... shows that a more immediate source of energy is creatine phosphate. Since, to perform its role, each creatine phosphate molecules splits into its constituent parts, one creatine and one phosphate, it seemed most reasonable to suppose that it was the bond that holds these two components together that ‘contains’ and ‘stores’ the energy originally derived from glucose or other food materials. And so the high-energy phosphate bond concept was launched.”
1935: ATP discovered: creatine phosphate + ADP < - > Creatine + ATP. Catalyzed by the enzyme creatine kinase - the Lohmann reaction shows why creatine kinase is one of the main markers to show rapid muscle breakdown (rhabdomyolysis).
“The Lohmann reaction is the only way for creatine phosphate to be synthesized, and also the only way for it to break down in living cells.”
1941: “[E]nergy stored in [ATP] ‘high-energy’ phosphate bonds was suggested to be the immediate source of energy for biological work performance, including ‘osmotic’ work or ionic pumping.”
1941: “[F]ree energy made available in metabolic reactions is
stored in the form of energy-rich phosphate bonds, represented as ~P to distinguish them from ordinary low-energy phosphate bonds, -P... its free energy could be released and utilized upon the hydrolysis of these bonds.”
~P free energy of hydrolysis was found to be ~11 - 12kcal/mole
3.2. The Postulation of the Na+ Pump
Theory: K+ enters cells via passive diffusion, and Na+ is continually pumped out.
3.3. Arguments and Evidence in Support of the Na+ Pump Theory
3.3.1. The Dependence of Ionic Distribution on Continued Metabolic Activities and Normal Temperature
“If ion accumulation and exclusion depend on continual pumping, the levels of ions in the cell should be dependent on metabolism.”
“Sensitivity of ion accumulation and exclusion to cooling also would be expected on the basis of the pump model, because inward leakage, a diffusion process, has a low temperature coefficient, while metabolic pumping, a chemical process, may be expected to have a high temperature coefficient. In agreement with this hypothesis, cooling causes loss of K+ and gain of Na+ in a variety of plant and animal tissues.” - This is one reason why I do not support the cold water emersion trend and have always been more of a proponent of using hot water (to toleration). Whenever I begin to feel “run-down” I increase the number of hot baths I take. I also consume warm foods and drinks, wear extra layers, etc. When I had CFS and recurring rhabdo, I would lay on heated seats and use heating pads and blankets. The arguments of nnEMFs are a consideration. However, I saw the heat as helping my cells "structure" themselves when I could not effectively.
3.3.2. The Energy Requirement of the Na+ Pump Appears to Be Adequately Met by Cell Metabolism
Calculations showed 18 - 46% of the cell's total energy would go to pumping Na+ out of the cell.
“This rate of energy consumption would be too high for the cell to cope with.”
Instead of dropping the pump idea, researchers “postulated a Na+-Na+’ exchange diffusion’ carrier within the membrane that shuttles Na+ back and forth without expending energy and without contributing to net Na+ movement in or out.”
3.3.3. Active Solute Transport by Epithelial Tissues and Giant Algal Cells
Active transport: pumping ions and other solutes against electrochemical gradients. Ling discusses this further in chapter 17.
3.4. The Further Development of the Membrane Theory of Cellular Electrical Potential in the Context of the Membrane Pump Theory: The Ionic Theory of Hodgkin, Katz, and Huxley
Future chapters will discuss action potentials further. In summary, our current understanding is that every neurotransmitter, hormone, etc., released from a cell requires the Ca+2-oscillation, which is tied to the action potential (early research primarily looked at Na+ and K+). These currents are also involved in current transmission down nerves - there is more to this, including the myelin sheath, nodes of Ranvier, etc. I can discuss these further in future Substacks if anyone is interested. Also, pacemaker cells in the heart, skeletal muscle, etc., use action potentials. Patch clamp electrodes measure the action potentials, usually via voltage clamp methods.
3.4.1. The Hodgkin-Katz-Goldman Equation
The discovery that cells are permeable to Na+ “forced a revolution in the theory of the cellular electrical potential.”
Up to 1943, "the electrical potential had been regarded primarily as an ionic equilibrium phenomenon." - imagine a libra scale with positive charge on one side and negative on the other.
1943: The ionic theory of electrical potential - “The ionic theory... closely harmonized with the biochemical view of the living cell as being in a state of dynamic equilibrium, or, more correctly, in a steady state. As is well known, the maintenance of an equilibrium state does not call for a continual energy expenditure while a steady state, by definition, is maintained only by a continued supply of energy.”
Action potentials, “the unit message of signal transmission in nerve and muscle,” were studied during this time (the 1930s - 1970s).
Resting potential depends on temperature - higher temperature leads to greater resting potential.
Resting potential depends on extracellular K+ concentration - less extracellular K+ leads to a greater resting potential. This occurs to a point due to Na+ "filling in" at low K+ extracellular concentration.
Resting potential is dependent on intracellular K+ concentration - the greater the intracellular K+, the greater the resting potential, to a point.
Resting and action potentials depend on extracellular Na+ - “the action potential is created, not merely as a result of a large gain of permeability to all solutes, but as a result of a large increase specifically in the permeability of the cell membrane to Na+.”
3.4.2. The Hodgkin-Huxley Theory of the Action Potential
My chemical engineering research included the Hodgkin-Huxley equations. Their equation is a partial differential equation. This means change depends on more than one independent variable. However, PDEs are still not analytically solvable without tricks, even with our best supercomputers. As such, one looks for ways to transform the PDE into an ordinary differential equation. ODEs only depend on one independent variable like time or space.
I did not look at the PDE for H-H but did look at Sel'Kov's reaction-diffusion glycolysis equation. Again, if anyone is interested, please let me know and I will do write a Substack about this topic.
The Hodgkin-Huxley model of the neuron expanded on the core conductor model, which was developed to describe transatlantic phone cables. My MEng thesis - “The cell can be modeled as a capacitor - anions outside the membrane and cations within the cell... Zooming in on an infinitesimal length of the cylindrical core conductor model allows the axon to be made into an equivalent circuit. The circuit includes an internal and external impedance, internal and external current, transmembrane current, and a membrane voltage leading to four nodes. Kirchhoff’s current law is then applied to these nodes. Kirchhoff’s current law states that the sum of the currents into the junction must equal the sum going out. The model also uses Kirchhoff’s voltage law for inside and outside the cable (or cell), which states that the sum of the voltage drops around a loop equals zero. This leads to four differential equations that relate the current, resistance, and voltage inside and outside the cell.”
Thesis - “Hodgkin and Huxley modeled this equivalent circuit as a membrane capacitance in parallel with three branches, each a resistor, and Vmq (q for quiescent). One branch describes the flow of potassium, the other sodium, and the third leakage, which is for the other ions that may flow into or out of the cell. The resistance of each branch is variable. The three Vmq are the Nernst resting potential due to potassium, sodium, and leakage ions. The leakage ions are the anions and cations other than sodium and potassium… The potassium conductor closes, the sodium opens, and the quiescent membrane potential will go from -65mV to +20mV (due to sodium inside the cell). Then the sodium conductor will close, and the potassium conductor opens, causing the potential to go back to -65mV. The Hodgkin-Huxley circuit models what occurs at the membrane as described above. The model reduces to the cable model for the passive process.”
In the plot above, you can see the transient inward Na+ current and the delayed outward K+ current.
3.4.3. The Hodgkin-Huxley Theory of Permeability Changes during the Action Potential
The change in cell permeability to K+ and Na+ generates the currents that lead to the action potential seen in nerve and muscle tissue.
3.4.4. Experimental Confirmation of the Membrane Theory of the Resting and Action Potentials
“The Hodgkin-Huxley theory provided the basis for a great deal of research on the action potential of a variety of excitable cells.”
The H-H model’s eight predictions were experimentally confirmed, lending more credence to the theory. If anyone is interested in this subject, I can discuss it further in a future Substack.
https://www.researchgate.net/publication/327250904/figure/fig1/AS:664286170984454@1535389573632/Hodgkin-Huxley-Model-circuit-representation-of-the-neuronal-membrane-The-circuit.png
Ling, G. (1984). In Search of the Physical Basis of Life. Springer Publishing. - From Hodgkin (1958), by permission of Proceedings of the Royal Society of London.