Chapter 4: The Reemergence of the Bulk Phase Theories
Transporters and pumps are not needed when there are fixed-charge sites.
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 4, The Reemergence of the Bulk Phase Theories, 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:
The 1940s and 50s research showed many things were permeable to cells - not only K+. These things did not only stay near the surface but made their way into the cell which goes against the understood equilibrium theories. This would require pumps and energy to run said pumps under the cell membrane theory. The amount of energy needed to run these pumps would be in excess of what metabolism can produce - assuming the energy is primarily coming from ATP’s “high-energy phosphate bond”. This led to a further split between membrane and bulk phase theories.
The two bulk phase theories were Troshin’s sorption theory and Ling’s fixed-charged hypothesis. Both theories postulate solutes like K+, are held on fixed areas of larger molecules, like amino acids, within cells. The solutes that are mostly kept out of the cell, like Na+, has to do with their hydrated cell diameter - so how well they dissolve in water. These fixed-charge sites are not only on the surface of the cell but are throughout. This would allow for movement of ions, etc. throughout the cell. Also, the resting potential then arises from the fixed charge at the cell membrane. Some experiments showed Na+ was adsorbed over K+. This led to not only considering the hydrated diameter, but the fixed charges - specifically, where the charge is located. This is the next evolution of Ling’s LFCH and known as the Association Induction hypothesis.
4.1. Kamnev's Study of Sugar Distribution in Frog Muscle
Uptake of galactose and sucrose into frog muscle cells increases after cell death. There is about equal amounts of each in the cell as in the extracellular environment.
4.2. Troshin's Sorption Theory
Another great read is Troshin’s “Problems of Cell Permeability”
Troshin used simple methods to show experimental evidence disproving the membrane theory - “this very simplicity of experimental approach often helps scientists to pursue truly fundamental issues.”
4.2.1. Osmotic Behavior of Living Cells
Rabbit erythrocytes in galactose and frog muscles in urea lose water and gain the galactose and urea - contradicts membrane theory as only things that cannot enter the ell should cause continuous cell shrinking.
Troshin: “The increase or decrease in the water content of a cell in solutions of different substances is a colloidal phenomenon connected with the penetration of molecules of the solute into the cell.”
4.2.2. Cells as Colloidal Coacervates
Colloid chemistry was studied in the USSR; the West went in a different direction - Troshin uses coacervates (a colloidal state) as an analogy for the living protoplasm.
4.2.3. Solute Exclusion and Accumulation
Troshin “considered that any solute in living cells can exist either dissolved in the cell water or in an adsorbed or chemically bound state.” - adsorb here means held on the surface of something.
When the tension at an interface is reduced (increased), more (less) adsorption occurs.
Troshin noticed that the “degree of exclusion” of solutes increased as the “saturation of adsorption sites” occurred - remember playing musical chairs?
Troshin’s theoretical equations (see the section) “accurately described the distribution of sugars and a variety of other solutes in coacervates as well as living cell… Killing the living cell destroys this property of solute exclusion.”
Troshin postulated there needs to be an “uninterrupted flow of energy metabolism” to maintain cellular processes.
Troshin - “The rate of penetration of substances into a cell and their distribution between cell and medium are determined by the rate of enzymatic process directly or thanks to the fact that metabolism maintains the sorptional activity of live matter on a definite level.”
Troshin’s 1958 paper shows the Na+ and Cl- experimental data from frog muscle cells follows his equations. See below.
4.3. Rekindled Doubts about the Revised Membrane Pump Theory
4.3.1. Discovery of the Non-Donnan Distribution of Many Permeant Substances
Multiple amino acids like carnosine were shown to enter cells without a net charge.
The theory postulated charged anionic phosphates (like ATP) are what caused the charge differential to bring K+, etc. in.
Na+ was, at the time, the one cation to go against the Donnan equilibrium theory causing the “need” for the Na-pump. However, Cl-, glutamate, and hexosephosphate (analog of glucose-6-phosphate) were shown to not follow the Donnan equilibrium as well - “The postulation of a Na+ pump does not provide a general solution to the problem, and many more pumps are required. Moreover, energy sources must be found for these pumps as well.”
4.3.2. Reinvestigation of the Question of Whether or Not Cells Have Enough Energy
to Operate the Postulated Na+ Pump
“Thus cooling, which of itself should slow down the pump and bring about K+ loss and Na+ gain, does just the opposite.” - this is because diffusion has a low-temperature coefficient, whereas pumping has a higher. As such, cooling should have less effect on the diffusing inward Na+ than the pumping outward Na+. So a cooled cell should have more Na+ and fewer K+.
Cooling the cells (0C and room temperature) was also shown to protect them against metabolic poisons - this should perk people’s ears about lowering body temperature and thyroid issues.
4.4. Ling's Fixed-Charge Hypothesis
Support for the Na+ pump came primarily from research looking at isolated frog sartorius muscle.
4.4.1. A New Molecular Mechanism for the Selective Accumulation of K+ over Na+ in Living Cells
“Ling (1952) pointed out that not all proteins were expected to adsorb K+ over Na+ selectively. Even a protein that possesses an inherent propensity for K+ adsorbtion would do so only when it exists in a specific conformation, and, to assume this conformation, proteins may require at the same time interaction with, for example, a key metabolic product like ATP.”
“It was then considered that the interaction of certain suitable intracellular proteins with ATP and other essential agents would prevent the internal neutralization of fixed anionic sites (in the form of beta- and gamma-carboxyl groups) by fixed cationic sites (in the form of epsilon-amino groups and guanidyl groups) on the same and other proteins (Ling, 1952). The free beta- and gamma-carboxyl groups can then serve as the seats of selective K+ adsorption. This basic theory provides a simple explanation for past failures, cited above, to demonstrate K + adsorption in isolated proteins.” - basically, “electrons” do not stay “still” and instead cluster around certain areas of compounds (like amino acids). This creates a local charge. The beta- and gamma-carboxyl groups of proteins become negatively charged and attract positively charged ions. And the epsilon-amino and guanidyl groups become positively charged and attract negatively charged ions.
Original research looked at the selective accumulation of K+ due to its small hydrated diameter. However, once Na+ was shown to permeate the cell, the ideas were dropped. The researchers did not consider a phase change, etc. possibility instead.
Ling’s fixed-charge hypothesis (LFCH): consider a muscle cell that contains a large amount of the protein myosin. The “myosin, alone contains enough free beta- and gamma-carboxyl groups… to provide fixed anionic adsorption sites for all of the cell K+.”
“The basic concept was that, owing to the intense electrical field near an anionic
site, the probability of finding a countercation in its immediate vicinity rises sharply as the distance between the fixed anions and the free cations decreases. This sharp increase in the probability of finding a cation in close proximity to a fixed anion occurs not only because of the exponential increase in the electrical forces as the distance of separation decreases but also because of the phenomenon of dielectric saturation in the immediate neighborhood of the ion.”
“K+ is accumulated over Na+ in living cells because its smaller size permits K+ to enter into the spherical shell where the highest probability of finding a countercation is located. As a result, most of the fixed anionic sites are associated with K+.”
4.4.2. Some Distinctive Features of Ling's Fixed-Charge Hypothesis
“(1) Ling's theory proposed the presence of fixed charges throughout the cell and not only on the cell membrane; (2) Ling's theory proposed extensive association of one counterion with one fixed ion; and (3) Ling's theory proposed selective K+ adsorption as the result of favorable electrostatic adsorption energy for K+ over Na+ associated with the fixed negatively charged sites. Without close association, the basic differences between hydrated K+ and hydrated Na+ cannot be perceived, since these differences are differences in short-range attributes. The long-range attributes (e.g., Couiombic forces) of the monovalent cations are not distinguishable.”
[T]he living cell is an amphoteric fixed-charge system carrying both fixed anions and fixed cations. These fixed cations and fixed anions tend to form electrostatic bonds or salt linkages. It is only when salt linkages are prevented from forming
or are dissociated that fixed anions are available for selective K+ adsorption… One example is the involvement of the formation of salt linkages and the displacement and liberation of adsorbed K + from muscle cells during contraction.”
“Metabolism ultimately is essential for the continued maintenance of selective K+
accumulation and in many cells ATP is the metabolic product that plays the key role. In the LFCH, this dependence of selective K+ accumulation on metabolism
is not due to the hydrolysis of ATP, but to its presence per se. The amphoteric nature of the living cell fixed-charge system endows it with a natural ability to assume either one of two alternate states, one in which the fixed negative charges are neutralized by fixed positive charges in the formation of salt linkages and another in which the fixed negative charges selectively adsorb K+ or Na+ and the fixed positive charges selectively adsorb anions.” - adsorbed anions: e.g. glutamate, Cl-, creatine phosphate, etc.
“In particular, ATP plays a special role in keeping the proteins from forming salt
linkages and assuming a contracted conformation. In this role ATP acts by adsorbing onto the proteins at key sites, thereby causing them to assume an expanded conformation because of long-range electrostatic repulsion. In addition, ATP causes a selective preference for K+ over Na+. Although this specific mechanism of ATP action was revised in the later and complete version of the AI hypothesis, in both hypotheses, in order to maintain selective K+ accumulation, ATP functions as an intact adsorbed molecular ion and not through its hydrolytic cleavage to liberate energy stored in postulated ‘highenergy’ phosphate bonds.” - AI: Ling’s Association Induction Hypothesis that comes after his LFCH. ATP is no longer needed for its “high-energy” phosphate bond. Please continue to follow along to find out what it is used for in Ling’s AI hypothesis.
4.5. Molecular Mechanisms of Selective Ionic Permeability
1904: Devaux “showed that plant cells of diverse types can rapidly and reversibly exchange their ionic contents with ions of different types in the environment… he was able to demonstrate that such ion exchanges follow a mass action law.” - this exchange occurred throughout the entire cell and is notably different than the conventional view of ion exchange today.
Ion exchange led to the “need” for membrane carriers in the cell membrane theory.
4.5.1. The Membrane Carrier Model
Membrane carriers: like boats transporting ions “back and forth between the outer and inner boundaries.” - at the inner boundary, “the binding compound or carrier is chemically altered by metabolic processes so that the ions are set free.”
K+, Rb+, and Cs+ compete for cell entry - hydrated (unhydrated) diameters: 160pm (300pm), 180pm (250pm), and 210pm (250pm) respectively - theory was used to show this is possible in the membrane theory.
4.5.2. Ling's Fixed-Charge Hypothesis
“The cell surface is endowed with fixed anionic sites with properties similar
to those envisaged inside the cells. Then the cell surface can, in essence, be seen as a two-dimensional replica of the three-dimensional cell. As such, a momentary snapshot of the cell surface would show most of the surface anionic sites to be occupied by K+ (or Rb+), which is preferred over Na+ by the same mechanism.” - the same equations for the cell membrane carrier model apply for Ling’s theory, “thus both the carrier model and the LCFH can predict the… ion permeability data.”
“The LFCH of selective K+ permeation is in fact merely another aspect of the
LFCH of selective K+ accumulation by the cell. No additional assumption need be
made, while in the carrier model additional assumptions are required.”
The carriers require resynthesis as they are destroyed once intracellular - this requires additional considerations to the carrier model (e.g. more energy than only pumping Na+ out).
The LFCH only requires “the presence of fixed anionic sites on the cell surface".” - “virtually all proteins contain anionic side chains in the form of beta- and gamma-carboxyl groups and since the presence of hydrophilic charged groups at the cell surface is also indicated by its low surface tension, it is hard not to assume that the surfaces of living cells are as a rule endowed with fixed ionic groups.”
“The LFCH of ionic permeation is based on the electrostatic field of the surface
fixed ions and their counterions.”
“These findings confirm a very basic and important tenet of the theory: The degree of ionic association increases sharply if one species of the ion is fixed in space.” - the fixed species is the beta- and gamma-carboxyl groups.
4.6. The Surface Adsorption Theory of the Cellular Resting Potential
4.6.1. Three Historical Models: Glass, Oil, and Collodion
Membrane theory: K+ is assumed to exist in a free state - like KCl → K+, Cl-, etc. when in water.
Questions remained as to how bound K+ could lead to a resting potential.
Experiments with glass, oil, and collodion models provided a model for the living cell.
4.6.2. The Surface Adsorption Theory of Cellular Electrical Potentials
Ling theorized aspartate and glutamate were the main amino acids providing the beta- and gamma-carboxyl groups in muscle and nerve cells.
These fixed anionic sites allow for the surface adsorption of K+, etc.
Another big divergence is the cell resting potential is not dependent on the intracellular concentrations of K+, Na+, or Cl- (see my Substack on Chapter 3 for a description of the Hodgkin-Huxley model, etc.). More on what is in upcoming chapters.