Chapter 2: Evolution of the Membrane and Bulk Phase Theories
The importance of whether intracellular K+ is bound or unbound.
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 2, Evolution of the Membrane and 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.
Before I continue, I understand many will point to the electron microscope, etc., images of the cell membrane. Please shelve this for now to continue to see the story unfold. We will look closer at this important argument in the future.
Starting with an overall summary of the chapter:
Into the 1940s, research conflicted as to the nature of intracellular potassium (K+). Some experiments supported bound K+ and others showed K+ was unbound. This is an important idea because if K+ is bound in something like a colloid (think jello), there is no need for a cell membrane. Also, this would account for the resting cellular potential, exclusion and inclusion criteria, cell swelling, etc. On the other hand, if the K+ is unbound, this would support more of a need for a cell membrane - think a water balloon - with the associated pumps, etc. Bound vs. unbound K+ and water also has implications on cell swelling, how the K+ and water got there in the first place, are they static or do they oscillate in and out of the cell, what about other ions, proteins, glucose, etc.
Note: many may not “care” about these early chapters and want to understand the later ones which deal with muscle, metabolism, etc. However, I think understanding the following background is important for gaining a grasp of the later chapters. Specifically, the nature of bound vs. unbound K+ is needed to understand muscle function; how water structure leads to ATP production, protein folding/ unfolding, DNA structure, etc.
2.1 Concepts of the Nature of the Plasma Membrane
2.1.1 Lipoidal Theory of Overton
Increasing polar groups (when “electrons” are not shared equally by atoms in a molecule - e.g., carboxyl or amino groups) to substances made them less permeable to cells.
When these substances’ carbon chains were lengthened, they became more permeable.
Comparing the solubility of these modified compounds showed lengthened carbon chain compounds are more soluble in lipids than water. And those with increased polar groups are more soluble in water than lipids.
This led to the lipoidal theory of passive permeability - the cell membrane is composed of lipids.
2.1.2. Mosaic Membranes with Pores
The lipodal theory did not account for substances like water and urea that are “insoluble or poorly soluble in lipids”, but are passively permeable to cells.
This led to the mosaic membrane containing pores theory - the membrane is a patchwork of lipid-containing and precipitate (a solid that is out of solution) pores.
The lipid areas allow for lipid-soluble and the pores for lipid-insoluble transport.
This became known as the molecular-sieve model. Each sieve had different characteristics (e.g., size, charge, lipid-containing, etc.) that determined what was “allowed” through it. Many know these ideas as membrane channels.
Gradient differences were not seen though - e.g., they did not see a build up of urea in only specific places around the cell.
2.1.3. Membranes with Charged Pores and Selective Ionic Permeability - for passive diffusion.
The molecular sieve model primarily stated that ion permeability was due to electrical charge differences found along the various membrane pores.
The pores were believed to have fixed charges “due to ion absorption onto the walls of the pores (which will make this type of charged ion immobile, leaving he oppositely charged ions free to move)” - basically the channels were maintained at a fixed charge that allowed other ions of a different charge to be attracted or repelled due to basic electric and magnetic principles.
The theory required protein entry to depend on “an H+-concentration-dependent ionization of ionic side chain functional groups.” - the amino acids’ side chains either gained or lost electrons to ionize them, which attracted them through specific pores.
Due to electrostatic repulsion, the membrane becomes less permeable to other anions when anions (negative charge) are adsorbed onto the pore walls. This leads to “selective permeability toward cations because they have different frictional resistances owing to their different degrees of hydration. Thus an ion with a thicker hydration shell has a higher frictional resistance because its’ sphere of attraction’ for water.” This would lead to “the low permeability of the collodion membrane (again, like gelatin) to anions in general, as well as its higher permeability toward the smaller, less hydrated K+, than toward the larger, more hydrated Na+.”
2.1.4. The Paucimolecular Membrane of Davson and Danielli
The surface tension of a lipid layer exposed to water is much higher than that of a living cell. This was one reason Overton suggested the lipid layer “is not directly exposed to water but is covered on each side with a layer of protein” (the phospholipid bilayer is born = paucimolecular model).
2.2. Cellular Electrical Potentials and Swelling in the Context of the Membrane Theory
2.2.1. Early History of Cellular Electrical Potentials
1757: electrical involvement during muscle contraction is first seen.
1786: experiments electrically stimulating a spinal cord → muscle twitching
1841: stimulate nerves → muscle contraction
1841: the intact end of a cut nerve is positively charged, and the cut end is negative.
1843/1848: during tetanus (intense muscle spasms), there is decreased current between the muscle and tendon.
The largest currents in muscles and nerves are seen after injury or cut.
Membrane potential = the negative potential that runs along the surface of a cell.
Cells have an internal negative charge.
There were numerous theories as to whether the membrane potential always existed or only during times of injury, when resting, etc.
2.2.2. Bernstein’s Membrane Theory and the Diffusion Potential
1890: Copper ferrocyanide membranes were used to study “muscle and nerve cell potentials due to similarities in semipermeability.”
1900: nerve cells have more intracellular electrolytes than extracellularly.
1912: Bernstein proposed an ionic semipermeability of cell membranes and postulated resting cell potentials due to the intracellular K+ and extracellular Na+ ratios = preexistence theory: “the electrolyte corresponding to [K+] existed in the intact cell.”
2.2.3. The Cremer-Haber-Klemensiewicz Theory for Glass Electrodes
Glass, collodion, and oil layer models were used to study bioelectric membrane potentials - multiple things were trialed to find which one most resembled the potential behavior of a cell membrane.
2.2.4. Phase Boundary Potentials and the Baur-Beutner Controversy
1892: “Nernst pointed out that the law of electroneutrality dictates that there be no net electric charge in either phase; in other words, in each phase the positive ions and negative ions occur in equivalent amounts. It is only at the phase boundary that a spatial separation of charges occurs, generating an electric double layer.” - An electric double layer is when anions form a layer on top of a positively charged surface to cancel the charge disparity. After this layer, there should be an equivalent anion:cation with fluctuations, of course.
1949: “Beutner and Barnes argued that the structural specificities of the actions of drugs "are nothing more than additional factors determining lipoid solubility and degree of ionization in the oil.” - drug mechanisms of action are still debated.
2.2.5. Michaelis’s Theory of the Cation-Permeable Collodion Membrane
The 1920s: well known by now that intracellular K+ “plays a major role in the creation of the cellular resting potential.” Also, “ionic concentrations inside and outside cells are roughly equal, though they exist largely as K+ salts in the cell and as Na+ salts outside the cell.”
1926: Michaelis using the electric double layer idea argued “since the side containing the lower concentration of salt is electrically positive, the membrane is more permeable to cations.” - basically, the cations want to stack up along the negative membrane potential for entry.
An important note is “it took many days for a minute amount of K+ to diffuse across a membrane, while the electrical potential measurements in the same membrane took no more than 15 min to reach a steady equilibrium value. This difference suggests that the relation between potential and permeability is much less direct than Michaelis envisaged.” - this was using a collodion model, not a cell.
2.2.6. The Donnan Theory of Membrane Equilibrium
Cell swelling research showed “the ability of neutral salt to suppress swelling in acid… [neutral salts] fail to suppress the swelling of collagen in alkali.” Also, “a swollen gel should remain unchanged in volume. Yet, as Procter described in his’ method,’ high concentrations of salts actually dehydrate the relating and shrink it.” - this is why many in a low ATP state crave salt and feel better with increasing intake.
Cell swelling research brought up the following discrepancies:
“If swelling and membrane potentials both originate from the same cause, it is difficult to see how the pH maxima of the membrane potential (3.7 - 4.1 *this is an important note that high energy resting cells are acidic) and of swelling (3.2 - 3.3 *but not too acidic) differ considerably in the same gelatin preparation.” - consider claims about “alkalizing food and water” as body pH is tightly regulated and is dependent on location.
“A considerable specific anion effect [on cell swelling] does indeed exist.” - this seems to follow the Hofmeister series: “a series of salts that have consistent effects on the solubility of proteins and on the stability of their structure. Anions have a larger effect than cations on proteins.” This is important for showing cell structure is dependent on proteins.
2.3 Cellular Ionic Distribution in the Context of the Membrane Theory
Rb+ and Cs+ can replace K+ intracellularly - this has to do with the hydration diameter and charge.
Resting muscle fibers are impermeable to all anions and the following cations - Ca+2, Li+, and Na+ - but permeable to K+ and Cs+ → postulated pores have fixed negative charges (so electrostatic repulsion of the anions).
“[The] cell membrane is permeable to K+ and other cations that are equal in size to or smaller than (hydrated) K+, but impermeable to ions equal in size to or larger than (hydrated) Na+.”
2.3.1. Boyle and Conway’s Theory of Membrane Potentials, Ionic Distribution, and Swelling
1941: Muscle in a KCl bath absorbed a large amount of K+ and Cl- without losing the ability to exclude Na+. Because Cl- was absorbed, the idea that there the pores are negatively charged and anions were excluded due to electrostatic repuslion was dropped.
“Pore size would seem to explain not only the permeability to K+ and H+, and the impermeability to Na+, Ca2+, and Mg2+… but also the permeability to some anions (e.g., CI-) but not to others.”
Boyle and Conway used their analysis to justify “the assumptions on which the membrane theory was based-that the interior of the cell, and the physical states of its water and ions, are essentially those of a dilute aqueous solution via membrane voltage.” - their work was published before it was known cells are sometimes permeable to Na+. Ling discusses further flaws in future chapters.
2.4. Early Criticisms of and Experimental Evidence against the Membrane Theory
The 1900s: “K+, Na+, PO4-3, and Cl-… are asymmetrically distributed across the cell surface. Moore and his colleagues found it difficult to understand
how a cell can contain a constant high concentration of K+ and yet be covered by a membrane impermeable to this ion… Moore and Roaf suggested that ‘cell protoplasm combines or fixes in some chemical or physical way the potassium and phosphatic ions, while the plasma similarly holds the sodium and chlorine ions’.”
The 1930s: “Fischer and Suer also rejected the membrane theory in general and the lipoid membrane theory in particular…: Such a surface layer has never been seen, is not demonstrable analytically (tissues do not ‘grease’ but ‘wet a bibulous paper and makes the life of all cells impossible by preventing ingress and egress of water, of most of their foods, and of many of the products of their metabolism).”
1930: Observations showed the surface thickness “could not be greater than 0.0001um, that is the diameter of one hydrogen ion.” - the thickness of the membrane is too small to be a lipid.
1913: puncturing egg cells with a glass needle only allowed dyes to penetrate “the swollen area near the cut to varying depths but never enter the normal unswollen cytoplasm.” - when a cell is “stabbed” its contents do not come spilling out like a water balloon.
2.5. Inquiries into the Nature of Protoplasm
The generally accepted view “that the physical basis of life is protoplasm and that protoplasm, whether in plant or animal cells, is gelatinous in nature was universally acknowledged in the middle of the nineteenth century.”
2.5.1 Protoplasm as a Structural Substance
“If the surface envelope of a plant or protozoan is broken, the protoplasm can often flow or be squeezed out. Furthermore these protoplasmic droplets respond to hypotonic solution by swelling, and to hypertonic solution by shrinking. The protoplasm thus isolated did not mix with the surrounding water but remained as discrete droplets.”
The 1880s/90s: “Biitschli believed that living protoplasm was like a foam in which the more liquid components were dispersed in many microcells of a heavier consistency. Flemming suggested that protoplasm is a tangle of minute fibrils lying in a fluid matrix.” - so some areas of unbound water dispersed in mostly bound water.
2.5.2. Fischer’s Theory of Protoplasm
“Protoplasm is a hydrated colloidal system. Water in the protoplasm is not free but in a chemically combined form. Salts in protoplasm cannot be easily leached out and they too must be held in combination.”
The 1930s: “Fischer and Suer suggested that protoplasm represents ‘a union of protein, salt and water in a giant molecule’ and wrote that ‘this compound when made synthetically actually repeats the physical properties of living matter (its gelatinous feel, high electrical resistance, nonmiscibility with water and neutrality).’ The ‘synthetic protoplasm’ to which these authors referred is derived from casein. Pure casein is hardly soluble in water or dilute salt solution. But if casein reacts with a small amount of alkali or acid, the acid-or base-caseinate formed is a homogeneous gel which on titration to neutrality will not precipitate out but remain in the transparent gel state.”
The 1900s: “Fischer’s study stressed the basic similarity of the swelling of living tissues in water and salt solutions and the swelling of fibrin and gelatin. The characteristics of swelling that Fischer found to be shared by both living and nonliving systems include the following:
They both swell more in acid than in distilled water; they swell more in some acids (e.g., HCl, HN03) than in others (e.g., H2S04). Fischer’s data indicate that the specific nature of the anionic component of acids with the same valency is very important in contradiction to the concept of swelling derived from the theory of Donnan.
Addition of salts reduces the degree of swelling produced by acid. The effectiveness of a salt in reducing swelling is the sum of the effectiveness of each of its ionic components.
Nonelectrolytes have much less or no ability to suppress the swelling induced by acid.”
Cell swelling is a marker of the cellular water becoming unbound or unstructured.
2.5.3. Lepeschkin’s Vitaid Theory
The 1930s: Lepeschkin believed lipoids are “a major constituent of living protoplasm or vitaid [based] on the experimental observations… that lipoid-soluble and water-soluble narcotics are readily taken up by living cells and suggest that lipoids must be present in them. Fischer and Suer, on the other hand, did not accept lipoids as an important component of protoplasm, pointing out that many living cells contain hardly any lipoids at all.”
Lepeschkin stated, “as is well known, living protoplasm possesses the so-called selective permeability ... the well-known German botanist, Pfeffer [proposed] twenty years ago that the surface of protoplasm is covered by a membrane, by the so-called ‘plasma membrane,’ which only possesses the selective permeability while the inside of protoplasm is as permeable to all substances as gelatine jelly. According to recently published investigations, Pfeffer’s theory proved to be wrong; it was based on an incorrect interpretation of experiments. Protoplasm has no ‘plasma membrane’ and all its parts possess the selective permeability.”
2.5.4. Nasonov’s Phase Theory of Permeability and Bioelectric Potentials - the 1960s
“[D]enaturation theory of excitation emphasizes proteins as the central components in both physiological and non physiological activities of living cells.”
“Nasonov also believed that ‘the main reason for the appearance of both action currents and resting currents is the release of electrolytes bound to proteins, and the loss of phase properties of protoplasm’.”
2.5.5. Bungenberg de Jong's Concept of Protoplasm as a Coacervate
Coacervate: “colloid rich viscous liquid phase that may separate from a colloidal solution on addition of a third component.”
“Bungenberg de Jong was not certain whether coacervates similar to these models constitute the entire cell or only a part of the living cell. Nevertheless he believed that coacervates must play an important role in biology. In support he quoted the following evidence:
Coacervates and living protoplasm are both fluid and yet immiscible with the surrounding fluid medium.
Vacuolization in coacervates can be brought about by a variety of circumstances. Vacuolization also is a common feature of living substances.
Both living protoplasm and coacervates can take up oil droplets.
When coacervates are vigorously shaken, an air bubble appears in many of the droplets. The same was described for living protoplasm.
Coacervates have a tendency to engulf solid particles (e.g., coal, indigocarmine); cells have the same tendency.
Under certain circumstances coacervates can engulf pollens, erythrocytes, and Euglena. Similar behavior of certain types of living cells is of course well known.
The movement of living cells in a constant-current electric field resembles that of coacervates.”
“Regarded coacervates as static models of living cells. He believed that the static models represent equilibrium models but that living cells represent states of ‘nonequilibrium.’ To maintain this state of nonequilibrium, the cells rely on their membranes.”
2.6. Early Inquiries into the Physical State of Water and Ions in Living Cells
2.6.1 Bound Water
“Graham attributed the selective permeability of membranes of, for example, gelatin and parchment to water chemically bound within the substance and thus without normal solvent properties.”
“Fischer and Suer and also Lepeschkin regarded protoplasmic water as bound in some way.”
“The originator of the membrane theory, Wilhelm Pfeffer, did not believe that the inside of a living cell is entirely filled with a simple aqueous solution… he was the first to point out that… swelling water could have accounted for the departure from osmotic behavior…”
1904: “Hamburger found that red blood cells transferred from 0.9% to 1.5% NaCl became only 17.5% smaller. If this volume were inversely proportional to the external osmotic pressure, they should have become 40% smaller.”
“In the 1920s and 1930s, a number of methods were developed in order to determine the amount of bound water in colloidal systems, including living cells. In one, the amount of water that does not freeze at -20°C was considered to be bound water.” - Dr. Albert Szent-Gyorgyi discusses some of the experiments he did (please see his “Bioenergetics” and other works).
2.6.2. Bound K+
“Moore, Roaf, Fischer, Suer, and their co-workers… envisaged some sort of chemical or physical binding of K+ in the protoplasm.”
“Meigs and Ryan… found that, while the voluntary muscle cells behaved as if they were covered with a semipermeable membrane impermeable to NaCI and sugars, the smooth muscle cells from the stomach exhibited no such impermeability. On the contrary, these cells appeared to be highly permeable to both sugars and salts. They concluded that smooth muscles are not covered with semipermeable membranes. To explain the continued selective accumulation of K+ in smooth muscle, they suggested that along with phosphorus, sulfur, and magnesium, K+ exists in the cell in a ‘nondiffusible form.’ In support of this idea they demonstrated that frog stomach muscles cut into small slices lose only a small fraction of their K+.”
“Ernst and Sheffer argued that the bulk of the muscle K+ is nondiffusible but becomes diffusible in response to functional activity.”
1935: “Thus far almost all the evidence suggested the possibility that K+ binding existed in muscle tissues. However, observations in other cell types led to a similar conclusion. Thus, Peters, in order to account for the osmotic behavior of red blood cells, suggested that the potassium “salt” of hemoglobin in red blood cells probably exists largely in ‘an unionized form’.”
2.7. Rejection of the Bulk Phase Theories
2.7.1. Evidence against the Bulk Phase Theories
1941: “Brooks and Brooks dismissed the theories of Fisher, Lepeschkin, and others on the ground that water-soluble dyes and salts diffuse freely and rapidly within the cell. They argued for the spontaneous formation of a new semipermeable membrane on freshly exposed protoplasmic surface which prevents free intermixture of cytoplasm and surrounding solution.”
“The findings of Kite (Section 2.4), which contradicted these statements, were not mentioned.”
“At that time, it was widely believed that the cut and exposed surface of cytoplasm rapidly regenerates a new cell membrane. However, it was not until many years later that methodology had evolved to a point that a definite experiment could be carried out to test this postulation.” - Ling discusses this further in chapter 5.
2.7.2. Evidence against the Concepts of Bound K+ and Bound Water
“From the foregoing, one realizes that the concepts that cell K+ and cell water are in some way bound have been repeatedly suggested but that they were not part of a coherent theory in the sense that the opposing membrane theories of Pfeffer, Overton, and Boyle and Conway were. It is therefore not altogether surprising that these bound K+ and bound water ideas, alongside the bulk phase or protoplasmic theories of the living cell, became all but extinct after the 1940s.”
“Hill confirmed the finding of Overton that osmotic swelling or shrinkage does not agree with the assumption that all the water in muscle cells is free.”
In section 2.6.1, it was noted that -20°C was used to study bound water. However, “Weismann pointed out that ice formation varies not only with how low the temperature is, but also with the speed of the cooling process. Since these factors were not uniformly controlled, a large scatter and inconsistency were present in the data obtained, making it difficult to use these data to establish a difference between free and bound water.” - Also, any addition of things like glucose and salt changed the results.
2.8 Summary
“Although Hill’s experiments played a critical role in the rejection of the bulk phase or protoplasmic theories, one must emphasize that Hill only triggered a decision that was probably inevitable because of the much more primitive state of development of the bulk phase theories when compared with the membrane theory. In the form of Boyle and Conway’s comprehensive [pump] model, the membrane theory and the Donnan equilibrium offered sophisticated quantitative explanations of many major cell functions, including selective ionic accumulation and exclusion, swelling and shrinkage, and the electrical potential.”
Basically, because the membrane theory had a robust mathematical model with predictive and explanatory power, while the protoplasmic model was incomplete, the membrane theory was accepted. This approach is commonly found in the sciences.
There is obviously a ton of information contained in this chapter. Please ask any questions in the comments for things you would like clarifications on. I included whole quotes in the latter sections as I thought the ideas needed to be expressed more completely.
Happy Thanksgiving! I will be back next week to share what I think are the most important parts of Chapter 3, “The Emergence of the Steady-State Membrane Pump Concept.”