Chapter 8: The Physical State of K+ and Na+ in Living Cells

Experimental evidence (e.g., electron microscopy and NMR) supports the adsorbed state

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Today’s Substack will continue with Chapter 8, The Physical State of K+ and Na+ in Living Cells. Thank you for the overwhelmingly positive comments about the new format change!

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Summary:

Multiple experimental evidence from microscopy to NMR support K+ ions in living cells are not freely diffusing but are localized or adsorbed to specific sites within the cells. This adsorption is crucial for maintaining ion gradients, electrical signaling, osmotic regulations, etc. “K+ and ions like Cs+, Rb+, and Li+ that can replace it - is localized to the COO- rich regions of the A band and Z line” in muscle.

8.1. A Reassessment of the Critical Experiments of Hill and Kupalov

Hill and Kupalov’s 1930s research was used to discard colloidal cell theory because they concluded: “virtually all water and K+ exist in the free[, not bound,] state in living cells.” Their conclusion was based on the unfounded assumption that the cell water is also free. Ling argues, “Since Hill’s arguments for free cell water are no longer tenable, the conclusion that cell K+ must all be free also becomes untenable.”

8.2. Experimental Proof That the Bulk of Muscle K+ Is in an Adsorbed State

Ling discusses the experimental evidence that the bulk of muscle K+ and water in living cells is in an adsorbed state. “The bulk of K+ in voluntary muscle cells must be localized in the A bands.” The A band corresponds to the region of the sarcomere (the basic unit of muscle contraction) where thick myosin filaments are located, overlapping with thin actin filaments. “Myosin alone provides enough beta- and gamma-carboxyl groups to adsorb all intracellular K+.”

Research in the early 1900s showed K+ was localized to the A bands in muscle. Microincineration in an oven or electron microscope demonstrated K+ localized to the A bands and the Z line. The Z line is located at the boundary between two adjacent sarcomeres, the repeating units of muscle fibers responsible for muscle contraction. The Z line comprises actin filaments and serves as an anchor point for the thin filaments (actin) of adjacent sarcomeres. During muscle contraction, the Z line moves closer together as the sarcomere shortens. Microincineration was criticized because it left a lot of artifacts like protein residue from incomplete incineration. This led to the development of other techniques.

For a short background on how biological materials are seen with microscopes: Light microscopes - most are dyed with a compound (e.g., methylene blue, heavy metals, etc.) that absorbs a specific wavelength of visible light. Electron microscopes usually use heavy metal dyes, passing an electron beam over the material. Electron microscopy is predominantly used over optical microscopes for imaging biological materials. There are other imaging modalities used as well. Ling showed that Cs+ and Tl+, two heavy metals used to dye, “compete for the same sites which normally adsorb K+.” The hydrated and unhydrated ionic diameters (pm) of K+, Cs+, and Tl+ are 160 and 300, 210 and 250, and 140 and 250, respectively. So when the cells are unhydrated as is the case in electron microscopy, it’s clear as to why Cs+ and Tl+ will replace K+ given their smaller diameter. The Cs+ and Tl+ were shown to precipitate and leech out of the A band, which indicates K+ was bound there.

“Autoradiography offers another possible way to test the prediction that beta- and gamma-carboxyl groups are the seat of K+ adsorption.” Autoradiography uses radioactive isotopes to visualize the distribution of a specific molecule or ion in tissues or cells using photographic film or an imaging system. However, K+ radionuclides “are either too short-lived (K+-42) or too expensive (K+-40).” As a side note, one would be amazed by how much K-40 is around; there’s even some naturally occurring in bananas. Cs+ and Tl+ are used as K+ substitutes. The muscle cells are incubated with the radioisotopes, then are fixed and sectioned, and the sections are placed in contact with a photographic film. The radioactive decay of the isotopes generates radiation that exposes the film, producing a visual record of the distribution of the ions within the cells. This process shows the radionuclides distributed in the A bands and Z lines.

Another imaging technique used is energy-dispersive X-ray microanalysis. X-rays are generated by the interaction of the electron beam with the sample, and these X-rays contain characteristic energy signatures that correspond to different elements (here, Cs+, Tl+, and K+). The emitted X-rays are detected and analyzed using an energy-dispersive X-ray detector. You can target the specific lines where K+ localization is expected. “The concentrations of Cs+, Tl+, and K+ in the A band are three times higher than those in the I band, the cation concentration is much high in the Z line than in the surrounding I band, and the concentrations of alkali metal ions accumulated in the two edges of the A band are higher than those in the middle of the A band. Thus, the x-ray microprobe technique also provides direct evidence for K+ (or Cs+ or Tl+) localization in the A band.”

The final method described for K+ localization is laser microprobe mass spectrometric analysis (LAMMA). By focusing the laser beam on specific areas of the muscle tissue, researchers can selectively vaporize and ionize the K+ ions present in those regions. The ionized K+ ions are then separated and analyzed using a mass spectrometer, which measures the mass-to-charge ratio of the ions. This allows for the quantification and identification of K+ ions specifically.

“The application of these four different methods in three different laboratories provides strong, mutually supportive evidence that K+ in striated muscle cells is localized in the A bands and Z lines. Moreover, the K+ - and ions like Cs+, Rb+, or Li+ that can replace it - are not simply hovering in the vicinity of fixed charges… but are adsorbed specifically onto fixed anions within the A bands and Z lines.” This means “intracellular K+ is not free in solution, but also that intracellular water cannot exist in a normal state.” Free K+ in the A band and Z line would create “a large osmotic pressure difference between the K+-rich A band and the K+-poor I band… since K+ is the only major solute in muscle cells. The result would be extensive swelling of the A band and shrinkage of the I band.” This idea is important when it comes to “the pump” many are after when bodybuilding.

8.3. X-Ray Absorption Edge Fine Structure of K+ in Frog Erythrocytes

The absorption edge fine structure, done using a monochromatic (one wavelength/ frequency) x-ray beam, differs for K+ salts and complexes compared to K+ in frog erythrocytes.

8.4. Secondary Evidence for K+ Adsorption in Living Cells

“Ions adsorbed on a chain or surface of fixed ionic sites do not necessarily have reduced mobility. The mobility of these ions may be equal to or even higher than that in free solution.” There are differences seen in conductance between axons and neurons which primarily led to disagreements on things like diffusion. Ling states this “can be reconciled by assuming that axons but not in cell bodies protein filaments that are oriented longitudinally provide evenly placed anionic sites that serve as a K+-conducting band.”

Ion-specific microelectrodes designed for measuring K+ activity are used to probe the concentration of K+ ions within living cells. These microelectrodes consist of a thin glass or metal tube filled with a solution containing an ion-selective membrane that selectively responds to K+ ions. To measure K+ activity, the microelectrode is inserted into a living cell, and the potential difference between the internal solution of the microelectrode and the cell interior is recorded. This potential difference is directly related to the K+ concentration or activity in the cell. By carefully calibrating the microelectrode and comparing the recorded potential with a calibration curve, the K+ activity in the living cell can be quantitatively determined. This allows researchers to assess changes in K+ activity under different conditions or stimuli and investigate its role in cellular functions. Ling states, “trauma brought about by the intrusion of the intracellular electrode liberates K+ and depolarizes water locally,” to account for the varying measurements seen during the procedure. He argues the divergence and variation in measurements “is not consistent with the membrane pump theory.”

Nuclear magnetic resonance, NMR, relaxation times were used to measure 23Na+ and 39K+ in living cells. The response of an atom to a magnetic field in NMR spectroscopy is primarily governed by the presence of nuclear magnetic moments and the phenomenon known as nuclear spin. Atoms that possess an odd number of protons or neutrons, such as hydrogen-1 (proton) or carbon-13, have an intrinsic property called nuclear spin. Nuclear spin arises from the fact that protons and neutrons themselves possess spin, which results in a non-zero net angular momentum for the atomic nucleus. When placed in a strong magnetic field, such as the magnetic field generated in an NMR spectrometer, these atoms with nuclear spin align themselves with or against the direction of the magnetic field. This alignment gives rise to two possible spin states: parallel (low energy) and antiparallel (high energy) with respect to the field. Relaxation time refers to the time it takes for the nuclear magnetic moments of certain atomic nuclei to realign after being perturbed by the external magnetic field - like a MRI. Relaxation times provide insights into the dynamics of these ions and their interactions with their cellular environment. In living cells, the relaxation times of 23Na+ and 39K+ are found to be different from their values in bulk water; they are longer which indicates they are subject to restricted mobility. This discrepancy suggests that these ions experience specific interactions or binding within the cellular environment.