Na⁺/K⁺-ATPase Is The Membrane Aldosterone Receptor

[Travis]

There exists an officially acknowledged mineralcorticoid receptor in the nucleus which binds both aldosterone and cortisol. This binding event induces the selective transcription of certain proteins in both the skin and renal tubules where they're most highly expressed, but the regulation of its primary function—the plasma and urine Na⁺/K⁺ ratios—has been demonstrated to occur nearly instantaneously; an event which happens on a time scale incompatible within the sum timescales of DNA to mRNA transcription, mRNA to protein translation, and the subsequent protein action—still undefined.

So not only has a cell membrane aldosterone receptor been discovered, it's necessitated by simple observations. The first person to describe the membrane aldosterone receptor as such was Dr Wehling (circa 1992).

• Wehling, M., M. Christ, and K. Theisen. "Membrane receptors for aldosterone: a novel pathway for mineralocorticoid action." American Journal of Physiology-Endocrinology And Metabolism 263.5 (1992): E974-E979.

➫ "Binding of ¹²⁵I-labeled aldosterone to plasma membranes of HML shares important features with these functional data. This includes a very low apparent dissociation constant (Kd) of 0.1 nM for both aldosterone and the effect on the Na⁺-H⁺-antiport, a high turnover rate, and the almost exclusive binding selectivity for aldosterone." ―Wehling​

Notice the extremely high binding affinity for aldosterone, a property confirmed two years later by Michael Christ:

• Christ, Michael, et al. "Non-classical receptors for aldosterone in plasma membranes from pig kidneys." Molecular and cellular endocrinology 99.2 (1994): R31-R34.

➫ "Rapid, nongenomic in vitro effects of ¹²⁵I-labeled aldosterone on intracellular electrolytes, cell volume and the sodium-proton antiporter have been found in human mononuclear leukocytes (HML), as have related membrane receptors. In the present study, binding of ¹²⁵I-labeled aldosterone to plasma membrane preparations from pig kidneys was studied, since nongenomic in vitro effects of aldosterone have also been described in cultured kidney cells. In this preparation, binding of aldosterone shares important features with both functional and binding data in HML. These include a very low apparent Kᵢ of ~0.1 nM for aldosterone, a high turnover rate and binding selectivity for aldosterone and fludrocortisone. Desoxycorticosterone acetate and corticosterone show intermediate affinity, with apparent Kᵢ values of ~1 and ~100 nM, with hydrocortisone even less active. Thus binding of aldosterone to kidney plasma membranes is compatible with the major features of its nongenomic renal effects." ―Christ​

The cell membrane aldosterone receptor has little affinity for cortisol, while the nuclear mineralcorticoid receptor has about equal affinity for the two. Aldosterone has both an immediate mineralcorticoid effect and a slower genomic effect. The binding affinity of aldosterone vs concentration curve is indicative of high-selectivity: Non-selective ligands show a straight line as they bind indiscriminately (or not at all), while selective ligands show steep curves at low concentrations which imply specific binding domains.

➫ "From a physiological standpoint, membrane receptors for aldosterone may be the effector system responding to the well-described immediate response of aldosterone plasma levels to postural changes, which are less appropriate in terms of the relatively slow-reacting, genomic effector system." ―Christ​

These are expressed both in the kidneys and skin. The skin in certain species must quickly respond to changing salinity levels of water. For this reason, amphibians are the classic model for skin mineralcorticoid activity.

• Nagel, Wolfram, and Jean Crabbe. "Mechanism of action of aldosterone on active sodium transport across toad skin." Pflügers Archiv European Journal of Physiology 385.3 (1980): 181-187.

➫ "The skin was divided into 2 fragments for incubation in the presence, or not, of aldosterone (≥ 0.1 μM). After incubation overnight, sodium transport by the hormone-treated piece was increased 2.7-fold on average, compared to the untreated control." ―Nagel​

Some species of fish—ostensibly never having evolved the need for such—will quickly die when placed in water with Na⁺/K⁺ concentrations which differ from its native environment.

The first indication that that the membrane "pump," or Na⁺/K⁺-ATPase, responds to mineralcorticoids comes from it's classic inhibitor ouabain—a molecule of central importance to the Na⁺/K⁺-ATPase mythology. A description of ouabain's role in the events leading to the recognition of Na⁺/K⁺-ATPase as the assumed "pump" can be found in Jean Skou's Nobel Lecture entitled "The Identification of the Sodium-Potassium Pump [sic]," but I will post an excerpt from https://www.geni.com... instead because the Nobel .pdf file will not allow copy–paste Ctrl + F keyboard function, a useful device which obviates the need for time-consuming transciption:

➫ "Post had not read Skou’s paper but was excited when Skou told him about his work with ATPase. Post asked whether the enzyme was inhibited by ouabain. At this stage Skou was unaware that ouabain inhibited the pump, but he immediately telephoned to his lab and arranged for the experiment to be done. Ouabain did indeed inhibit the enzyme, thus establishing a link between the enzyme and the sodium-potassium pump." ―Anonymous
​

Oubain exists as a glycoside, but it's well-established that glycosidic bonds are routinely and readily hydrolyzed in the body. This has been shown in the case of many types of glycosides, perhaps the most famous is the rutin to quercetin transition. Below is an image which demonstrates this:

Notice the similarities between ouabain aglycone, the synthetic antimineralcorticoid spironolactone, and the endogenous ligand aldosterone.

Perhaps it should be no great surprise that cortisone acetate was selected as substrate by chemist Brian H. Heasley for the partial synthesis of oubain aglycone.

Another classic antagonist of Na⁺/K⁺-ATPase is digoxin (digitalis), a molecule historically used to treat high blood pressure and cardiac arrhythmias—first isolated from Digitalis lanata (foxglove). Digoxin aglycone is quite similar in structure to the aforementioned mineralcorticoids and their inhibitors, a fact that you can easily verify here. So striking in their ability to affect sodium levels that some have speculated on the existence of an endogenous Na⁺/K⁺-ATPase agonist acting at the ouabain site.

• Kelly, Ralph A. "Endogenous cardiac glycosidelike compounds." Hypertension 10.5 Pt 2 (1987): I87.

➫ "The possibility that endogenous inhibitors of the sodium pump exist and bind to the cardiac glycoside binding site on Na⁺/K⁺-adenosine triphosphatase (ATPase) has been a source of much interest. A number of endogenous compounds that modulate the activity of the sodium pump have been identified, including catecholamines, insulin, thyroxine, mineralocorticoids, and other hormones. Most of these compounds affect the sodium pump indirectly by varying the intracellular sodium concentration, the rate-limiting substrate for the pump in vivo, or by increasing the number of pump units in the membrane. Evidence exists, however, that sodium pump activity may be directly affected by several hormones, possibly by altering the affinity of the pump for sodium, although the mechanism by which this occurs is not yet understood." ―Kelly​

In this article Dr. Kelly presents a wealth of evidence arguing for the existance of an endogenous ligand for Na⁺/K⁺-ATPase, the so-called "sodium membrane pump." However, the word "aldosterone" appears not once.

➫ "Nevertheless, the near-universal presence of high-affinity binding sites for these cardiac glycosides on the a-subunit of the enzyme has engendered intense speculation that native ligands for these receptors must exist." ―Kelly​

He does, in fact, seem a bit confused—looking for an endogenous glycoside despite the fact that they are readily hydrolized within the body.

➫ "In addition to the evidence that an endogenous inhibitor of Na⁺/K⁺-ATPase is present in the plasma of patients with primary hypertension and animals with some forms of experimental hypertension, several groups reported the presence in vertebrate species of an endogenous substance with immunological similarities to the digitalis glycosides." ―Kelly​

He should be looking for a molecules similar to ouabain aglycone. His search for the endogenous "sodium pump" ligand seems confounded by this.

➫ "J-F. Cloix (personal communication, 1987) suggests that certain ouabainlike compounds may be structurally similar to steroids." ―Kelly​

The endogenous ligand is staring him in the face. He has the answer, yet overlooks it.

➫ "In humans, several studies suggested that plasma levels of an inhibitor of Na⁺/K⁺-ATPaseare increasedin primary hypertension." ―Kelly​

➫ "They concluded that uremic patients have abnormally large quantities of a circulating sodium pump inhibitor." ―Kelly

➫ "Some investigators determined that unextracted plasma or urine from hypertensive animals or humans inhibits sodium pump function and crossreacts with digoxin-specific antibodies to a greater extent than control samples." ―Kelly

➫ "The recent observation that the affinity of Na⁺/K⁺-ATPase enzyme units for ouabain changes along the nephron, with the highest affinity occurring in the medullary collecting duct, lends credence to this supposition. Regardless, the evidence presented at this symposium indicates that a spirited search continues for a physiologically important inhibitor of sodium pump activity in vivo, an inhibitor that may be the true endogenous ligand for the cardiac glycoside binding site on Na⁺/K⁺-ATPase." ―Kelly​

The endogenous ligand for the protein currently known as Na⁺/K⁺-ATPase is, as will soon be shown, the corticosteroid known as aldosterone. This is implied by its structural similarity to ouabain aglycone, digoxin aglycone, and spirolactone; and also for the following reasons:

• Dostanic-Larson, Iva, et al. "The highly conserved cardiac glycoside binding site of Na, K-ATPase plays a role in blood pressure regulation." Proceedings of the National Academy of Sciences of the United States of America 102.44 (2005): 15845-15850.

➫ "We tested whether the cardiac glycoside binding site of the α1 and α2 isoforms of Na, K-ATPase has a biological function in vivo by using genetically engineered mice with modified cardiac glycoside binding affinity of the α1 and α2 Na⁺/K⁺-ATPase isoforms." ―Dostanic-Larson​

There are four isoforms of the protein currently known as Na⁺/K⁺-ATPase. The rat has a high-affinity α2 isoform and a low affinity α1 isoform. In humans, this is reversed.

➫ "The α2 isoform was converted to low affinity by introducing L111R and E122D amino acid substitutions, which abolished the high affinity binding of cardiac glycosides but without altering enzymatic activity." ―Dostanic-Larson
​

Although not actually a "pump" (Ling, 1960), the protein currently known as Na⁺/K⁺-ATPase has been both structurally elucidated by X-ray crystallography and sequenced. It's real, just misnamed.

​

Mice with the genetically-altered and low-affinity isoform of type α2 Na⁺/K⁺-ATPase are completely resistant to adrenocorticotropic hormone-induced changes in blood pressure—as analyzed by the method of tail cuff sphygmomanometry.

They also had genetically modified the type α1 variety of the protein currently known as Na⁺/K⁺-ATPase. They substituted amino acids in a manner intended to create a high-affinity receptor, and injected these mice with adrenocorticoid hormone as well.

➫ "The α1 isoform was converted to a high affinity subunit by introducing R111L and D122N amino acid substitutions, which enhanced binding of cardiac glycosides ≈100-fold without altering enzymatic activity." ―Dostanic-Larson​

The results were just as striking, though opposite in effect. In this experiment, the rats carrying the high-affinity α1-type "sodium pump", and with a low-affinity α2 isoform, responded with a far greater change in blood pressure than wild-type mice.

The implications are obvious: The adrenocorticoid releasing hormone released aldosterone which bound to the ouabain receptor of "Na⁺/K⁺-ATPase," raising blood pressure through the Na⁺/K⁺ ratio. Nonetheless, the author is under the same misconception as Dr. Ralph Kelly—looking for an actual glycoside in the blood. As previously noted, it is the aglycone of digoxin and ouabain which would be expected to exist within the body.

➫ "Taken together these results demonstrate that the cardiac glycoside binding site of the isoforms of the Na,K-ATPase have a physiological function and supports the hypothesis for a role of the endogenous cardiac glycosides." ―Dostanic-Larson​

The authors should instead be looking for the steroid hormone similar to the aglycones of digoxin released by the adrenocorticoid releasing hormone. The only thing which can fulfill both of these conditions is aldosterone—the very same ligand of the Aldosterone Membrane Receptor first discovered by Wehling.

➫ "Although our studies demonstrated an in vivo function of the cardiacglycoside binding site of the Na,K-ATPase and strongly suggested that an endogenous ligand for this site must exist, information on the exact nature of the ligand is unknown." ―Dostanic-Larson​

The Na⁺/K⁺-ATPase is The Membrane Aldosterone Receptor, and it works independently of ATP. This is highlighted by Dostanic-Larson when she states:

➫ "The α1 isoform was converted to a high affinity subunit by introducing R111L and D122N amino acid substitutions, which enhanced binding of cardiac glycosides ≈100-fold without altering enzymatic activity." ―Dostanic-Larson
​

And you wouldn't expect it to, even under the standard model. The ouabain binding domain exists on the outside of the membrane facing the lymph or plasma, while the site purportedly responsible for ATPase activity is cytosolic. The inability of ouabain to effect the apparent enzymatic hydrolysis of ATP had been often noted in the past.

Judging by my use of modifiers such as "purportedly" and "apparent," I think you can probably tell where I'm going with this. Not only is Na⁺/K⁺-ATPase not a "pump"—and really just the Membrane Aldosterone Receptor (Travis, 2017)—its ostensibly not even an enzyme.

• Cornelius, Flemming. "Modulation of Na, K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics." Biochemistry 40.30 (2001): 8842-8851.

To determine the kinetic rates of membrane protein such as this, they are reconstituted into phospholipid micelles or liposomes. This is the form phospholipids naturally take in wateras the hydrophobic regions (acyl tails) are not attracted by the polar water solution. Instead, the water molecules have affinity for the phophate heads—incorporating them in solution while forcing the nonpolar fatty tails to occupy spaces removed from the interface. The lowest energy state is when all fatty chains are towards the centre with the polar phosphates facing the water, the micelle. The similar thing happens when proteins initially fold—the nonpolar amino acids are found towards the center, and protein folding occurs spontaneously.

Cornelius reconstituted Na⁺/K⁺-ATPase in a micelle/liposome to analyze the rates of hydrolysis. Below is an excerpt of the summary of his article:

➫ "As seen from Table 2 the dephosphorylation rate constant is by far the lowest of the rate constants for the Na-ATPase reaction. Thus, for the Na-ATPase reaction the turnover number kcat will depend in principle on a single elementary step, the dephosphorylation reaction. In the Na,K-ATPase reaction the maximum turnover at 20 °C for shark enzyme can be calculated as the ratio between the maximum hydrolytic activity (Vmax) and the phosphorylation site concentration. In membrane-bound enzyme these values are found to be about 180 µmol·mg⁻¹·h⁻¹ at 50 µM ATP and 2.5 nmol/mg, respectively, giving a kcat ≈ 20 s⁻¹ at 25 °C. After reconstitution with di-C18:1 PC [oleic acid phosphotidylcholine] + 40 mol % cholesterol the values are found to 642 µmol·mg⁻¹·h⁻¹ and 5.6 nmol/mg giving a kcat ≈ 33 s⁻¹." ―Cornelius​

With kcat values of between 20 s⁻¹ and 33 s⁻¹ these enzymatic rates are low—very low. Consider the rates of a real enzyme: carbonic anyhydrase—the enzyme which transforms carbon dioxide into bicarbonate. This enzyme has a kcat of.. . . .

➫ "Carbonic anhydrases accelerate CO₂ hydration dramatically. The most active enzymes, typified by human carbonic anhydrase II, hydrate CO₂ at rates as high as kcat = 10⁶ s⁻¹, or a million times a second." ―Berg​

A metabolic enzyme involved in glycolysis, glyceraldehyde 3-phosphate dehydrogenase, has a kcat around 1,000 s⁻¹ (Wolfson-Stofko, 2013). The membrane-bound phospholipase A₂, another real enzyme, has a kcat of 400 s⁻¹ (Berg, 1991).

In fact, the rates of ATP hydrolysis by the so-called "sodium pump" are so low that they approach the rate of spontaneous, nonenzymatic hydrolysis. That is to say, they're not really enzymatic at all.

• Hulett, H. R. "Non-enzymatic hydrolysis of adenosine phosphates." Nature 225.5239 (1970): 1248-1249.

➫ "Also of interest is the extremely short half-lift of ATP—of the order of a few hours of less—at temperatures above 90°C, even though bacterial growth has been reported at these temperatures." ―Hulett
​

The terminal phosphoester bond in ATP is more labile than commonly realized. Besides the aforementioned temperature dependence, the direction of equilibrium is also highly dependent on pH, Mg²⁺, K⁺, and Ca²⁺ concentrations.

• Alberty, Robert A. "Effect of pH and metal ion concentration on the equilibrium hydrolysis of adenosine triphosphate to adenosine diphosphate." Journal of biological chemistry 243.7 (1968): 1337-1343.

➫ "The hydrolysis of adenosine triphosphate to adenosine dihosphate and inorganic phosphate,

ATP + H₂O ⥨ ADP + Pᵢ
​

➫ provides an excellent example because of its basic importance and the fact that a good deal is known about the proton and metal ion equilibria of the three reactants. The effects of pH on this reaction are well known, and the effects of Mg²⁺ concentration have been discussed to some extent, but it does not appear that these effects have been thoroughly treated together. It is important to know about the effect of metal ion concentrationtion on acid production, the effect of pH on metal ion production, and the effect, of one metal ion on the production of another." ―Alberty
​

He determined the equilibria, not the rates. But the rates are proportional to the equilibria and would vary in accordance with these curves. Seen below are the dependence of both pH and the log(Mg²⁺ concentration) on the ATP ⥨ ADP + Pᵢ equilibria:

At higher pH, the auto-hydrolysis of ATP occurs at a faster rate. Essentially all assays of Na⁺/K⁺-ATPase activity use histidine as a buffer. With a pH of 7.7, the use of histidine in these assays is driving the hydrolysis of ATP towards ADP. For example (from the Cornelius article):

➫ "Enzyme Preparation. Membrane bound Na,K-ATPase from the shark Squalus achantias was prepared as previously described. The specific hydrolytic activity measured at 37 °C was 30-33 U/mg of protein at standard conditions (120 mM Na⁺, 30 mM K⁺, 4 mM Mg²⁺, 3 mM ATP, and 30 mM histidine, pH 7.5) according to Ottolenghi." ―Cornelius​

The "standard ATPase conditions" result in a pH of 7.5, which is rather unphysiological. Maximum ATP stability occurs, according to MIT chemist Robert Alberty, at p[Mg²⁺] = ~2.5 and not the p[Mg²⁺] of −2.397 as used under "standard Na⁺/K⁺-ATPase conditions." The logarithm of the reported intracellular magnesium concentraion is 3, much closer the ATP stability nadir as graphed by Alberty. And since the purported enzymatic activity of Na⁺/K⁺-ATPase occurs on the cytosolic side of the membrane, you would want native intracellular ion levels to determine the kinetic rates. This apparently has never been done, and thus Na⁺/K⁺-ATPase has never truly been demonstrated to be be an enzyme. Even at supra-physiological levels of Na⁺ and K⁺, and far less Mg²⁺, they are only able to achieve dubious enzymatic rates of around 20 s⁻¹ to 33 s⁻¹. These values approach the rates of self-hydrolysis under these high sodium conditions (pH ~7.5). Below is a comparison of the "standard Na⁺/K⁺-ATPase conditions" vs the real intracellular concentrations.

_________________Assay__________Intracellular
________________________________________
pH________________7.5_____________6.8_____
_________________________________________
[Na⁺]_____________120·mM__________10·mM____
[K⁺]_______________30·mM_________140·mM____
[Mg²⁺]______________4·mM__________30·mM____
__________________________________________

According to Fujita, the difference in sodium alone doubles the nonenzymatic hydrolysis (and apparent ATPase activity).

You might also expect the 7.5× lower Mg²⁺ concentration, and higher pH, of the "standard ATPase conditions" to drive the equilibrium further towards ADP (Alberty, 1968) thereby inflating apparent "ATPase activity."

As you can see, the word "ATPase" now gets scare quotes since its identity as an enzyme is highly questionable.

There's been dozens of people to remark on the lability of ATPs terminal phosphate group, but only a few who've remarked upon what this means to presumed ATPase activity:

• Baginski, E. S., P. P. Foa, and B. Zak. "Determination of phosphate: study of labile organic phosphate interference." Clinica Chimica Acta 15.1 (1967): 155-158.

➫ "The determination of inorganic phosphate in biological materials is often carried out in the presence of organic compounds containing labile phosphate. The liberation of phosphate from these compounds during the analysis can lead to errors. This interference is commonly encountered in enzymatic procedures where phosphate liberated from a substrate is the measure of enzyme activity. Any non-enzymatic hydrolysis of the substrate would contribute to the over-all phosphate concentration and erroneously indicate higher than actual enzyme activity." ―Baginski​

Even with much greater Na⁺, histidine, and −Mg²⁺, biochemists can only tease-out dubious catalytic rates which are orders of magnitude smaller than common enzymes; rates which do, in fact, approach that of spontaneous hydrolysis. Also, the fact that two obvious mineralcorticoid agents are the classic ligands for this so-called "sodium pump" seems to have previously gone unmentioned.

The "Na⁺/K⁺-ATPase enzyme" is actually The Membrane Aldosterone Receptor, and not even an enzyme. This Aldosterone Pore also works independently of ATP concentration.

A Nobel Prize had been won for this particular unicorn, a mythical chimera in which Gilbert Ling had first recognized as such. But it took the recognition of its classic "inhibitor" ouabain as a mineralcorticoid to expose it further—not to drive the final nail into its coffin and bury it, but to remove it's spiral strap-on horn to show that it's actually the same species of horse originally discovered by Wehling in 1992.


Wolfson-Stofko, Brett, Timin Hadi, and John S. Blanchard. "Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis." Archives of biochemistry and biophysics 540.1 (2013): 53-61.
Berg, J. M., J. L. Tymoczko, and L. Stryer. "Biochemistry (5th International edition) WH Freeman and Co. New York (1995).
Berg, Otto G., et al. "Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants." Biochemistry 30.29 (1991): 7283-7297.

 

[Such_Saturation]

Lol, I will save this in case it goes down

I was just thinking about Orch OR and Hameroff trying to work around the "GTP hydrolysis" objection, and how Ling and Pollack are always saying how ATP has more energy in conformational changes rather than chemical reaction. And then Hameroff says in conference that it's closeness to mitochondria that powers microtubules, and seemed to imply a field effect mediated by water rather than a chemical action/molecular energy mediator (of course someone still needs to point him towards Pollack ). Do you think there is something there?

 

[Travis]

I was just thinking about Orch OR and Hameroff trying to work around the "GTP hydrolysis" objection, and how Ling and Pollack are always saying how ATP has more energy in conformational changes rather than chemical reaction. Do you think there is something there?

Well, GTP does accelerate the formation of microtubules in vitro. The rate of microtubule growth in the presence of GTP is so striking that it's become the canonical microtubule accelerator. Other drugs which do this, like taxol, function almost like a proxy for pregnenolone and simply stabilize the microtuble.

I don't think that GTP is incorporated into the structure. The way it does this remains illusive, but is something that I find interesting.

In vivo, GTP is found wherever microtubules are found. This must have a role in creating microtubule growth rates (learning? adaptability?) in the brain. I bet there's even a few articles by people who've taken this on directly, and have proposed mechanisms for GTP's catalytic role in microtubule growth.. . .[searching]. . ..I found a few, and with free full-text links!

➫Carlier, Marie-France, Terrell L. Hill, and Y. Chen. "Interference of GTP hydrolysis in the mechanism of microtubule assembly: an experimental study." Proceedings of the National Academy of Sciences 81.3 (1984)
➫Hyman, Anthony A., et al. "Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP." Molecular biology of the cell 3.10 (1992): 1155-1167
➫Stewart, Russell J., Kevin W. Farrell, and Leslie Wilson. "Role of GTP hydrolysis in microtubule polymerization: evidence for a coupled hydrolysis mechanism." Biochemistry 29.27 (1990): 6489-6498.
➫Hill, Terrell L., and Marie-France Carlier. "Steady-state theory of the interference of GTP hydrolysis in the mechanism of microtubule assembly." Proceedings of the National Academy of Sciences 80.23 (1983)
➫Carlier, Marie France, et al. "Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogs." Biochemistry 28.4 (1989): 1783-1791.
➫Dimitrov, Ariane, et al. "Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues." science 322.5906 (2008): 1353-1356.​

Evidence of "GTP remnants?" Looks like I could have been wrong about them being not incorporated into the final structure.

I haven't read these articles, but I perhaps their is already a highly-plausible mechanism for GTP's action?

I was just thinking about Orch OR and Hameroff trying to work around the "GTP hydrolysis" objection

What is this objection? Remember reading about that other guy's objection? His entire argument was basically "Hameroff can't be right because some textbook has an elaborate (yet actually impossible) explanation for photoreception." I did read the Hameroff article, but that was about a year ago—before I even knew about GTP's role in microtubule formation.

I've read some stuff about ATP. You'd might like to read Barbara Banks' objection to ATP as an energy molecule.

• Banks, Barbara EC, and C. A. Vernon. "Reassessment of the role of ATP in vivo." Journal of theoretical biology 29.2 (1970): 301-326.

It's good, and a bit different that Ling's angles.

• Ling, Gilbert N. "Oxidative phosphorylation and mitochondrial physiology: a critical review of chemiosmotic theory, and reinterpretation by the association-induction hypothesis." Physiol. Chem. Phys 13.1 (1981): 29-96.

The protons stripped-off of glucose (H⁺) must go somewhere, and the formation of ATP actually removes a proton from solution.

ADP + H⁺ ⟶ ATP​

So I think that Mitchell's articles on oxidative phosphoryation* were logical, and about as good as anyone can do in the 60s. I don't think that ATP is an "energy molecule," but a pH buffer which has roles in chelating Mg²⁺ and keeping phosphate groups in a configuration where they cannot bind calcium and precipitate. Look at what happens in vitamin K deficiency (lack of Ca²⁺-chelating γ-carboxyglutamate domains)? Calcium will find phosphate and precipitate hydroxyapatite crystals indiscriminately and everywhere. Maybe ATP is more about what it isn't (3 Ca²⁺-safe phosphates) that what it is?

Whatever the best way to describe ATP is, it certainly does not have "high-energy" phosphate bond. It's presence is correlated with energy in so much that it mirrors the rate of the electron transport chain, which in turn reflects the rate of metabolism. Many biochemists simply take the effect as the cause, while ignoring the energy that is flowing from the mitochondria down the centre of microtubules. There is no need for another soluble energy molecule besides NADH, flavin, CoQ₁₀, and the catechol–quinones. All of these molecules transfer electrons, the only realistic way to transfer energy through solution. The terminal phosphoester of ATP certainly isn't powering anything with ΔG and H⁺.

That Barbara Banks article talks about the experiment in which ATP entered into muscle contraction mythology. She said that it's a ability to cause muscle contraction was actually dubious and cites the study. I need to look at that original study before I look into muscle contraction further. I was interested in that in the past but had decided that I'd need to explain nerve transmission first. I can can explain much of that now, but get the feeling that muscle contraction will be more difficult to comprehend. But however it works, it can't possibly work anything like this:

• Huxley, Andrew F., and Ro M. Simmons. "Proposed mechanism of force generation in striated muscle." Nature 233.5321 (1971): 533-538. [CAUTION: This article can have a downright lobotomizing effect. However, it is the officially-recognized model at this point; the one-and-only Crossbridge Theory.]

*Mitchell, Peter. "Chemiosmotic coupling in oxidative and photosynthetic phosphorylation." Biological Reviews 41.3 (1966): 445-501.

 

[michael94]

Could you speak some more about A tp and what actually functions as cell energy? I have some tangential thoughts on the subject that need confirming. Anything you are willing to share would be appreciated, even if it's just a theory or what have you.

Edit: Skipped over your last comment. Makes a lot of sense.

 

[Such_Saturation]

What is this objection? Remember reading about that other guy's objection? His entire argument was basically "Hameroff can't be right because some textbook has an elaborate (yet actually impossible) explanation for photoreception." I did read the Hameroff article, but that was about a year ago—before I even knew about GTP's role in microtubule formation.

McKemmish et al. further assert that tubulin switching in Orch OR requires significant conformational structural change, and that the only mechanism for such conformational switching is due to GTP hydrolysis, i.e. conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) with release of phosphate group energy, and tubulin conformational flexing. McKemmish et al. correctly point out that driving synchronized MT oscillations by hydrolysis of GTP to GDP and conformational changes would be prohibitive in terms of energy requirements and heat produced. This is agreed. However, we clarify that tubulin switching in Orch OR need not actually involve significant conformational change, that electron cloud dipoles (London forces), or magnetic spin dipoles are sufficient for bit-like switching, superposition and qubit function (Figs. 5–7). We acknowledge tubulin conformational switching as discussed in early Orch OR publications and illustrations do indicate significant conformational changes. They are admittedly, though unintentionally, misleading. Discovery of gigahertz, megahertz and kilohertz BC in single microtubules supports dipole states providing a favorable signal with regard to the underlying ideas of Orch OR.

The only tubulin conformational factor required in Orch OR is superposition separation at the level of atomic nuclei, e.g. 2.5 Fermi length for carbon nuclei (2.5 femtometers; 2.5×10−15meters" role="presentation" style="box-sizing: border-box; display: inline-block; line-height: normal; font-size: 14.4px; word-spacing: normal; word-wrap: normal; white-space: nowrap; float: none; direction: ltr; max-width: none; max-height: none; min-width: 0px; min-height: 0px; border: 0px; padding: 0px; margin: 0px; position: relative;">2.5×10−15meters). This shift may be accounted for by electronic cloud dipoles with Mossbauer nuclear recoil and charge effects [90,91]. Tubulin switching in Orch OR requires neither GTP hydrolysis nor significant conformational changes, depending on collective London force dipoles, or magnetic spin dipoles in quantum channels of aromatic rings (Figs. 5–7).

Hameroff also claims mitochondria power microtubules and always sit near them. Remote powering through water?

I don't think that ATP is an "energy molecule," but a pH buffer which has roles in chelating Mg²⁺ and keeping phosphate groups in a configuration where they cannot bind calcium and precipitate. Look at what happens in vitamin k deficiency (lack of Ca²⁺-chelating γ-carboxyglutamate domains)? Calcium will find phosphate and precipitate hydroxyapatite crystals indiscriminately everywhere in the body. Maybe ATP is more about what it isn't (3 Ca²⁺-safe phosphates) that what it is?

Yes I think Ray Peat would like this view. Pollack says the ATP goes to the protein and attaches to it to "charge" it.

 

[Travis]

Could you speak some more about A tp and what actually functions as cell energy? I have some tangential thoughts on the subject that need confirming. Anything you are willing to share would be appreciated, even if it's just a theory or what have you.

Microtubules have been histologically-confirmed to run through mitochondria, and microtubules are the only molecular structure which can possible conduct electricity at 100 m/s (the velocity of nerve signals.)

I think that the Citric Acid Cycle, the electron transport chain, and oxidative phosphorylation are all suitably accurate but not the idea that ATP is shuttling energy throughout the body. While technically true—the terminal phosphoester has additional energy—I can't see how it can transduce energy onto a protein, and neither could Szent–Györgi (citation available upon request.) It's a ubiquitous molecule and you cannot deny that it has other functions besides.

Energy is fundamentally electrons flowing down the centre of microtubules, from oxygen lone-pairs (―Ö:) in catecholamines, oxygen lone-pairs (―Ö:) in quinols, and hydrides (H:) in flavins and NADH. I think Albert Szent–Györgi's book on Bioenergetics, which stresses electrons, is better than the ones that stress thermodynamics. Thermodynamics is characterized by heat (ΔH) and disorder (ΔS), two things that can't explain the more important energetic processes such as nerve conduction, vision, consciousness, and the electron transport chain.

There is a place for thermodynamics, but I don't think that it should be central to bioenergetics in the manner presented in most textbooks.

 

[meatbag]

Hameroff also claims mitochondria power microtubules and always sit near them. Remote powering through water?


Yes I think Ray Peat would like this view. Pollack says the ATP goes to the protein and attaches to it to "charge" it.

I think the cellular water is mostly absorbing infrared and far infrared wavelengths (like guenter albrecht buehler says)and that the near infrared wavelegnth is what is signalling to the MT network and that like Travis said the MTs are directly attached to the mito.

 

[Travis]

I think the cellular water is mostly absorbing infrared and far infrared wavelengths (like guenter albrecht buehler says)and that the near infrared wavelegnth is what is signalling to the MT network and that like Travis said the MTs are directly attached to the mito.

I just looked more into microtubule connectivity. I think I have pinpointed one specific protein as well as a few protein classes which are involved in mediating cytoskeletal information transfer between cells.

The G proteins are actually microtubule-associated proteins, and were originally discovered in the eye (of all places). The 'G' comes from GTP, the molecule essential for microtubule growth. There are many such G proteins; they are denoted by Roman subscripts such as Gs, Gi, and Gq. Each G protein exists as a three-unit complex, so each type is further differentiated and denoted using Greek subscripts; Gsα, Gsβ, Gsγ, Giα, Giβ, Giγ, would be examples of such. This would be a straighforward notation if it weren't for Gβ, leading to one class of G proteins having two Greek subscripts (when further subdivided). The G protein unit Gββ would be the second largest protein of the Gβ trimer complex, which can perhaps be denoted by Gβα·Gββ·Gβγ (with an interpunct (·) indicating connectivity). The G Protein Beta has been shown to promote microtubule growth.

Below is shown images created with fluorescent antibodies to Giα.

Not the most interesting G protein, as this one appears to be mostly intracellular. The most interesting G protein is probably Gs, which exists on the cell membrane:

The "s" stands for signal, and for good reason. It is these G proteins which appear to be connected to the transmembrane G-protein-coupled receptors. This type is also covalently-modified by saturated fatty acids such myristate and palmitate, perhaps forming a solubility region which aligns them with the cell membrane. I can almost picture these things being synthesized near the nucleus, prenylated and palmitated, and then diffusing up towards the membrane where it aligns perfectly—owing to these fatty acids attached (and prenyl groups).

I'm getting the feeling that Gs forms a junction between microtubules and G-protein-coupled receptors. The first receptors of this class were discovered in relation to epinephrine, followed by ones found in the eye. The ones in the eye turned-out to be actually rhodopsin, the protein which binds retinol through a lysine Schiff base—putting the head of retinol right near a tryptophan, of all things.

" The ionone ring of retinal makes direct contact with the analogous Trp residue in rhodopsin, whereas carazolol in β₂AR and cyanopindolol in β₁AR pack against aromatic residues that shield the residue from the binding site." ―Daniel M. Rosenbaum​

As you can see, the G-protein-coupled receptors have their own labeling scheme. The β₁AR is short for beta adrenergic receptor one, the first type discovered. What is highly interesting about these is how similar they all are.

"The superpositions of different receptors using the homologous transmembrane domains led to root mean squared deviation values of less than 3 Å. This degree of overlap indicates that these four proteins have a similar overall architecture, yet the divergences are still high enough to signify important differences in helical packing interactions." ―Daniel M. Rosenbaum​

The angstrom (Å) is of course one-tenth of a nanometer. Below is shown a few G-protein-coupled receptors superimposed over β₂AR:

Are they all so similar because the microtubule diameter is a near constant?

Serotonin appears to have a G-protein-coupled receptor, and I would imagine that histidine would as well. As far as I can tell, the connectivity appears to be microtubule➮Gs-protein➮G-protein-coupled receptor. . . or something like tubulin·Gs·β₂AR . . . in the case of the epinephrine receptor.

It would be tempting to view these G-protein-coupled receptors as forming the pore between the microtubule interior and the extracellular fluid. There also exists salt bridges, or carboxylate–amine interactions, in the G-protein-receptor pore. These interior 'crosslinks' are thought to exclude water, yet still allow binding of ligand when available. Carboxylate–amine salt bridges are noncovalent, relatively weak interactions.

Perhaps with the ligand bound–be it epinephrine, histidine, or serotonin–the light inside of the cytoskeleton formed through the mitochondria is allowed the enter the extracellular space? It would also be tempting to wonder if these fluorescent ligands change the frequency of this emitted light. You can find the fluorescent emission wavelengths of epinephrine, histidine, or serotonin online.

But there is another protein involved, one that allows direct mictrotubule-to-microtubule coupling. This is called connexin, and it allows one cell to couple its cytoskelton with another's directly. This protein would have probably been created later in evolution.

You can probably imagine that the insides of nerves are coupled with these direct links, with microtubule networks running between cells–connected by connexin. I think you'd almost be forced into believing such a thing, as the nerve transmission speed is 100 meters per second–far too quick for anything else.


[!] Rasenick. "Tubulin binds specifically to the signal-transducing proteins, Gs alpha and Gi alpha 1." Journal of Biological Chemistry (1990)
[2] Giepmans. "Gap junction protein connexin-43 interacts directly with microtubules." Current Biology (2001)
[3] Rosenbaum."The structure and function of G-protein-coupled receptors." Nature (2009)
[4] Xie, Tao. "β cell-specific deficiency of the stimulatory G protein α-subunit Gsα leads to reduced β cell mass and insulin-deficient diabetes." Proceedings of the National Academy of Sciences (2007)

 

[meatbag]

The ones in the eye turned-out to be actually rhodopsin, the protein which binds retinol through a lysine Schiff base—putting the head of retinol right near a tryptophan, of all things.

Given what you've posted about Tryptophan-Tryptophan Forster resonance transfer that seems to be a sensible position

Very interesting about the G-proteins, I'll need to read more about what you've posted, thanks! I think you're right, that is best explanation for the propagation rates

 

[Travis]

Given what you've posted about Tryptophan-Tryptophan Forster resonance transfer that seems to be a sensible position

Very interesting about the G-proteins, I'll need to read more about what you've posted, thanks! I think you're right, that is best explanation for the propagation rates

You might like to read this. This is really bizarre:

[He is explaining the interaction of interferon‐γ with its cell membrane receptor.]

“The phosphorylated receptor-ligand complex is internalized, enters an acidified endosomal compartment, and dissociates. Free IFNγ eventually traffics to the lysosome where it is ultimately degraded. In many cells, such as fibroblasts, the uncoupled receptor enters a large intracellular pool of mature receptor and eventually recycles back to the cell surface. The size of the intracellular receptor pool is generally 2-4 times greater than the pool of receptors expressed at the cell surface.” ―Farrar​

I didn't think that cytokines could be internalized like that. He doesn't explain what happens next, so you'll have to use your imagination, this article was written in 1993.

What I found interesting about interferon‐γ is that it's N‐terminal region is a pyroglutamate. This is a cylcic glutamate, believe it or not, and may be fluorescent when oxidized (−2H). Of the three amino acids on the receptor found necessary for interferon‐γ binding effect, two were fluorescent.

“A point mutational analysis of this region demonstrated that only three residues are functionally important. These are tyrosine at position 440, aspartic acid at position 441, and histidine at position 444. Alteration of any one of these residues to alanine produced a receptor which was unable to induce a variety of IFNγ-dependent biological responses in murine fibroblasts that contained human chromosome 2l. [...] The particular functional importance of tyrosine‐440 was confirmed by two additional observations. First, substitution of phenylalanine for tyrosine‐440 also resulted in generation of a functionally inactive receptor. [...] Second, mutation or deletion of any of the other tyrosine residues within the receptor's intracellular domain did not ablate receptor activity." ―Farrar​

This doesn't appear to be a G‐protein‐coupled receptor, but this could have importance. The difference between tyrosine and phenylalanine is only one hydroxyl group; the entire function of this entire ~300 amino acid receptor·dimer complex can abrogated by the removal of just two atoms in one specific location.

Farrar, Michael A. "The molecular cell biology of interferon-gamma and its receptor." Annual review of immunology (1993)​