lp6a Three sets of independent disproofs against the membrane-pump theory

According to the membrane-pump theory, the fundamental ability of living cells to maintain a chemical composition different from that of the surrounding medium is due to the ceaseless activities of a battery of hypothetical pumps located in the cell membrane. Thus the much lower concentration of the sodium ion (represented by the symbol Na+ ) seen in almost all living cells than in the surrounding medium is due to a specific pump called the sodium pump.

Short Version:

The membrane-pump theory in general and the sodium pump theory in particular have been disproved because (i) the cells do not have enough energy to run the pumps; (ii) a healthy membrane sac (without cell content or cytoplasm) does not extrude sodium ion as the theory predicts; (iii) a cell assembly without functional cell membrane (and postulated sodium pump) maintains a steady low sodium-ion concentration like its normal intact counterpart.

Longer Version:

(1)Insufficiency of energy to operate the sodium pump

The complete original data were presented first in "A Physical Theory of the Living State; the Association-Induction Hypothesis", Blaisdell Publishing Co, A Branch of Random House, Waltham, 1962, pp. 195-212.

In the 1950's during which most of the following experiments were performed, it was widely believed that living (frog) tissues had three major sources of energy: (1) respiration--- producing carbon dioxide (CO2), (2) glycolysis ---producing lactic acid and (3) the store of energy in the high-energy-phosphate bonds of the ultimate metabolic products, ATP and creatine phosphate (CrP).

First, it was clearly established that isolated frog tissues including muscle, kidney , testes retained their normal potassium-ion (K+) and sodium-ion (Na+) contents after incubation at zero degree Centigrade in a Ringer solution containing 5 mM sodium iodoacetate or IAA (which suppresses glycolysis) and bubbled with 99.99% pure nitrogen in addition to 1 mM sodium cyanide (which together assured the suppression of respiration). This finding shows that respiration and glycolysis per se are not indispensable to the maintenance of the high K+ and low Na+ concentration in the cell. This exclusion leaves the store of ATP and CrP as the only possible energy source for the postulated sodium pump of the muscle under study (ibid, Table 8.4, pp. 200-201).

In other experiments, the decline of ATP and of CrP as well as the minute amount of lactic acid formed (despite the inhibition of glycolysis by IAA) in the poisoned muscle were also measured during a period of time when its respiration and glycolysis were suppressed. From the data obtained and the assumption that all processes involved have 100% efficiency and that the cells used all their energy for nothing else than pumping Na+, the maximum energy that could have been used to drive the postulated sodium pump was calculated and compared with the minimum energy needed to pump out the Na+ at the rate measured (against both an electrical potential gradient and a concentration gradient, also measured throughout the experiment). In the three sets of experiments performed between 9-12, 9-20, and 9-26, 1956, the minimum energy needed for the postulated sodium pump were respectively 30.60 times, 15.42 times and 18.00 times the respective maximum available energy. Six more sets of fully-completed between 3-20-1953 and 8-9-1955 gave corroborating results (ibid, table 8.9, pp.202-212).

In the three sets of experiments and computations, by far the largest sources of the (maximum) available energy to the surviving muscle cells came from the decline and presumed consumption of energy in ATP and creatine phosphate (CrP). Not long afterward, the revolutionary findings of Podolsky and Morales ( J. Biol. Chem. vol. 218, p.945, 1956) and of George and Rutman (Progr. Biophys. and Biophys. Chem. Vol 10, p.1, 1960) showed unequivocally that there is no high-energy-phosphate-bond energy to speak of. Or put it differently, the concept of high-energy phosphate bond was a mistake. By taking this new knowledge into consideration, the energy required would no longer be only 15 to 30 times but from 600 to 1200 times greater than the energy available--- even though the earlier lower set of figures of 15 to 30 times were already more than enough to disprove the membrane-pump theory.

However, in order to understand the untenable situation created by the energy deficiency, the reader is well advised to take into account some additional relevant facts.

Thus the Na+ pump is but one of many energy-consuming pumps postulated, (for a partial list of other pumps proposed by 1973, see Ling et al, Ann. N.Y. Acad. Sci., 204, pp.6-50, 1973, Table 2 on p. 9). Each of these pumps requires additional energy.

And that beside these pumps postulated to exist at the plasma membrane--- covering the outer surface of the cell---, there must also be pumps at the membranes of all the subcellular particles like the mitochondria and sarcoplasmic reticulum (SR). For an otherwise similar pump, the energy requirement is directly proportional to the surface of the membrane. Since the sarcoplasmic reticulum of muscle cells (SR) has a membrane 50 times bigger than the plasma membrane of muscle cells (Peachy, J. Cell Biology, vol 25, p. 209, 1965), the energy requirement of the SR pump would be 50 times higher than the corresponding pump at the plasma membrane (Ling, A Revolution in the Physiology of the Living Cell. Krieger Publish. Co., Melbourne, Fl. 1992, pp.19-20).

Adding still more difficulty to the membrane-pump concept is that there must be pumps for chemical compounds which did not exist in Nature and were first created by organic chemists in the laboratory. Since the organism's genome could not possibly have been exposed to these compounds non-existent before, to expect pumps anticipating the organic chemist's new creation is also beyond reason.

Since there is no limit to the number of new compounds that organic chemists can create in the future, even if we could accept---against all reason--- the non-genetic creation of more and more pumps, a new problem arises. How can a cell membrane with finite dimensions accommodates an infinity of pumps? That too would be beyond reason.

(2)Failure to demonstrate potassium-ion accumulation and sodium-ion extrusion in an ideal cell-membrane preparation without cytoplasm.

In 1961, two laboratories in Europe simultaneously announced the successful developments of techniques for removing the cytoplasm from the giant nerve fiber or axon of squids without impairing the normal functional activities of the remaining cell membranes (Baker, Hodgkin and Shaw, Nature 190:8851961; Oikawa, Spyropoulos, Tasaki and Teorell, Acta Physiol. Scand. 52:195, 1961). Since according to the membrane-pump theory, it is the postulated sodium pump located in the cell membrane which maintains both the low level of Na+ and high level of K+ in living cells, isolated squid axons with its cytoplasm removed offers an ideal preparation for the testing of the membrane-pump theory.

Thus if the open ends of a segment of such an axoplasm-free axon are tied after the inside of the sac has been filled with sea water containing the right nutrients, incubation of the closed axon-sac in a medium of normal sea water should lead to the gradual rise of K+ concentration along with a fall of the Na+ concentration in the fluid within the closed axon-sac. Experiments of this nature were carried out in the hands of some of the most skilled workers in this field. However, it was Professor Richard Keynes from the Cambridge University of England who announced in a seminar given in the spring of 1963 at the Johnson Foundation of the University of Pennsylvania---a seminar which I attended --- that these efforts all failed (for written record of this announcement, see Ling , Perspect. Biol. Med.9:87, 1965).

Professor Keynes and his coworker did not publish their unsuccessful attempt to verify the sodium pump hypothesis with the cytoplasm-free squid-axon sac. Brinley and Mullins who also failed to demonstrate transport of sodium ion against concentration gradients in a perfused giant squid axon, did publish their negative results (Brinley and Mullins, J. Gen. Physiol. 52: 181, 1968; Mullins and Brinley J. Gen. Physiol, 53: 704, 1969), leaving little doubt that the failed experiments of Keynes and coworkers were valid expression of the truth.

(3)Success in demonstrating potassium ion accumulation and sodium ion exclusion in the absence of an intact functional cell membrane and (postulated) sodium pump.

The original work was published by myself in J. Physiol. (London) 280:105 (1978).

An isolated frog sartorius muscle, a flat and thin muscle, contains about 1000 single muscle cells each no wider than a human hair but 2 to 3 centimeters in length. Each one of these muscle fiber or cell runs the full length from one end of the muscle to the other end. A razor-blade cut across the muscle at a locus proximal to the tapering tibial end produces a sartorius-muscle preparation containing approximately a thousand single muscle cells each with one end open. And the open end stays open as revealed by electron-microscopy (Cameron, Physiol. Chem.Phys. & Med. NMR 20:221, 1988).

When such a preparation is suspended in moistened air and only the cut end of the muscle cells exposed to a Ringer solution which contains radioactively labeled K+ and Na+, we have what is called an effectively membrane-less open ended cell or EMOC preparation. In this preparation, the cut end of the muscle has no membrane and hence pumps; the remaining intact portion of the muscle cell membrane is suspended in air. Since air does not take up Na+, there is no "sink"in this EMOC preparation to take up the Na+ pumped out by the hypothetical pump. Similarly, the surrounding air does not contain K+ and thus cannot be a "source" for the postulated inward potassium pumping. Without the needed "sink" or "source", the pumps---if they exist--- cannot function as postulated.(For cause of involvement of potassium ion here, see linked page lp6c.)

Yet despite these crucial deficiencies, radioactively labeled K+ enters and continues to rise in the intact portion of the muscle cells to concentration much higher than that in the medium bathing the cut end of the muscle, and the radioactively labeled Na+ also enters the intact end of the muscle but stays low at a concentration equal to only a fraction of the concentration of the sodium ion in the source solution despite continued incubation---just as one find in normal frog muscle cells.

Theoretically speaking, the possibility exists that labeled Na+ was actually pumped out of the cells but found its way back to the source solution (where it came from) via the "extracellular-space fluid". If indeed such a backward flow of labeled sodium did take place, it could only have done so by building up a diffusion head of sodium ion in the intact end of the extracellular space. To account for the amount of labeled Na+ that must have been transported back to satisfy this explanation, the concentration of labeled Na+ in the extracellular fluid must be built up to a level so high that it would approach the limit of NaCl solubility, ca. 5 Molar.

The actual analyses showed that the labeled Na+ concentration in the extracellular fluid recovered from an EMOC preparation after an incubation of about 5 days was actually slightly below that in the solution bathing the cut end of the muscle. Thus there is no indication whatsoever that the muscle cells had made efforts to transport back labeled Na+ to the source solution bathing the cut end.

The conclusion of this set of experiment is that the selective accumulation of K+ and selective exclusion of Na+ in living cells do not depend on the presence of a functional cell membrane and its postulated pumps. Since the only reason for postulating these pumps is to provide a mechanism for maintaining a high K+ and low Na+ concentration in the cell, the demonstration that these phenomena persist in the absence of a functional cell membranes ( and the postulated Na+ pump) shows also that the theory is wrong. No such pumps exists in the normal cell membrane.