I need a reference (for the article "Steady-state physiology…" I have just written) that proves the pumping role of Na,K-ATPase. I have tried to locate any publication that demonstrates the pumping function of Na,K-ATPase, but have found nothing. Please help me with references (even one would be enough). To make it clear what I need, let me to give the following clarification.
We believe that the ionic asymmetry between the cell and its environment is provided by a plasma membrane pump, Na-K-ATPase. Because of this pump, the concentration of K+ in the cell is above that in the medium and the concentration of Na+ is lower than in the medium. The pump works continuously, using energy. Once the pump stops working, K+ leaves the cells, and Na+, conversely, enters them. If we turn on the pump again, it will start to pump Na+ out of the cells, and K+ will be pumped into the cell. The question is, what experiments should be designed to show that the membrane pump is real?
Let us consider the squid giant axon. (Since the work done on this cell was honored with a Nobel Prize), this axon and other similar preparations have become a favorite subject of numerous studies]. Remove its axoplasm to obtain the axon ghost (axon without axoplasm), then fill the axon ghost with natural or artificial seawater. The composition of the solution inside the axon will serve us as a reference point (the sodium and potassium concentrations in it will be the same as in the washing solutions). Now add ATP (and an ATP generating system, e.g. phosphoenolpyruvate plus pyruvate kinase) to the interior of the axon ghost and securely tie the ends of the axon so that its contents are not mixed with the external medium. Prepare a sufficient number of such ghosts, and take one axon after another at various time points to determine the ionic composition of their contents. Take the first axon after 10 minutes after the start of the experiment (analyze the contents, record the data), a second axon after 20 minutes, the third after an hour, and so on. If Na,K-ATPase does indeed function as the pump, the amount of Na+ in the axon ghost should gradually decrease, and K+ should increase. As a result of the experiment, we should obtain curves that clearly demonstrate the work of the membrane pump.
Instead of experiments such as the one I have described, the literature is full of articles about the activity of Na,K-ATPase, how its activity can be changed, and how its activity affects membrane permeability and other properties. The authors of these articles have constructed a lot of graphs and created a mass of equations. But all of these are irrelevant to the experiment that I described above.
In addition, there are many articles in the literature describing experiments along the following lines: the authors load lipid vesicles containing embedded Na,K-ATPase with Na+, with ATP or without ATP (control), and then separate the vesicles from the mother liquor. The result of such an experiment is normally: control vesicles (no ATP) contain lower Na+ than the experimental ones (with ATP). From this observation the authors conclude as a rule that the Na,K-ATPase in the presence of ATP acts as a pump and pumps sodium into the vesicles. However, can we consider such experiments as an evidence of the physiological pumping role of Na,K-ATPase? I think, not.
The fact is that as soon as the vesicles are separated from the mother liquor, Na+ starts to leave them down the chemical potential gradient. The type of experiment discussed shows us only one thing: Na+ leaves control vesicles FASTER than vesicles with ATP. The authors of these studies explain this difference by the fact that in the presence of ATP, Na-K-ATPase works as a pump: sodium ions are initially pulled out of the vesicles and ATPase grabs them, and once again pumps them into the vesicles. However, there is another possible explanation: ATP, being a hydrophobic polyanion, interacts with the lipid membrane and changes its physical properties, affecting the properties of the Na,K-ATPase. As a result, the lipid membrane-Na,K-ATPase become LESS permeable to Na+ in the presence of ATP. If we really wish to examine the role of Na,K-ATPase as a "pump" we should adopt the negative hypothesis: Na,K-ATPase is not really a pump, but simply serves as a barrier to Na+ when it passes from the vesicles into the surrounding medium. To the best of my knowledge, nobody has checked such a counter-interpretation. If so, we have TWO possible explanations of the experiment with lipid vesicles. The existence of two explanations means the absence of proof.
It is quite possible that there are other experimental approaches to proving Na,K-ATPase is a pump. It is important that we assume that the pumping function of Na-K-ATPase is not proven. If, however, an experimenter holds a priori the idea that Na,K-ATPase is a membrane pump, his experiments cannot be correct. As we well know, when we seek to prove something we must proceed by contradiction.
Could you please give me even ONE reference with definitive proof of the pumping role of Na,K-ATPase? I cannot find it.