Like a basement in a flood plain, a cell needs a good pump. Cells must maintain a particular mix of ions inside their membrane walls, with low concentrations of sodium and high concentrations of potassium. The only problem is that cells are leaky, and sodium constantly rushes into the cell while potassium rushes out. To fight against this tide, the cell uses a very important and peculiar membrane protein called the sodium-potassium pump.
Since its discovery in the 1970’s, cell biologists have been baffled by the strange features of this powerful pump. Rather than an even one-to-one swap of potassium for sodium, in each cycle the pump transports three sodium ions out for every two potassium ions it takes in. Later, scientists discovered that both sodium and potassium could bind to the same locations on the pump, a fickle temperament that is unusual among membrane proteins typically very picky about the type of ion they bind. That presented an intriguing molecular engineering problem — how could the pump modify itself to bind sodium when it’s accessible to one side of the membrane and potassium when it’s accessible to the other?
Some biologists have suggested that this riddle could only be answered by analyzing the highly precise geometry of the binding sites. Thus, many predicted that the model could not be solved until the most minute details of the structure of the sodium-potassium pump was fully captured in both its sodium-bound and potassium-bound states. So far, only the latter pictures (taken by X-ray crystallography) exist. But Benoit Roux, professor of biochemistry and molecular biophysics, decided that half the information was good enough to form a new theory of how the pump pulls double duty.
“Biologists have swept this under the rug, saying we need to know the structure of both the sodium and potassium bound forms with a sub-angstrom accuracy to address this issue,” Roux said. “Our point of view is that proteins are flexible macromolecules and that the mechanism of ion selectivity ought to be fairly robust, even when there are small sub-angstrom thermal fluctuations.”
Roux’s group, which included Haibo Yu of UChicago and Ian Ratheal and Pablo Artigas from Texas Tech, applied a computational method called molecular dynamics to the two existing crystal structures of the pump – isolated, strangely, from the rectal gland of a shark. For a paper published in Nature Structural & Molecular Biology last week, the team ran computer simulations that tested the possibilities of how four important amino acids in the binding sites mediate the pump’s change in selectivity under normal conditions. Instead of a complicated transformation from sodium-binding to potassium-binding mode, Roux’s model identified a small change that could account for the pump’s changed loyalties.
Protonation is a chemical reaction that adds a single hydrogen atom to a molecule. The four binding site amino acids of interest happen to carry negatively-charged acidic side chains that may or may not bind an extra proton. Roux’s group found that when the four acidic residues lose that extra proton (called deprotonation), they strongly prefer sodium to potassium. In their protonated state, the preference reverses to potassium over sodium.
“At this point it’s speculation because we do not know the structure of the sodium-bound state. But perhaps protonation and deprotonation play a more active role on modulating selectivity of these sites during the functional cycle of the pump,” Roux said. “It’s a provocative idea, nobody has ever proposed something like that to the best of our knowledge. Some people might be a bit shocked.”
A simple, if non-physiological, way to test that theory is to adjust the pH around the cell. In a high pH, more basic environment, fewer protons would be available to “switch” the binding site preference from sodium to potassium, decreasing the amount of potassium that is brought into the cell. Experimental measurements under these conditions were in line with these expectations, supporting the new theory. Previously published work by other laboratories that mutated the crucial binding site amino acids also fit the proposal that protonation is important for selectivity.
“One critical residue, Asp811, that we predicted as one that has to be deprotonated was mutated to a neutral asparagine, and it completely killed the pump,” Roux said. “With this mutation, the pump does not carry any ions any more, which is consistent with our calculations.”
But because the crystallography currently tells only half the story, a complete test of the protonation theory is not yet possible. When detailed information about the structure of the inward-facing sodium-bound pump is available, scientists can test whether deprotonation completes the story of how the sodium-potassium pump pulls off its unusual change of allegiance.
“What we have now is a new proposition based on our careful study of the potassium binding site, and we think that it is worth seeing if it’s true,” Roux said. “At this point we really wish to have a crystal structure of other states of the pump.”
Yu, H., Ratheal, I., Artigas, P., & Roux, B. (2011). Protonation of key acidic residues is critical for the K+-selectivity of the Na/K pump Nature Structural & Molecular Biology DOI: 10.1038/nsmb.2113