The ion-transporting activities of many membrane proteins, such as the Na+,K+-pump (or Na+,K+-ATPase), and Na+- and K+-channels, are sensitive to the transmembrane electrical potential difference. In experimental electrophysiological studies, this can be relatively easily controlled via electrodes in the aqueous solutions on each side of the membrane. However, all transported ions must bind to sites of the protein embedded within the membrane and, therefore, the crucial parameter for any rate or equilibrium constant of an ion transport protein’s mechanism is in fact the local electric field strength within the membrane. This is extremely difficult to measure experimentally. The image on the September 16 issue of the Biophysical Journal shows theoretical calculations of the contribution to the electric field strength from K+ ions (above) and Na+ ions (below) bound to the Na+,K+-ATPase. The calculations are based on recently published x-ray crystallographic structures of the protein in K+- and Na+-bound states.
The calculations show that as one moves away from the ion binding sites the field strength decays rapidly through the protein matrix and its surrounding membrane, such that the field strength originating from the bound ions is negligible at the membrane/aqueous interface adjacent to the protein. However, voltage-sensitive fluorescent membrane probes, such as RH421, situated in the membrane adjacent to the Na+,K+-ATPase, respond with large fluorescence changes on addition of Na+ or K+ ions to the protein and have often been used to resolve the kinetics of the enzyme’s partial reactions. What is it then that RH421 is responding to? By studying the interaction of the Na+,K+-ATPase with the large cation benzyltriethylammonium (BTEA), which is capable of binding to the protein’s ion transport sites but, in contrast to Na+ and K+, can’t be occluded within the protein interior, we isolated the conformational change of the protein necessary ion occlusion, rather than ion binding per se, as the origin of the RH421 response. Because RH421 is known to be sensitive to membrane dipole potential, if our model of field strength emanating from ions bound to the Na+,K+-ATPase is correct, a likely explanation for the probe’s response is that the protein conformational change disturbs the lipid packing around the protein. This would lead to a local electric field strength change at the membrane/protein/aqueous solution interface.
The activity of the Na+,K+-ATPase is known to be particularly sensitive to the lipid composition of the membrane. If occlusion reactions perturb the surrounding membrane, as our results indicate, it seems logical that the flexibility and polarisability of the membrane should influence the protein’s activity. Ion occlusion reactions of ion pumps, such as the Na+,K+-ATPase, can be seen as analogous to gating reactions of ion channels. Factors such as the membrane dipole potential, lipid packing and membrane hydrophobic thickness could, therefore, also be important determinants of channel activity and its voltage dependence.
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– Laura Mares, Alvaro Garcia, Helge Rasmussen, Flemming Cornelius, Yasser Mahmmoud, Joshua Berlin, Bogdan Lev, Toby Allen & Ronald Clarke