Steady-state measurements of Escherichia coli sodium and proton potentials at alkaline pH support the hypothesis of electrogenic antiport.

31P and 23Na NMR spectroscopy was applied to the measurement of proton and sodium potentials in endogenously respiring Escherichia coli cells over an external pH (pHex) range from mildly acidic (6.4) to fairly alkaline (8.4). Stable maintenance of alkaline pHex in the face of metabolic acidification was achieved by use of a perfusion system. In the acidic to neutral pHex range, the sodium chemical potential followed the proton chemical potential quite closely, although always exceeding it slightly, as has been reported previously (Castle, A. M., Macnab, R. M., and Shulman, R. G. (1986) J. Biol. Chem. 261, 7797-7806). Above pHex 7.4, the sodium potential changed abruptly from a decreasing to an increasing function of pHex, whereas the proton potential continued to decrease. As a consequence, the apparent stoichiometry (i.e. the ratio between the sodium and proton electrochemical potentials) took on progressively higher values, increasing from approximately 1.1 at pH 7.4 to approximately 1.3 at pH 7.8. Thereafter, the sodium chemical potential started to decrease again; however, since the decrease was less steep than that of the proton potential, the apparent stoichiometry continued to increase. At the highest pHex examined (8.4), it had reached a value of approximately 1.4. These results strongly support the hypothesis of an electroneutral (1:1) H+/Na+ antiporter operating virtually alone under acidic to neutral conditions and then being supplemented to an ever increasing degree by an electrogenic (for example, 2:1) antiporter under more alkaline conditions.