Evidence for the allosteric regulation of the mitochondrial K+/H+ antiporter by matrix protons

It is well accepted that the mitochondrial K+/H+ antiporter is regulated by matrix Mg2+; however, this is not the only factor controlling its activity. The precise conditions used to deplete divalent cations have profound effects on the subsequent activity of the antiporter in a KOAc assay medium. Examination of the proton fluxes during both pretreatment and subsequent assay of K+/H+ antiport reveals that differences in K+/H+ antiport activity correlate very well with differences in matrix pH. Thus, inhibition of the K+/H+ antiporter following depletion of Mg2+ appears to result from inhibition by matrix protons. To test this hypothesis, we have examined the effect of modulating matrix pH in three different ways on the activity of the K+/H+ antiporter: 1) lowering the pH of the K+ pretreatment medium to 6.7 leads to inactivation of the K+/H+ antiporter; 2) adding NH4+ to the assay medium eliminates the lag in activity induced by depleting Mg2+ in a pretreatment medium containing NH4+; 3) permitting mitochondria to respire in a tetraethylammonium(+)-containing pretreatment medium activates the K+/H+ antiporter. Each one of these procedures leads to a change in matrix pH and an effect on K+/H+ antiport which appears to require regulation of the K+/H+ antiporter by matrix protons. This finding is not only physiologically significant but also provides a useful definition of conditions required for unmasking the K+/H+ antiporter in a reproducible manner.