Abstract: The epithelial Na /H  exchanger, NHE3, was found to activate

slowly following an acute cytosolic acidification. The sigmoidalcourse of activation could not be explained by the conventionaltwo-state model, which postulates that activation results fromprotonation of an allosteric modifier site. Instead, mathematicalmodeling predicted the existence of three distinct states of the exchanger: two differentinactive states plus an active form.The interconversion of the inactive states is rapid and dependent on pH,whereas the conversion between the second inactive state and theactive conformation is slow and pH-independent but subject toregulation by other stimuli. Accordingly, exposure of epithelialcells to hypoosmolar solutions activatedNHE3by accelerating thislatter transition. The number of surface-exposed exchangers andtheir association with the cytoskeleton were not affected byhypoosmolarity. Instead, NHE3 is activated by the membranedeformation, a result of cell swelling. This was suggested by thestimulatory effects of amphiphiles that induce a comparable positive(convex) deformation of the membrane. We conclude thatNHE3 exists in multiple states and that different physiological

parameters control the transitions between them.