A slow pH-dependent conformational transition underlies a novel mode of activation of the epithelial Na+/H+ exchanger-3 isoform.

Allosteric control of Na(+)/H(+) exchange by intracellular protons ensures rapid and accurate regulation of the intracellular pH. Although this allosteric effect was heretofore thought to occur almost instantaneously, we report here the occurrence of a slower secondary activation of the epithelial Na(+)/H(+) exchanger (NHE)-3 isoform. This slow activation mode developed over the course of minutes and was unique to NHE3 and the closely related isoform NHE5, but was not observed in NHE1 or NHE2. Activation of NHE3 was not due to increased density of exchangers at the cell surface, nor was it accompanied by detectable changes in phosphorylation. The association of NHE3 with the cytoskeleton, assessed by its retention in the detergent-insoluble fraction, was similarly unaffected by acidification. In contrast to the slow progressive activation elicited by acidification, deactivation occurred very rapidly upon restoration of the physiological pH. We propose that NHE3 undergoes a slow pH-dependent transition from a less active to a more active state, likely by changing its conformation or state of association.