Na+/H+ exchanger isoform 3 (NHE3) is predominantly expressed in the apical membrane of intestinal and renal epithelia, where it plays a pivotal role in NaCl absorption and acid-base homeostasis. Transport activity of NHE3 is sensitive to intracellular pH (pHi). At resting pHi, a large fraction of transporters resides in an inactive state. When the H+ concentration of the cytosol rises, the transporters are converted into an active state. There are two proposed models to explain this kinetic regulation of transporters, one is the H+ sensor model and the other is the symmetrical dimer model. We have previously shown that NHE3 is slowly activated over the course of minutes, implying involvement of a conformational change of NHE3 such as dimerization in heterogenous expression systems (1). Under physiological conditions, NHE3 is thought to be coupled with other transporters in a concerted manner. One example is with Cl-/HCO3- exchanger in electroneutral NaCl absorption in the intestine. Another is with H+-coupled peptide transporter whose driving force is supported by NHE3 (2). To gain insight into the activation mechanism of NHE3, we used the NHE3 specific inhibitor tenapanor, which has a symmetrical structure with two proposed binding sites, and compared it with another NHE3 inhibitor, S3226. We examined the effect of inhibitors on NHE3 activity in different NHE3 transport modes, which involve coupling to other transporters, in NHE3 expressing cell lines and isolated mouse intestinal tissue. NHE3 was stably expressed in PS120 cells, which are deficient in endogenous NHE activity. pHi was measured by microfluorometry of the emission of BCECF. The activity of NHE3 was determined as the rate of Na+-induced pHi recovery after acid loading. Mice (C57Bl/6jjcl) were anaesthetized with a mixture of drugs (10 µl/g B.W., i.p.) consisting of 30 µg/ml medetomidine, 0.4 mg/ml midazolam and 0.5 mg/ml butorphanol. The isolated intestinal segments were mounted on Ussing chambers. In NHE3 expressing cells, tenapanor inhibited NHE3 activity dose-dependently with IC50 values of 1.26 ± 0.40 nM (n=4) and 3.02 ± 0.53 nM (n=3) for 1 min and 10 min acidification, respectively. In native epithelia, peptide-induced short-circuit current was inhibited dose-dependently by tenapanor and S3226 with an IC50 value of 6.3 ± 3.4 nM (n=3) and 5.9 ± 1.0 µM (n=3), respectively. However, S3226 completely inhibited transepithelial 22Na+ flux but tenapanor did not. It is of interest to note that pretreatment of tenapanor (no tenapanor during NHE3 activity measurement) dose-dependently inhibited acid activated NHE3 activity. Together, these results suggested that tenapanor may irreversibly bind to NHE3 and recognize the different NHE3 transport modes.