Functional characteristics of a cloned epithelial Na⁺/H⁺ exchanger (NHE3): Resistance to amiloride and inhibition by protein kinase C

We previously cloned an isoform Na⁺/H⁺ exchanger (NHE3), which was expressed only in intestine, kidney, and stomach. We show here the functional characteristics of NHE3 as a Na⁺/H⁺ exchanger by stably transfecting NHE3 cDNA into PS120 cells, a fibroblast cell line that lacks endogenous Na⁺/H⁺ exchangers. NHE3 was 39- and 160-fold more resistant to inhibition by amiloride and ethylisopropyl amiloride, respectively, than NHE1, the housekeeping Na⁺/H⁺ exchanger isoform. Although both exchangers were stimulated by serum, NHE3 was inhibited by phorbol 12-myristate 13-acetate (PMA), which stimulated NHE1. Mechanistically, serum and PMA stimulated NHE1 by an increase in the apparent affinity of the exchanger for intracellular H⁺. In contrast, serum stimulated and PMA inhibited NHE3 by a V_max change. When NHE3 was stably expressed in Caco-2 cells, an intestinal epithelial cell line, NHE3 was functionally expressed in the apical membrane. Thus, NHE3 is a good candidate to be an epithelial brush border Na⁺/H⁺ exchanger. Furthermore, Na⁺/H⁺ exchangers can be rapidly regulated by mechanisms that change either the V_max or the affinity for intracellular H⁺, depending on the Na⁺/H⁺ exchanger subtype.