Kinetics and regulation of three cloned mammalian Na⁺/H⁺ exchangers stably expressed in a fibroblast cell line

The kinetics and second messenger regulation of three cloned mammalian intestinal Na⁺/H⁺ exchangers were studied using fluorometric techniques. These exchangers, NHE1, NHE2, and NHE3, were stably expressed in PS120 fibroblasts, which lack an endogenous Na⁺/H⁺ exchanger. H⁺ kinetic data indicated cooperativity by internal protons, with Hill coefficients of ∼2 for all three isoforms. In contrast, Na⁺ kinetic data fit Michaelis-Menten kinetics, with Km(Na⁺) 15-18 mM and a Hill coefficient of ∼1. The exchangers were all activated by growth factors and thrombin; in NHE1 these agonists increased the apparent affinity for intracellular H⁺, but did not change V_max, while for NHE2 and NHE3 the effect was on V_max alone. Phorbol ester stimulated NHE1 and NHE2, but inhibited NHE3 with a decrease in V_max. ATP-depletion decreased F_max and the apparent affinity for H+ for all three isoforms, and reduced the Hill coefficient to ∼1, suggesting that a basal level of phosphorylation was required for the cooperatively. The differences in kinetics and second messenger regulation suggest that the NHE isoforms may serve different cellular functions. The up- and down-regulation of NHE3 by kinases indicates that this isoform may be involved in a specialized function such as Na⁺ absorption.