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

Abstract

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 approximately 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 approximately 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 Vmax, while for NHE2 and NHE3 the effect was on Vmax alone. Phorbol ester stimulated NHE1 and NHE2, but inhibited NHE3 with a decrease in Vmax. ATP-depletion decreased Vmax and the apparent affinity for H+ for all three isoforms, and reduced the Hill coefficient to approximately 1, suggesting that a basal level of phosphorylation was required for the cooperativity. 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.