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

S. A. Levine, M. H. Montrose, C. M. Tse, M. Donowitz

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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 K(m)(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 V(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 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.

Original languageEnglish (US)
Pages (from-to)25527-25535
Number of pages9
JournalJournal of Biological Chemistry
Issue number34
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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