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

doi:10.1016/s0021-9258(19)74423-8

S A Levine, M H Montrose + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(19)74423-8

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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.

Highlights

The Na+/H+ exchanger isoforms are regulated differently bysecondmessenger [8]
This study describes in detail the kinetics and regulation by hypothesized that secondmessenger regulation of Na+/H' several growth factors and protein kinases,including protein exchangers occursvia changes in the affinitfyor intracellular kinase C, of three cloned rabbit ileal Na'/H' exchangers
The variability in Na'/H' exchange rate among clonal lines and concern over possible inadvertent selection of nonrepresentative characteristics in certain clones led us to perform the remainder of the reported studies of kinetics and regulation on mixed populationsof cells (PS120/NHE1, /NHE2, /NHE3) which had been recently acid selected to provide maximal activity

Summary

No effect

Cell Culture-Transfection of cDNAs forNHE1,NHE2, and NHE3 into PS120 fibroblasts was previously described [23,24,25] and produced three stably transfected “mixed population” fibroblast cell lines designated PSlZO/NHEl, PS120/NHE2, and PS120/NHE3. Data from the Na+ activation experiments were analyzed in a similar fashion to the H+kinetic data, but results have been expressed as a percent of the maximal Na+/H+ exchange rate obtained during the experiments, reducing the effect of variation in V,,, on analysis of the kinetics of the response. For these experiments, the data fit Michaelis-Menten kinetics, and the K,(Na+) value obtained should be an accurate estimate of the [Na+]necessary to generate 1/2 V,, and of the affinity of the transporter for external [Na+]. BCECF-AM and nigericin were from Molecular Probes (Eugene, OR) and H7 from Seikagaku America, Inc. (Rockford,MD), while other reagents were purchased from Sigma

RESULTS

ATP Depletion

DISCUSSION