Since the cloning of the first member of the Na+/H+ exchanger (NHE) family, termed NHE1, four NHE isoforms have been cloned (NHE2, NHE3, NHE4, and the trout beta-NHE) and expressed in exchanger-deficient cell lines. All these isoforms exhibit significant identity to NHE1 and possess a similar hydropathy profile with two highly conserved transmembrane segments presumably involved in ion transport. These isoforms are allosterically activated by intracellular H+, regulate intracellular pH in a Na(+)-dependent manner, and are inhibited by amiloride and 5-amino derivatives with distinct Ki values. NHE1 is the amiloride-sensitive, growth factor-activatable, and ubiquitously expressed NHE known to regulate intracellular pH and cellular volume. NHE2, NHE3, and NHE4 are, however, restricted in their tissue distribution, suggesting roles in specialized functions of these epithelial tissues. In this review we present and discuss the most recent advances in the molecular and biochemical features, hormonal and growth factor activation, specific expression, and membrane sorting of the members of this NHE family.

Migration and morphology of human melanoma cells (MV3) depend on extracellular pH (pHe) and the activity of the Na+/H+ exchanger NHE1. To distinguish effects of NHE1 activity per se from effects of pHe we compared an NHE1-deficient mutant with rescued and wild-type cells. Time lapse video microscopy was used to investigate migratory and morphological effects caused by pHe and NHE1 activity, and a membrane-bound fluorescein conjugate was employed for ratiometric pH measurements at the outer leaflet of the cell membrane. As long as NHE1 remained inactive due to deficiency or inhibition by cariporide (HOE642) neither migration nor morphology was affected by changes in pHe. Under these conditions pH at the outer leaflet of the plasma membrane was uniform all over the cell surface. The typical pH dependence of MV3 cell migration and morphology could be reconstituted by restoring NHE1 activity. At the same time the proton gradient at the outer leaflet of the plasma membrane with the higher proton concentration at the leading edge and the lower one at the cell rear was re-established as well. Hence, NHE1 activity generates a proton gradient at the cell surface accompanied by the cells' ability to respond to changes in pHe (bulk pH). We conclude that NHE1 activity contributes to the generation of a well-defined cell surface pH by creating a proton gradient at the outer leaflet of the plasma membrane that is needed for (i) the development of a variety of morphologies including a distinct polarity and (ii) migration. A missing proton gradient at the cell surface cannot be compensated for by varying pHe.

Trinh NT, Privé A, Maillé E, Noël J, Brochiero E. EGF and K ϩ channel activity control normal and cystic fibrosis bronchial epithelia repair.

Extracellular pH and the Na⁺/H⁺ exchanger (NHE1) modulate tumor cell migration. Yet, the pH nanoenvironment at the outer surface of the cell membrane (pH_em) where cell/matrix interaction occurs and matrix metalloproteinases work was never measured. We present a method to measure this pH nanoenvironment using proton-sensitive dyes to label the outer leaflet of the plasma membrane or the glycocalyx of human melanoma cells. Polarized cells generate an extracellular proton gradient at their surface that increases from the rear end to the leading edge of the lamellipodium along the direction of movement. This gradient collapses upon NHE1 inhibition by HOE642. NHE1 stimulation by intracellular acidification increases the difference in pH_em between the tips of lamellipodia and the cell body in a Na⁺ dependent way. Thus, cells create a pH nanoenvironment that promotes cell migration by facilitating cell adhesion at their front and the release of cell/matrix contacts at their rear part.

Na؉ uptake in response to ionomycin and thrombin was observed in N3N1, accompanied by an alkaline shift of pH i sensitivity (ϳ0.2 pH units). Deletion of the cytoplasmic calmodulin-binding domain within N3N1 resulted in a constitutive alkaline shift of pH i sensitivity and abolished the activation by ionomycin and thrombin. Together, these data reinforce our concept of Ca 2؉ -induced activation of NHE1. Furthermore, they provide evidence for a functional interaction of the autoinhibitory domain of NHE1 with the H ؉ -modifier site of a different isoform, NHE3.Calcium ion is an important second messenger in mammalian cells, regulating various cell functions including muscle contraction, secretion, cell cycle progression, and a large variety of nerve cell functions. In many of these processes, elevation of the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and subsequent Ca 2ϩ -dependent activation of a ubiquitous regulator protein calmodulin (CaM) 1 have been recognized as a major mechanism of signal transduction in response to hormonal stimulation or membrane depolarization (1, 2).The electroneutral plasma membrane Na ϩ /H ϩ exchanger isoform 1 (NHE1) has been shown to be one of the targets regulated by intracellular Ca 2ϩ (3-8). NHE1 (9) is a ubiquitous amiloride-sensitive transporter that regulates pH i and cell volume (10, 11), and its structure-function relationship has been studied extensively (7,(12)(13)(14)(15)(16)(17). We have recently shown that NHE1 is a CaM-binding protein containing high and low affinity CaM-binding sites in the middle of the carboxyl-terminal cytoplasmic domain (17). Based on the analysis of function of NHE1 mutant molecules that do not bind CaM, we proposed that Ca 2ϩ -induced activation of NHE1 occurs via direct binding of Ca 2ϩ /CaM to the high affinity site that has an autoinhibitory function (7). However, further experiments were required to unambiguously confirm this hypothesis because of lack of evidence for the direct effect of Ca 2ϩ /CaM on the exchange activity.When NHE1 is activated in response to various stimuli such as growth factors, calcium, and hyperosmotic stress, it is generally accepted that pH i sensitivity of Na ϩ /H ϩ exchange increases without an apparent change in V max (18 -20). This is thought to result from increased affinity of the allosteric modifier site of the exchanger for the intracellular H ϩ (21). However, recently cloned other exchanger isoforms (NHE2, NHE3, and NHE4) (22-25) differ greatly from NHE1 in their regulation. Growth factors activate the epithelial isoforms NHE2 and NHE3 by increasing V max (26,27). Phorbol ester stimulates NHE1 and NHE2 but inhibits NHE3 (26,27). Hyperosmolarity stimulates NHE1, NHE2, and NHE4 but inhibits NHE3 (28 -30). These differences appear to be attributable to sequence divergence of the cytoplasmic domains of these NHE isoforms. The amiloride-resistant NHE3 that is expressed in the apical membrane of epithelial cells in kidney or intestine is the least related isoform among four mammalian NHEs. The NHE3 cy...

Several respiratory diseases are associated with extensive damage of lung epithelia, and the regulatory mechanisms involved in their regeneration are not clearly defined. Growth factors released by epithelial cells or fibroblasts from injured lungs are important regulators of alveolar repair by stimulating cell motility, proliferation, and differentiation. In addition, K(+) channels regulate cell proliferation/migration and are coupled with growth factor signaling in several tissues. We decided to explore the hypothesis, never investigated before, that K(+) could play a prominent role in alveolar repair. We employed a model of mechanical wounding of rat alveolar type II epithelia, in primary culture, to study their response to injury. Wound healing was suppressed by one-half upon epidermal growth factor (EGF) titration with EGF-antibody (Ab) or erbB1/erbB2 tyrosine-kinase inhibition with AG-1478/AG-825. The addition of exogenous EGF slightly stimulated the alveolar wound healing and enhanced, by up to five times, alveolar cell migration measured in a Boyden-type chamber. Conditioned medium collected from injured alveolar monolayers also stimulated cell migration; this effect was abolished in the presence of EGF-Ab. The impact of K(+) channel modulators was examined in basal and EGF-stimulated conditions. Wound healing was stimulated by pinacidil, an ATP-dependent K(+) channel (K(ATP)) activator, which also increased cell migration, by twofold, in basal conditions and potentiated the stimulatory effect of EGF. K(ATP) or KvLQT1 inhibitors (glibenclamide, clofilium) reduced EGF-stimulated wound healing, cell migration, and proliferation. Finally, EGF stimulated K(ATP) and KvLQT1 currents and channel expression. In summary, stimulation of K(+) channels through autocrine activation of EGF receptors could play a crucial role in lung epithelia repair processes.

Cell biological approaches were used to examine the location and function of the brush border (BB) Na(+)/H(+) exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as approximately 400- and approximately 900-kDa standards, whereas surface NHE3V was enriched in the approximately 900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from approximately 400 to approximately 900 kDa.

We report the characterization in the human genome of 966 pseudogenes derived from the four human Y (hY) RNAs, components of the Ro/SS-A autoantigen. About 95% of the Y RNA pseudogenes are found in corresponding locations on the chimpanzee and human chromosomes. On the contrary, Y pseudogenes in mice are both infrequent and found in different genomic regions. In addition to this rodent/primate discrepancy, the conservation of hY pseudogenes relative to hY genes suggests that they occurred after rodent/primate divergence. Flanking regions of hY pseudogenes contain convincing evidence for involvement of the L1 retrotransposition machinery. Although Alu elements are found in close proximity to most hY pseudogenes, these are not chimeric retrogenes. Point mutations in hY RNA transcripts specifically affecting binding of Ro60 protein likely contributed to their selection for direct trans retrotransposition. This represents a novel requirement for the selection of specific RNAs for their genomic integration by the L1 retrotransposition machinery. Over 40% of the hY pseudogenes are found in intronic regions of protein-coding genes. Considering the functions of proteins known to bind subsets of hY RNAs, hY pseudogenes constitute a new class of L1-dependent non-autonomous retroelements, potentially involved in post-transcriptional regulation of gene expression.