Regulation of Intestinal Epithelial Brush Border Na+/H+ Exchanger Isoforms, NHE2 and NHE3, in C2bbe Cells

Until recently, studies to characterize the intestinal epithelial Na⁺/H⁺ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na⁺/H⁺ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH _i only approximate Na⁺/H⁺ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used ²²[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca⁺⁺ involves changes to both K _Na and V _max . In contrast, the same two second messengers inhibit NHE3 by a decrease in V _max exclusively. Phorbol ester activation of protein kinase C alters both V _max and K _Na of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways. Received: 28 January 1999/Revised: 18 May 1999     

Half-lives of plasma membrane Na+/H+ exchangers NHE1-3: plasma membrane NHE2 has a rapid rate of degradation

The Na⁺/H⁺ exchangers NHE2 and NHE3 areinvolved in epithelial Na⁺ and HCOabsorption. To increase insights into the functions of NHE2 vs. NHE3,we compared their cellular processing with each other and with thehousekeeping isoform NHE1. Using biotinylated exchanger, we determinedthat the half-life of plasma membrane NHE2 was short (3 h) comparedwith that of NHE1 (24 h) and NHE3 (14 h) in both PS120 fibroblasts andCaco-2 cells. NHE2 transport and plasma membrane levels were reduced by3 h of Brefeldin A treatment, whereas NHE1 was unaffected. NHE2was degraded by the lysosomes but not proteosomes, as demonstrated byincreasing levels of endocytosed NHE2 protein after inhibition of thelysosomes, but not with proteosome inhibition. Unlike that of NHE3,basal NHE2 transport activity was not affected by phosphatidylinositol 3-kinase inhibition and did not appear to be localized in the juxtanuclear recycling endosome. Therefore, for NHE2, protein degradation and/or protein synthesis probably play important roles inits basal and regulated states. These results suggest fundamental differences in the cellular processing and trafficking of NHE2 andNHE3. These differences may underlie the specialized roles that theseexchangers play in epithelial cells.

     

IFN-gamma downregulates expression of Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells

Diarrhea associated with inflammatory bowel diseases has traditionally been attributed to stimulated secretion. The purpose of this study was to determine whether chronic stimulation of intestinal mucosa by interferon-gamma (IFN-gamma) affects expression and function of the apical membrane Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells expressing NHE2 and NHE3 were treated with IFN-gamma for 2, 24, and 48 h. Adult rats were injected with IFN-gamma intraperitoneally for 12 and 48 h. NHE2 and NHE3 activities were measured by unidirectional (22)Na influx across C2 cells and in rat brush-border membrane vesicles. NHE protein and mRNA were assessed by Western and Northern blotting. IFN-gamma treatment of C2 monolayers caused a >50% reduction in NHE2 and NHE3 activities and protein expression. In rats, region-specific, time- and dose-dependent reductions of NHE2 and NHE3 activities, protein expression, and mRNA were observed after exposure to IFN-gamma. Chronic exposure of intestinal epithelial cells to IFN-gamma results in selective downregulation of NHE2 and NHE3 expression and activity, a potential cause of inflammation-associated diarrhea.     

SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells

Short-chain fatty acids (SCFA), produced by colonic bacterial flora fermentation of dietary carbohydrates, promote colonic Na absorption through mechanisms not well understood. We hypothesized that SCFA promote increased expression of apical membrane Na/H exchange (NHE), serving as luminal physiological cues for regulating colonic Na absorptive capacity. Studies were performed in human colonic C2/bbe (C2) monolayers and in vivo. In C2 cells exposed to butyrate, acetate, proprionate, or the poorly metabolized SCFA isobutyrate, apical membrane NHE3 activity and protein expression increased in a time- and concentration-dependent manner, whereas no changes were observed for NHE2. In contrast, no significant changes in brush-border hydrolase or villin expression were noted. Analogous to the in vitro findings, rats fed the soluble fiber pectin exhibited a time-dependent increase in colonic NHE3, but not NHE2, protein, mRNA, and brush-border activity. These changes were region-specific, as no changes were observed in the ileum. We conclude that luminal SCFA are important physiological cues for regulating colonic Na absorptive function, allowing the colon to adapt to chronic changes in dietary carbohydrate and Na loads.     

Protein kinase C activates the renal apical membrane Na+/H+ exchanger

Summary Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na⁺/H⁺ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm ²²Na⁺ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of²²Na⁺ taken as a measure of the activity of the Na⁺/H⁺ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of²²Na⁺ uptake TPA did not affect the amiloride-insensitive component of²²Na⁺ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other active phorbol esters stimulated the rate of Na⁺/H⁺ exchange, but phorbol esters of the 4 configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na⁺/H⁺ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with -[³²P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.     

Alpha 2-adrenergic receptors and the Na+/H+ exchanger in the intestinal epithelial cell line, HT-29

alpha 2-Adrenergic receptors (alpha 2-AR) are negatively coupled to adenylyl cyclase via the GTP-binding protein Gi. However, inhibition of adenylylcyclase does not account for many effector cell responses to alpha 2-AR agonists, suggesting that the receptor can couple to other signal transduction pathways. One potential pathway may be the stimulation of Na+/H+ exchange elicited by alpha 2-AR activation in renal proximal tubule cells, platelets, and the NG-10815 cell line. To determine whether the various receptor-effector coupling mechanisms operate in a tissue-specific manner, we studied the effect of alpha 2-AR activation on basal and stimulated Na+/H+ exchange in epithelial cells isolated from human colon (HT-29 adenocarcinoma cells). Na+/H+ exchange was measured by quantitation of intracellular hydrogen ion concentration (acetoxymethyl ester 2,7-biscarboxyethyl-5(6)carboxyfluorescein) and 22Na+ uptake. HT-29 cells expressed an amiloride-sensitive Na+/H+ exchanger that was activated by reduction of intracellular pH (pHi) to 6.0 but was quiescent at a physiological pHi. The rapid alkalinization observed after acid loading (0.57 +/- 0.07 pH units/min/10(4) cells) was dependent on external sodium and was blocked by amiloride (Ki approximately 2.1 microM). Although epinephrine and the selective alpha 2-AR agonists clonidine and UK-14304 inhibited forskolin-activated adenylylcyclase, these compounds did not alter basal Na+/H+ exchange. Stimulated Na+/H+ exchange was similarly unaffected by epinephrine. In contrast, stimulated Na+/H+ exchanger activity was completely inhibited by the selective alpha 2-agonists clonidine, UK-14304, and guanabenz. This inhibitory effect was not blocked by the alpha 2-AR antagonist rauwolscine, and it is likely due to a direct interaction with the exchanger molecule itself. Structure/activity studies indicated that the compounds inhibiting exchanger activity possess either an imidazoline or guanidinium moiety. Although these molecules bear structural similarity to amiloride, they did not inhibit the amiloride-sensitive epithelial sodium channel in toad urinary bladder, suggesting that these compounds may be useful as "amiloride-like" ligands selective for the Na+/H+ exchanger. These data indicate that in the HT-29 intestinal cell line, in contrast to observations in other tissues, alpha 2-adrenergic receptors are not coupled to the Na+/H+ exchanger, suggesting that the cell-signaling mechanisms utilized by the alpha 2-AR are tissue specific.     

Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange

Intestinal neutral NaCl absorption, which is made up ofbrush-border (BB)Na⁺/H⁺exchange linked to BBCl/HCO₃exchange, is up- and downregulated as part of digestion and diarrhealdiseases. Glucocorticoids stimulate ileal NaCl absorption and BBNa⁺/H⁺exchange. Intestinal BB contains twoNa⁺/H⁺exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbitileal BBNa⁺/H⁺exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BBNa⁺/H⁺exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ~50% to ilealNa⁺/H⁺exchange. Glucocorticoids (methylprednisolone) increase BBNa⁺/H⁺exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times),without an effect on NHE2 activity. Thus ileal BBNa⁺/H⁺exchange in animals treated with glucocorticoids is 69% via NHE3. Aquantitative Western analysis for NHE3 was developed, using as aninternal standard a fusion protein of the COOH-terminal 85 amino acidsof NHE3 and maltose binding protein. Glucocorticoid treatment increasedthe amount of BB NHE3. The quantitative Western analysis showed thatNHE3 makes up 0.018% of ileal BB protein in control rabbits and0.042% (2.3 times as much) in methylprednisolone-treated rabbits.Methylprednisolone treatment did not alter the amount of ileal BB NHE2protein. NHE3 turnover number was estimated to be 458 cycles/s underbasal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thusmethylprednisolone stimulates ileal BBNa⁺/H⁺exchange activity only by an effect on NHE3 and not on NHE2; it does soprimarily by increasing the amount of BB NHE3, although it alsoincreases the NHE3 turnover number.

     

Effect of angiotensin-II on renal Na+/H+ exchanger-NHE3 and NHE2

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na+/H+ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of 22Na+ study in the presence of 50 microM HOE-694 revealed that Na+ uptake mediated by NHE3 was increased approximately 88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na+ uptake by stimulating NHE3 mRNA and protein content.     

Plasma membrane Ca2+ pumps and Na+/Ca2+ exchangers

Membrane-intrinsic transport systems play an essential role in intracellular Ca2+ homeostasis. ATP-driven Ca2+ pumps and carrier-mediated Na+/Ca2+ exchangers are the two specific Ca2+ transporting systems mainly responsible for Ca2+ extrusion across the plasma membrane. Ca2+ pumps operate in all eukaryotic cell types and are characterized by their high Ca2+ affinity and their specific regulation by direct interaction with Ca2+/calmodulin. Na+/Ca2+ exchangers are particularly abundant in excitable tissues and are responsible for the bulk Ca2+ efflux in these tissues. Recent success in the molecular characterization of the pumps has led to the determination of complete amino acid sequences for several isoforms and has allowed the identification and topological assignment of important functional and regulatory domains. Genetic evidence indicates that mammalian Ca2+ pump diversity is generated from a multigene family and via alternative RNA splicing. Different isoforms may vary in their regulatory properties, presumably reflecting different physiological requirements of the tissues of their expression. Although the molecular characterization of Na+/Ca2+ exchangers is not as far advanced as that of the pumps, recent studies have established detailed kinetic, stoichiometric and regulatory properties of these systems. Together with advances in expression cloning methods these studies promise to result in a rapid improvement of our knowledge of the functional properties of these ion transporters on a molecular level.     

Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1

The mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, play a major role in the regulation of pivotal cellular processes such as cell death/survival balance, cell cycle progression, and cell migration. MAPK activity is regulated by a three-tiered phosphorelay system, which is in turn regulated by a complex network of signaling events and scaffolding proteins. The ubiquitous plasma membrane Na(+)/H(+) exchanger NHE1 is activated by, and implicated in, the physiological/pathophysiological responses to many of the same stimuli that modulate MAPK activity. While under some conditions, NHE1 is regulated by MAPKs, a number of studies have, conversely, implicated NHE1 in the regulation of MAPK activity. Here, we discuss the current evidence indicating the involvement of NHE1 in MAPK regulation, the mechanisms by which this may occur, and the possible physiological and pathophysiological relevance of this phenomenon.     

Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface

NHE1, NHE2, andNHE3 are well-characterized cloned members of the mammalianNa⁺/H⁺exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbersof NHE1, NHE2, and NHE3 measured in the same cell system: PS120fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitopetagged with vesicular stomatitis virus glycoprotein (VSVG). Thefollowing characteristics were determined on the same passage of cellstransfected with NHE1V, NHE2V, or NHE3V:1) maximal reaction velocity(V_max) by²²Na⁺uptake and fluorometery, 2) totalamount of NHE protein by quantitative Western analysis with internalstandards of VSVG-tagged maltose-binding protein, and3) cell surface expression by cellsurface biotinylation. Cell surface expression (percentage of totalNHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despitethese divergent cell surface expression levels, turnover numbers forNHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and99.2 ± 9.1 s¹, whenV_max wasdetermined using ²²Na uptake at22°C and 742 ± 47, 459 ± 16, and 609 ± 39 s¹ whenV_max wasdetermined using fluorometry at 37°C). These data indicate that, inthe same cell system, intrinsic properties that determine turnovernumber are conserved among NHE1, NHE2, and NHE3.

     

Membrane curvature alters the activation kinetics of the epithelial Na+/H+ exchanger, NHE3

The epithelial Na(+)/H(+) exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them.     

Evidence for involvement of the putative first extracellular loop in differential volume sensitivity of the Na+/H+ exchangers NHE1 and NHE2

We studied hyperosmolarity-induced changes in cell volume and cytoplasmic pH in PS120 cells expressing Na(+)/H(+) exchanger (NHE) isoforms and their mutants. Change in cell volume was estimated by measuring change in cell height by means of confocal microscopy. Regulatory volume increase (RVI) and cytoplasmic alkalinization were observed in cells expressing NHE1 but not in cells expressing NHE2 or NHE3. Studies using chimeric exchangers revealed that the membrane domain of the exchanger is responsible for the difference in volume sensitivity between NHE1 and NHE2. Although deletion or point mutation within the first extracellular loop of NHE1 did not affect RVI and alkalinization, point mutations within the corresponding region of NHE2, particularly a region containing aa 41-53, as well as replacement of the N-terminus of NHE2 with the corresponding region of NHE1, rendered NHE2 responsive to the activating effect of cell shrinkage. Thus, the membrane domain plays an important role in the response of the exchanger to cell shrinkage. The data suggest that the putative first extracellular loop of NHE2, but not that of NHE1, may exert an inhibitory influence on hyperosmolarity-induced activation of the exchanger and thereby block RVI.     

Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Na+/H+ exchanger NHE3 activity and trafficking are lipid Raft-dependent

A previous study showed that approximately 25-50% of rabbit ileal brush border (BB) Na(+)/H(+) exchanger NHE3 is in lipid rafts (LR) (Li, X., Galli, T., Leu, S., Wade, J. B., Weinman E. J., Leung, G., Cheong, A., Louvard, D., and Donowitz, M. (2001) J. Physiol. (Lond.) 537, 537-552). Here, we examined the role of LR in NHE3 transport activity using a simpler system: opossum kidney (OK) cells (a renal proximal tubule epithelial cell line) containing NHE3. approximately 50% of surface (biotinylated) NHE3 in OK cells distributed in LR by density gradient centrifugation. Disruption of LR with methyl-beta-cyclodextrin (MbetaCD) decreased NHE3 activity and increased K'(H+)(i), but K(m)((Na+)) was not affected. The MbetaCD effect was completely reversed by repletion of cholesterol, but not by an inactive analog of cholesterol (cholestane-3beta,5alpha,6beta-triol). The MbetaCD effect was specific for NHE3 activity because it did not alter Na(+)-dependent l-Ala uptake. MbetaCD did not alter OK cell BB topology and did not change the surface amount of NHE3, but greatly reduced the rate of NHE3 endocytosis. The effects of inhibiting phosphatidylinositol 3-kinase and of MbetaCD on NHE3 activity were not additive, indicating a common inhibitory mechanism. In contrast, 8-bromo-cAMP and MbetaCD inhibition of NHE3 was additive, indicating different mechanisms for inhibition of NHE3 activity. Approximately 50% of BB NHE3 and only approximately 11% of intracellular NHE3 in polarized OK cells were in LR. In summary, the BB pool of NHE3 in LR is functionally active because MbetaCD treatment decreased NHE3 basal activity. The LR pool is necessary for multiple kinetic aspects of normal NHE3 activity, including V(max) and K'(H+)(i), and also for multiple aspects of NHE3 trafficking, including at least basal endocytosis and phosphatidylinositol 3-kinase-dependent basal exocytosis. Because the C-terminal domain of NHE3 is necessary for its regulation and because the changes in NHE3 kinetics with MbetaCD resemble those with second messenger regulation of NHE3, these results suggest that the NHE3 C terminus may be involved in the MbetaCD sensitivity of NHE3.     

Inhibition and redistribution of NHE3, the apical Na+/H+ exchanger, by Clostridium difficile toxin B

NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.     

Unique Regulation of Human Na+/H+ Exchanger 3 (NHE3) by Nedd4-2 Ligase That Differs from Non-primate NHE3s

Na⁺/H⁺ exchanger NHE3 expressed in the intestine and kidney plays a major role in NaCl and HCO₃⁻ absorption that is closely linked to fluid absorption and blood pressure regulation. The Nedd4 family of E3 ubiquitin ligases interacts with a number of transporters and channels via PY motifs. A comparison of NHE3 sequences revealed the presence of PY motifs in NHE3s from human and several non-human primates but not in non-primate NHE3s. In this study we evaluated the differences between human and non-primate NHE3s in ubiquitination and interaction with Nedd4-2. We found that Nedd4-2 ubiquitinated human NHE3 (hNHE3) and altered its expression and activity. Surprisingly, rat NHE3 co-immunoprecipitated Nedd4-2, but its expression and activity were not altered by silencing of Nedd4-2. Ubiquitination by Nedd4-2 rendered hNHE3 to undergo internalization at a significantly greater rate than non-primate NHE3s without altering protein stability. Insertion of a PY motif in rabbit NHE3 recapitulated the interaction with Nedd4-2 and enhanced internalization. Thus, we propose a new model where disruption of Nedd4-2 interaction elevates hNHE3 expression and activity.     

Endosomal Na+/H+ exchanger NHE5 influences MET recycling and cell migration

Increased recycling and elevated cell surface expression of receptors serve as a mechanism for persistent receptor-mediated signaling. We show that the neuron-enriched Na⁺/H⁺ exchanger NHE5 is abundantly expressed in C6 glioma cells and plays an important part in regulating cell surface expression of the receptor tyrosine kinases MET and EGF receptor. NHE5 is associated with transferrin receptor (TfR)- and Rab11-positive recycling endosomal membranes, and NHE5 knockdown by short hairpin RNA significantly elevates pH of TfR-positive recycling endosomes. We present evidence that NHE5 facilitates MET recycling to the plasma membrane, protects MET from degradation, and modulates HGF-induced phosphatidylinositol-3-kinase and mitogen-activated protein kinase signaling. Moreover, NHE5 depletion abrogates Rac1 and Cdc42 signaling and actin cytoskeletal remodeling. We further show that NHE5 knockdown impairs directed cell migration and causes loss of cell polarity. Our study highlights a possible role of recycling endosomal pH in regulating receptor-mediated signaling through vesicular trafficking.     

Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney

We present evidence that tissue distribution of two highlyconservedNa⁺/H⁺exchanger isoforms, NHE2 and NHE4, differs significantly from previously published reports. Riboprobes unique to each of these antiporters, from 5' (noncoding and coding) and 3' codingregions, were used to analyze mRNA from adult rat kidney and intestine by ribonuclease protection assay and in situ hybridization. In contrastto earlier work that concluded that both NHE2 and NHE4 were expressedthroughout the intestine and in the kidney, our data show that there isno NHE2 message in the kidney and NHE4 is not expressed in small orlarge intestine. Analyses of intestinal epithelial and kidney membraneproteins by an NHE2-specific antibody identified a doublet at <90 kDain intestine but not in kidney. NHE2 is highly expressed in theNa⁺-absorptive epithelium ofjejunum, ileum, and ascending and descending colon. NHE4 mRNA messageis found in the inner medulla of the kidney as previously reported (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao,and E. B. Chang. J. Biol. Chem. 269:29704-29709, 1994) and not in the intestine. From these data, wespeculate that neither NHE2 nor NHE4 has a role in renalNa⁺ absorption. NHE2 is likelyinvolved in gut Na⁺ absorption,whereas NHE4 may have a specialized role in cell volume rectificationof inner medullary collecting duct cells. Knowledge of the correcttissue and cell-specific distribution of these two antiporters shouldhelp significantly in understanding their physiological roles.

     

Casein kinase 2 binds to the C terminus of Na+/H+ exchanger 3 (NHE3) and stimulates NHE3 basal activity by phosphorylating a separate site in NHE3

Na(+)/H(+) exchanger 3 (NHE3) is the epithelial-brush border isoform responsible for most intestinal and renal Na(+) absorption. Its activity is both up- and down-regulated under normal physiological conditions, and it is inhibited in most diarrheal diseases. NHE3 is phosphorylated under basal conditions and Ser/Thr phosphatase inhibitors stimulate basal exchange activity; however, the kinases involved are unknown. To identify kinases that regulate NHE3 under basal conditions, NHE3 was immunoprecipitated; LC-MS/MS of trypsinized NHE3 identified a novel phosphorylation site at S(719) of the C terminus, which was predicted to be a casein kinase 2 (CK2) phosphorylation site. This was confirmed by an in vitro kinase assay. The NHE3-S719A mutant but not NHE3-S719D had reduced NHE3 activity due to less plasma membrane NHE3. This was due to reduced exocytosis plus decreased plasma membrane delivery of newly synthesized NHE3. Also, NHE3 activity was inhibited by the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole DMAT when wild-type NHE3 was expressed in fibroblasts and Caco-2 cells, but the NHE3-S(719) mutant was fully resistant to DMAT. CK2 bound to the NHE3 C-terminal domain, between amino acids 590 and 667, a site different from the site it phosphorylates. CK2 binds to the NHE3 C terminus and stimulates basal NHE3 activity by phosphorylating a separate single site on the NHE3 C terminus (S(719)), which affects NHE3 trafficking.