Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.     

A photoactivatable probe for the Na+/H+ exchanger cross-links a 66-kDa renal brush border membrane protein

Earlier studies on LLC-PK1 cells have demonstrated two pharmacologically distinct Na+/H+ exchangers in renal epithelia. In addition, the cDNA clone for the human Na+/H+ antiporter which is growth factor activatable has been isolated and expressed (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280). We report here the synthesis of an amiloride analogue that can be photoactivated and labeled with 125I. This analogue covalently cross-links a 66-kDa protein of bovine renal brush border membranes. A rabbit polyclonal antibody that was directed against a 20-amino acid peptide of the cytoplasmic domain of its human Na+/H+ antiporter also gives a positive Western against 66-kDa protein of bovine brush border membranes. Thus, the photoactive probe may be helpful in the isolation and purification of the brush border Na+/H+ exchanger.     

Purification of the cardiac Na+-Ca2+ exchange protein

We have used fractionation procedures to enrich solubilized cardiac sarcolemma in the Na+-Ca2+ exchange protein. Sarcolemma is extracted with an alkaline medium to remove peripheral proteins and is then solubilized with decylmaltoside. Next, the exchanger is applied to DEAE-Sepharose and eluted with high salt. The DEAE fraction is applied to WGA-agarose, and a small fraction of protein, enriched in the exchanger, can be eluted by changing the detergent to Triton X-100. This fraction is reconstituted into asolectin proteoliposomes for measurement of Na+-Ca2+ exchange activity and gel electrophoresis. The purified fraction has a Na+-Ca2+ exchange activity of 600 nmol Ca2+/mg of protein per s at 10 microM Ca2+ and a purification factor of about 30 as compared with control reconstituted sarcolemmal vesicles. Ca2+-Ca2+ exchange and Na+-Ca2+ exchange activities were both present in the same final reconstituted vesicles indicating that the same protein is responsible for both transport activities. SDS-PAGE reveals two prominent protein bands at 70 and 120 kDa. After mild chymotrypsin treatment (1 microgram/ml), there is no loss of exchange activity, but the 120 kDa band disappears and the 70 kDa band becomes more dense. This suggests that the 70 kDa band is due to an active proteolytic fragment of the 120 kDa protein. Under non-reducing gel conditions, only a single protein band is seen with an apparent molecular weight of 160 kDa. Antibodies to the purified exchanger preparation are able to immunoprecipitate exchange activity and confirm that the 70 kDa protein derives from the 120 kDa protein. We propose that both the 70 and 120 kDa proteins are associated with the Na+-Ca2+ exchanger.     

Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter

We present the complete sequence of a cDNA encoding the human amiloride-sensitive Na+/H+ antiporter. After functional complementation of a mouse fibroblast mutant by gene transfer, we isolated a 0.8 kb genomic probe from a third-cycle mouse transformant. The probe detects gene amplification in Na+/H+ antiporter "overexpressers" and a single class of mRNA of ca. 5.6 kb in human, mouse, and hamster cells. With this probe we isolated a 4 kb cDNA from a library constructed from a mouse transformant in which the transfected human gene was amplified. This cDNA includes a noncoding leader of 407 bp, a 2682 bp open reading frame, and a 3' noncoding sequence containing a mouse B1 repeated element. The amino acid sequence predicts a protein of Mr = 99,354 with an N-terminal amphipathic domain that contains 10 putative transmembrane-spanning segments and two potential glycosylation sites, followed by a hydrophilic stretch of 395 residues, presumably cytoplasmic. Stable expression of the transfected cDNA in Na+/H+ antiporter-deficient cells restored the key functional features of this transporter: H+i-activated Na+ influx, amiloride sensitivity, and pHi regulation.     

Vasopressin elevation of Na+/H+ exchange is inhibited by genistein in human blood platelets.

The regulation of intracellular Na+ and pHi in human blood platelets is known to be controlled by the function of the Na+/H+ exchanger. The phosphorylation state of the Na+/H+ exchanger which determines the exchanger activity in human blood platelets is regulated by the activities of protein kinases and protein phosphatases. Observations in this study indicate that arginine vasopressin (AVP) that interacts with a V1 receptor, activates the Na+/H+ exchange in human blood platelets through a genistein-inhibited mechanism. The AVP-activated Na+/H+ exchange is probably not regulated by protein kinase C (PKC), since this activation is not inhibited by staurosporine. The multiple ways in which platelet Na+/H+ exchange can be modulated may indicate the critical role played by this exchanger in the homeostasis control of pHi in human blood platelets.     

Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa.

Na+/H+ exchanger catalyzes the countertransport of Na+ and H+ across membranes. We isolated a rice cDNA clone the deduced amino acid sequence of which had homology with a putative Na+/H+ exchanger in Saccharomyces cerevisiae, NHX1. The sequence contains 2330 bp with an open reading frame of 1608 bp. The deduced amino acid sequence is similar to that of NHX1 and NHE isoforms in mammals, and shares high similarity with the sequences within predicted transmembrane segments and an amiloride-binding domain. The expression of the gene was increased by salt stress. These results suggest that the product of the novel gene, OsNHX1, functions as a Na+/H+ exchanger, and plays important roles in salt tolerance of rice.     

Na+/H+ exchange inhibitors: a new class of cardioprotectors

The Na+/H+ exchanger (NHE) extrudes intracellular H+ in exchange for Na+ in an electroneutral process. Of the 6 mammalian exchanger isoforms identified to date, the NHE-1 is believed to be the molecular homologue of the sarcolemma Na+/H+ transporter. The exchanger is activated primarily by a reduction in intracellular pH, although such activation is subject to modulation by a variety of endogenous mediators (catecholamines, thrombin, endothelin) through receptor-mediated mechanisms. A large body of animal studies using both in vitro and in vivo models indicates that the inhibition of the sarcolemma NHE-1 attenuates myocardial injury in ischemia and reperfusion. Cardioprotective effects of NHE-1 inhibition involve a reduced susceptibility to severe ventricular arrhythmia, augmentation of contractile function recovery, and limitation of infarction size during reperfusion. Such protection is likely to arise partly from attenuation of "Ca2+ overload" in ischemic cardiomyocytes, which has been causally linked with all these pathologic phenomena. A marked benefit that has been observed with cariporide (HOE-642) and its structurally related congener HOE-694 in patients with acute myocardial infarction and in cardiac surgery demonstrates that selective NHE-1 inhibitors represent a novel and effective class of cardioprotectors.     

Cloning and expression of the bovine cardiac sodium-calcium exchanger.

Two clones (p17 and p13), each containing the complete coding sequence for the bovine cardiac Na+/Ca2+ exchanger, were obtained from a lambda gt10 cDNA library by screening with cDNA probes from the canine exchanger. The coding sequence of clone p17 was 92 and 98% identical to the canine cDNA at the nucleotide and amino acid levels, respectively. Nine of the 21 amino acid differences between the two exchangers were found within the 32-amino acid signal sequence. The sequenced portions of the 3' untranslated regions of the cow and dog clones were 88% identical. Na+/Ca2+ exchange activity was expressed in Xenopus laevis oocytes injected with cRNA from clone p17, and in COS cells transfected with expression vectors containing p17. Immunoprecipitation of 35S-labeled proteins from transfected cells with an antibody against the N-terminal portion of the bovine exchanger showed the presence of a 120-kDa protein corresponding to the intact cardiac exchanger. The second bovine clone (p13) did not express exchange activity in either of the above expression systems, presumably because it contained a 300-bp insert with multiple stop codons which interrupted the coding sequence. Comparison of the 5' untranslated regions of p13 and p17 revealed a 156-bp segment in p17 that was apparently spliced out of p13. This segment contained a short open reading frame. A chimera encoding the 5' untranslated region of p13 and the coding sequence of p17 exhibited only a modest (74%) increase in expressed exchange activity in transfected cells compared to p17, suggesting that the presence of the upstream open reading frame in p17 did not greatly reduce translation efficiency. The results suggest that alternate splicing mechanisms may be involved in processing mRNA for the bovine cardiac exchanger.     

Covalent modification of the renal Na+/H+ exchanger by N,N'-dicyclohexylcarbodiimide

We studied the effect of the carboxyl group-specific reagent N,N'-dicyclohexylcarbodiimide on the Na+/H+ exchanger present in microvillus membrane vesicles isolated from rabbit renal cortices. Pretreatment of membrane vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of Na+/H+ exchange which was not due to vesicle disruption or collapse of imposed pH gradients. Inhibition by dicyclohexylcarbodiimide followed pseudo-first-order kinetics, resulted primarily from a decrease in binding affinity for substrate, was pH-dependent in a manner consistent with reaction with carboxyl groups, and was greater than inhibition by hydrophilic carbodiimides. Substrates Na+ and Li+ and the competitive inhibitor amiloride protected against inhibition by dicyclohexylcarbodiimide in a pH-dependent fashion. Finally, we demonstrated amiloride-sensitive covalent binding of radiolabeled dicyclohexylcarbodiimide to a 100-kDa protein. In conclusion, a catalytically important carboxyl group is located in a relatively hydrophobic microenvironment at or near the external transport site of the renal Na+/H+ exchanger; and the transporter itself, or a subunit thereof, may be a 100-kDa protein.     

Characterization of a Histidine Rich Cluster of Amino Acids in the Cytoplasmic Domain of the Na+/H+ Exchanger

We examined the function of a highly conserved Histidine rich sequence ofamino acids found in the carboxyl-terminal of the Na⁺/H⁺exchanger (NHE1). A fusion protein containing the sequenceHYGHHH (540–545) and the balance of the carboxyl terminalof the protein did not bind calcium but bound to an immobilizedmetal affinity column and could be used to partially purify theexchanger protein. Mutation of the sequence to either HYGAAA orHYGRRR did not affect activity of the intact protein. Mutationto HHHHHH did not affect proton activation of the Na+/H+exchanger or localization but caused a decreased maximal velocitysuggesting that this conserved sequence is important in maximalactivity of the Na⁺/H⁺ exchanger.     

Properties of the Na+/H+ exchanger protein. Detergent-resistant aggregation and membrane microdistribution.

The Na+/H+ exchanger is a ubiquitous membrane protein of bacteria, plants and mammals. The first isoform discovered (NHE1) is present on the mammalian plasma membrane and transports one H+ out of cells in exchange for one extracellular Na+. With solubilization in standard SDS/PAGE buffer, this protein had a high tendency to aggregate when subjected to elevated temperature. The aggregates were stable and did not dissociate in high concentrations of SDS or 2-mercaptoethanol. We examined the distribution of the Na+/H+ exchanger within membrane subfractions. The Na+/H+ exchanger was found both in caveolin-containing fractions and, in lesser amounts, in higher density membrane fractions where the bulk of proteins were contained. Treatment with cytochalasin D caused only a minor reduction of the amount of Na+/H+ exchanger present in caveolin-enriched fractions suggesting an intact cytoskeleton was not important for NHE1 localization to these microdomains. Treatment of cells with methyl beta-cyclodextrin had a small stimulatory effect on Na+/H+ exchanger activity and reduced the amount of Na+/H+ exchanger in low density membrane fractions. Our study demonstrates that SDS cannot maintain the protein in a monomeric state suggesting that strong hydrophobic interactions are responsible for this temperature dependent aggregation behavior. In addition a large proportion of the Na+/H+ exchanger protein is found to be enriched in low density caveolin-containing fractions.     

Sphingosine differentially inhibits activation of the Na+/H+ exchanger by phorbol esters and growth factors

The role of protein kinase C in activation of the plasma membrane Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. The basic lipid, sphingosine, was used to block enzymatic activity of protein kinase C. Na+/H+ exchange was activated by phorbol 12-myristate 13-acetate (PMA), diacylglycerols, platelet-derived growth factor (PDGF), thrombin, or by osmotically-induced cell shrinkage. Intracellular pH and Na+/H+ exchange activity were measured using the intracellular pH indicator, 2',7'-bis(carboxyethyl)-5(6) carboxyfluorescein. Acting alone, both crude sphingosine and pure, synthetic C18 D-(+)-erythro-sphingosine raised pHi in a dose-dependent manner (from 6.95 +/- 0.02 to 7.19 +/- 0.09 over 10 min for 10 microM sphingosine). This alkalinization was not due to Na+/H+ exchange as it was not altered by t-butylamiloride (50 microM) nor by replacement of the assay medium with a Na(+)-free solution. Sphingosine-induced alkalinization did not require protein kinase C activity, since it was fully intact in protein kinase C-depleted cells. It was also not due to a detergent action of sphingosine on the cell membrane, since both ionic and non-ionic detergents caused cell acidification. Rather, alkalinization induced by sphingosine appeared to be due to cellular uptake of NH3 groups since N-acetylsphingosine showed no alkalinization. After the initial cell alkalinization, cellular uptake of [3H]sphingosine continued slowly for up to 24 h. The ability of PMA or dioctanoylglycerol to activate Na+/H+ exchange fell to 20% of control after 24 h of sphingosine exposure. At all times, C11 and N-acetylsphingosine failed to block PMA-induced activation of the exchanger. Activation of the Na+/H+ exchanger by sucrose, which does not depend on protein kinase C activity, was unaffected by sphingosine. Activation of Na+/H+ exchange by thrombin and PDGF was partially inhibited by 30 and 20%, respectively. These data indicate that both thrombin and PDGF activate Na+/H+ exchange by pathway(s) that are primarily independent of protein kinase C.     

Identification of the Na+/CA2+ exchanger of calf heart sarcolemma with the help of specific antibodies

The Na+/Ca2+ exchanger of calf heart sarcolemma has been identified in solubilized membrane preparations with the help of specific antibodies as a molecule of approximate Mr of 30 KDa. The conclusion supports the previous proposal by Soldati et al. (J. Biol. Chem. 260, 13321-13327, 1985) that the exchanger is a molecule of Mr about 33 KDa. Antibodies (IgG) were raised in rabbits by injecting proteins electroeluted from different regions of preparative SDS gels of solubilized heart sarcolemma. After purification the IgG against the proteins of the 30 KDa region recognized the 33 KDa component but also proteins of Mr about 70 and 140 KDa. Conversely, antibodies against the 140 KDa protein(s) also recognized the 70 and the 33 KDa proteins. However, if the solubilized sarcolemma extract was treated with DTT prior to the transfer to nitrocellulose the 140 KDa protein was not seen. Both the antibodies against the 30 KDa and those against the 140 KDa proteins inhibited the Na+/Ca2+ exchange activity of sarcolemma vesicles. It is proposed that the basic unit of the Na+/Ca2+ exchanger of heart sarcolemma is a monomer of Mr about 33 KDa, the functionally active exchanger being a tetramer in which the four 33 KDa subunits are held together by disulfide bonds. In the monomer-tetramer transition an intermediate dimeric state of Mr 70 KDa is also formed.     

Na+/H+ exchange activity in the plasma membrane of Arabidopsis

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.     

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.     

Effect of temperature on sodium-calcium exchange in sarcolemma from mammalian and amphibian hearts

We have investigated temperature dependence of Ca2+ uptake by the cardiac sarcolemmal Na(+)-Ca2+ exchanger from dog, rabbit and bullfrog. In native rabbit sarcolemmal vesicles, Ca2+ affinity of the Na(+)-Ca2+ exchanger is unchanged from 7 to 37 degrees C; however, the initial velocity of Ca2+ uptake declines much more steeply below 22 degrees C than above 22 degrees C. In native dog sarcolemma, the temperature dependence of Na(+)-Ca2+ exchange velocity is similar to that of native rabbit. However, in frog heart the velocity of Na(+)-Ca2+ exchange declines much more slowly with decreasing temperature at both temperature ranges. Reconstitution of the Na(+)-Ca2+ exchanger into artificial lipid vesicles consisting of either asolectin or phosphatidylserine, phosphatidylcholine, and cholesterol has little effect on temperature dependence of Na(+)-Ca2+ exchange velocity in any of the three species. We conclude that the lesser temperature sensitivity of the cardiac sarcolemmal Na(+)-Ca2+ exchanger of a poikilothermic species is at least partly an intrinsic property of the transport protein.     

Modulation of Na+/H+ exchange and intracellular pH by protein kinase C and protein phosphatase in blood platelets.

Phosphorylation of the Na+/H+ exchanger in human platelets is apparently controlled by the balancing activities of protein kinase C (PKC) and protein phosphatase (PP). To explore cellular expressions of these activities, we have examined the impact of modulation of PKC and PP on Na+/H+ exchange activity, its pHi set point and intracellular pH (pHi). These parameters were followed spectrofluorimetrically in BCECF-loaded platelets. Phorbol 12-myristate 13-acetate (PMA) and dihexanoylglycerol (DHG), which stimulate PKC, and okadaic acid, which inhibits PP 1 and 2A, elevate the measured parameters in concert, while staurosporine, which inhibits protein kinases, had opposite effects. The stimulatory and inhibitory effects are similarly very rapid, being discerned within seconds. It is concluded that: (a) phosphorylation of the Na+/H+ exchanger is the common origin of the diverse effects of PMA, DHG, okadaic acid and staurosporine, (b) Na+/H+ exchange properties are tightly regulated by phosphorylation and dephosphorylation, and (c) the exchanger plays a major role in pHi regulation in platelets.     

Two Histidine Residues in the Juxta-Membrane Cytoplasmic Domain of Na+/H+ Exchanger Isoform 3 (NHE3) Determine the Set Point

Histidine residues in Na+/H+ exchangers are believed to participate in proton binding and influence the Na+/H+ exchanger activity. In the present study, the function of three highly conserved histidines in the juxtamembrane cytoplasmic domain of NHE3 was studied. His-479, His-485, and His-499 were mutated to Leu, Gln or Asp and expressed in an Na+/H+ exchanger null cell line and functional consequences on Na+/H+ exchange kinetics were characterized. None of the histidines were essential for NHE3 activity, with all mutated NHE3 resulting in functional exchangers. However, the mutation in His-475 and His-499 significantly lowered NHE3 transport activity, whereas the mutation in H485 showed no apparent effect. In addition, the pH profiles of the H479 and H499 mutants were shifted to a more acidic region, and lowered its set point, the intracellular pH value above which the Na+/H+ exchanger becomes inactive, by approximately 0.3-0.6 pH units. The changes in set point by the mutations were further shifted to more acidic values by ATP depletion, indicating that the mechanism by which the mutations on the histidine residues altered the NHE3 set point differs from that caused by ATP depletion. We suggest that His-479 and His-499 are part of the H+ sensor, which is involved in determining the sensitivity to the intracellular H+ concentration and Na+/H+ exchange rate.