Identification of a mitochondrial Na+/H+ exchanger

The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.     

Human esophageal epithelial cells possess an Na+/H+ exchanger for H+ extrusion

Chronic progressive renal function loss is a main cause of long-term graft loss after initially successful renal transplantation. Transplanted kidneys share some risk factors for renal function loss, such as hypertension or proteinuria, with diseased native kidneys. Recently, it has been shown that renal function loss is influenced by the angiotensin-converting enzyme (ACE) (insertion/deletion [I/D]) genotype in renal disease in diseased native kidneys. This study examines whether donor or recipient ACE (I/D) genotype is a risk factor for graft loss after renal transplantation. To avoid bias by acute events, graft survival was studied, with patients dying with a functioning graft censored, starting at 12 mo after transplantation in a cohort of 367 patients transplanted between 1987 and 1994 with at least 2 yr of follow-up. Mean follow-up was 58 mo. ACE (I/D) genotype was determined by PCR on stored donor and recipient lymphocytes. Neither donor nor recipient ACE (I/D) genotype was associated with graft survival. However, Cox proportional hazards analysis identified recipient, but not donor, ACE (I/D) genotype D-allele to be independently associated with a shorter time to graft loss in subgroups of patients at high risk for graft loss defined by a creatinine clearance <50 ml/min (n = 108, P = 0.017) or proteinuria > or =0.5 g/24 h at 12 mo (n = 97, P = 0.0051) after transplantation. In conclusion, recipient ACE (I/D) genotype was associated with time to graft loss in a specific high-risk subgroup of the study population. This suggests that the effect of ACE (I/D) genotype on graft survival only becomes apparent when other risk factors are simultaneously present.     

Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1.     

Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway

The NHE3 isoform of the Na+/H+ exchanger localizes to both the plasmalemmal and endosomal compartments in polarized epithelial and transfected Chinese hamster ovary (AP-1) cells. It is unclear how the distribution of NHE3 between these compartments is regulated. In this study, we examined the potential involvement of phosphatidylinositol 3'-kinase (PI3-K) in regulating the activity and distribution of NHE3, as this lipid kinase has been implicated in modulating vesicular traffic in the endosomal recycling pathway. Wortmannin and LY294002, both potent inhibitors of PI3-K, markedly inhibited NHE3-mediated H+ extrusion across the plasma membrane in a concentration- and time-dependent manner. The subcellular distribution of the antiporters was monitored by transfecting epitope-tagged NHE3 into AP-1 cells. In parallel with the inhibition of transport, PI3-K antagonists induced a pronounced loss of NHE3 from the cell surface and its accumulation in an intracellular compartment, as assessed by immunofluorescence microscopy and enzyme-linked immunosorbent assays. Further analysis using cells transfected with antiporters bearing an external epitope tag revealed that the redistribution reflected primarily a decrease in the rate of recycling of intracellular NHE3 to the cell surface. The wortmannin-induced inhibition and redistribution of NHE3 were prevented when cells were incubated at 4 degreesC, consistent with the known temperature dependence of the endocytic process. These observations demonstrate that NHE3 activity is controlled by dynamic endocytic and recycling events that are modulated by PI3-K.     

Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils

The ubiquitous isoform of the Na+/H+ exchanger (NHE1) is essential for the regulation of cellular volume. The underlying molecular mechanism, which is poorly understood, was studied in human polymorphonuclear leukocytes (PMN). Suspension of PMN in hypertonic media induced rapid cellular shrinkage and activation of NHE1, which is measurable as a cytosolic alkalinization. Concomitantly, hypertonic stress also induced extensive tyrosine phosphorylation of several proteins. Pretreatment of PMN with genistein, a tyrosine kinase inhibitor, prevented not only the tyrosine phosphorylation in response to a hypertonic shock but also the activation of NHE1. The signal elicited by hyperosmolarity that induces activation of tyrosine kinases and NHE1 was investigated. Methods were devised to change medium osmolarity without altering cell volume and vice versa. Increasing medium and intracellular osmolarity in normovolemic cells failed to activate tyrosine kinases or NHE1. However, shrinkage of cells under iso-osmotic conditions stimulated both tyrosine phosphorylation and NHE1 activity. These findings imply that cells detect alterations in cell size but not changes in osmolarity or ionic strength. The identity of the proteins that were tyrosine-phosphorylated in response to cell shrinkage was also investigated. Unexpectedly, the mitogen-activated protein kinases SAPK, p38, erk1, and erk2 were not detectably phosphorylated or activated. In contrast, the tyrosine kinases p59(fgr) and p56/59(hck) were phosphorylated and activated upon hypertonic challenge. We propose that cells respond to alterations in cell size, but not to changes in osmolarity, with increased tyrosine phosphorylation, which in turn leads to the activation of NHE1. The resulting changes in ion content and cytosolic pH contribute to the restoration of cell volume in shrunken cells.     

Demonstration of an Na+/H+ exchanger in mouse keratinocytes measured by the novel pH-sensitive fluorochrome SNARF-calcein

In many cell types cytoplasmic alkalization is an early marker for cell activation. An amiloride-sensitive Na+/H+ exchanger is an important regulator of this process. However, in keratinocytes the existence of a Na+/H+ exchanger nor a proliferation-associated increase in intracellular pH (pHi) has been demonstrated. The aim of this study was to investigate whether or not keratinocytes, derived from the BALB/MK cell line, contain a Na+/H+ exchanger and whether cytoplasmic alkalization is proliferation-associated in these cells. This mouse keratinocyte cell line can easily be switched between a proliferative and a quiescent state under defined culture conditions. The novel pH-sensitive dye seminaphthorhodafluor (SNARF)-calcein proved to be very suitable for flow cytometric pHi measurements in BALB/MK cells. Initial measurements of the pHi using a cocktail of the established fluorochromes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and SNARF-1 failed because of the differential uptake and binding kinetics of these pH-sensitive dyes. Using SNARF-calcein we were able to show proliferation to be associated with increased pHi. However, culture conditions were critical for these measurements. Our data indicate that the Na+/H+ exchanger is involved in this process, since acid load and pHi-recovery experiments showed the alkalization to be amiloride-sensitive.     

Comparative molecular analysis of Na+/H+ exchangers: a unified model for Na+/H+ antiport?

There is increasing evidence to suggest that free radical generation is central to a variety of pathological processes, including drug toxicity. Studies demonstrating the ability of gentamicin to facilitate the generation of radical species suggest that this process plays an important role in aminoglycoside-induced ototoxicity. Because transition metals, particularly iron, play an important role in the production of free radicals and the generation of reactive oxygen species, we sought to determine whether gentamicin-induced ototoxicity is exacerbated by increases in serum iron levels. To this end, we assessed the effects of supplemental iron administration (2 mg/kg/day and 6 mg/kg/day) on changes in auditory function induced by co-administration of gentamicin (100 mg/kg/day for 30 days). Experiments were carried out on pigmented guinea pigs initially weighing 250-300 g. Changes in cochlear function were characterized as shifts in compound action potential (CAP) thresholds, estimated every third day throughout the treatment period by use of chronic indwelling electrodes implanted at the round window, vertex, and contralateral mastoid. Results showed that animals receiving iron in combination with gentamicin demonstrated a more rapid and profound elevation in CAP thresholds compared with animals receiving gentamicin alone. This effect occurred in a dose-dependent manner. Animals receiving supplemental iron alone maintained normal CAP thresholds throughout the treatment period. There was no statistically significant difference in serum gentamicin levels between groups receiving gentamicin alone or gentamicin plus iron. These results provide further evidence of the recently reported intrinsic role of iron in aminoglycoside ototoxicity, and highlight a potential risk of aminoglycoside administration in patients with elevated serum iron.     

Effect of long-term hyperosmolality on the Na+/H+ exchanger isoform NHE-3 in LLC-PK1 cells

Communication is an important cornerstone to the physician-patient relationship when considering advance directives. Discussing advance directives with patients is a process best initiated in routine, well-adult care that can be made more daunting when the patient is critically ill; yet, when patients are afflicted with cancer, communication on advance directives can be optimized when the primary care physician and oncologist together work with the patient. The need to counsel patients on advance directives regardless of the venue (whether inpatient or outpatient) highlights that an ongoing alliance between the oncologist and the primary care physician can help facilitate consent to, and allow periodic review of, advance directives by cancer patients. This process ensures that the patient's preferences are respected at life's end.     

Role of conserved glycosylation site unique to murine class I MHC in recognition by Ly-49 NK cell receptor

The recognition of class I MHC molecules on target cells by the Ly-49 family of receptors regulates NK cytotoxicity. Previous studies have suggested that carbohydrates are involved in the recognition of class I MHC by Ly-49, although their precise role remains unclear. Here, we examined the role of asparagine-linked carbohydrates of the murine class I MHC in the binding to Ly-49A and Ly-49C. We have generated H-2Dd mutants that lack the highly conserved glycosylation sites at amino acid residues 86 in the alpha1 domain and 176 in the alpha2 domain, respectively. These mutant Dd cDNAs were transfected into leukemic cell lines, and the binding of the transfected cells to COS cells expressing Ly-49A or Ly-49C, as well as their susceptibility to lysis by Ly-49A+ NK cells, was examined. Only the mutation of the alpha2 domain glycosylation site significantly reduced the binding of Dd to Ly-49A and Ly-49C. Cells expressing Dd with the mutation at this site were partially resistant to killing by Ly-49A+ NK cells. These results suggest that, while carbohydrates linked to residue 176 seem to function as a part of the ligand structure for the Ly-49 family of NK receptors, there are additional structural features involved in this recognition. This glycosylation site is highly conserved among murine class I MHC but is not found among those of other species, suggesting that its role is unique to the murine immune system. It further suggests that murine class I MHC and Ly-49 gene families may have evolved in concert.     

Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.     

Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger

Na+/H+ exchanger isoform 3 (NHE3), an epithelial brush border isoform of the Na+/H+ exchanger gene family, plays an important role in reabsorption of Na+ in the small intestine, the colon, and the kidney. In several cell types, phorbol 12-myristate 13-acetate (PMA) acutely inhibits NHE3 activity by changes in Vmax, but the mechanism of this inhibition is unknown. We investigated the role of subcellular redistribution of NHE3 in the PMA-induced inhibition of endogenous brush border NHE3 in a model human colon adenocarcinoma cell line, Caco-2. Subcellular localization of NHE3 was examined by confocal morphometric analysis complemented with cell surface biotinylation and compared with NHE3 activity evaluated by fluorometric measurement of intracellular pH. PMA inhibited NHE3 activity by 28% (p < 0.01), which was associated with a decrease of the ratio of the brush border/subapical cytoplasmic compartment of NHE3 from approximately 4.3 to approximately 2.4. This translocation resulted in 10-15% of the total cell NHE3 being shifted from the brush border pool to the cytoplasmic pool. These effects were mediated by protein kinase C, since they were blocked by the protein kinase C inhibitor H7. We conclude that inhibition of NHE3 by protein kinase C in Caco-2 cells involves redistribution of the exchanger from brush border into a subapical cytoplasmic compartment, and that this mechanism contributes approximately 50% to the overall protein kinase C-induced inhibition of the exchanger.     

Functional analysis of amino acid residues essential for activity in the Na+/H+ exchanger of fission yeast

We identified amino acid residues important for activity of sod2, the Na+/H+ antiporter of Schizosaccharomyces pombe. We mutated all eight His residues of sod2 into Arg. Only His367-->Arg affected function and resulted in complete inability of sod2 to allow growth of S. pombe in LiCl-containing medium. Mutant S. pombe (H367R) could not expel sodium in acidic (pH 4.0) medium and were defective in their ability to alkalinize external medium. When His367 was replaced by Asp, sodium export of S. pombe was suppressed at acidic pH while the sodium-dependent proton influx at pH 6.1 was increased compared to wild type. We also mutated three residues conserved in putative membrane regions of various eukaryotic and prokaryotic Na+/H+ exchangers. S. pombe containing Asp241-->Asn and Asp266, 267-->Asn mutations had greatly impaired growth in LiCl-containing medium. In addition, sodium-dependent proton influx at external pH 6. 1 was impaired. Sodium export from S. pombe cells at external pH 4.0 was also almost completely abolished by the D266,267N mutation; however, the D241N mutant protein retained almost normal Na+ export. The results demonstrate that His367, Asp241, and Asp266,267 are important in the function of the eukaryotic Na+/H+ exchanger sod2.     

Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells

Clinical follow-up data of 276 colorectal adenocarcinoma patients treated in Kuopio University Hospital between 1976 and 1986 and followed up for a mean of 14 years were analysed. The clinical findings were correlated with tumour-infiltrating lymphocytes (TILs) and with histological and quantitative factors including nuclear parameters and volume-corrected mitotic index. In univariate survival analysis, TNM classification, Dukes' stage, histological grade, and TILs were significant predictors of survival. TNM classification, Dukes' stage, and TILs also predicted recurrence-free survival. In multivariate analysis, TILs were an independent prognostic factor of survival in all cases, as well as in patients with T1-4N0-3M0 and T1-4N1-3M1. TILs also independently predicted recurrence-free survival. TILs can provide important prognostic information in colorectal cancer to be used in evaluating for adjuvant therapy in different tumour stages.     

Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.     

Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. Insights into mechanisms of sodium tolerance

Sodium tolerance in yeast is disrupted by mutations in calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, which is required for modulation of Na+ uptake and efflux mechanisms. Five Na+-tolerant mutants were isolated by selecting for suppressors of calcineurin mutations, and mapped to the PMA1 gene, encoding the plasma membrane H+-ATPase. One mutant, pma1-alpha4, which has the single amino acid change Glu367 --> Lys at a highly conserved site within the catalytic domain of the ATPase, was analyzed in detail to determine the mechanism of Na+ tolerance. After exposure to Na+ in the culture medium, 22Na influx in the pma1 mutant was reduced 2-fold relative to control, consistent with a similar decrease in ATPase activity. Efflux of 22Na from intact cells was relatively unchanged in the pma1 mutant. However, selective permeabilization of the plasma membrane revealed that mutant cells retained up to 80% of intracellular Na+ within a slowly exchanging pool. We show that NHX1, a novel gene homologous to the mammalian NHE family of Na+/H+ exchangers, is required for Na+ sequestration in yeast and contributes to the Na+-tolerant phenotype of pma1-alpha4.     

Rapid computerized analysis of Na+/H+ exchange flux

The regulation of intracellular pH (pHi) is mediated by membrane transporters whose activity is directly controlled by pHi. Therefore, transport rates must be compared at identical pHi values in functional studies of these transporters. This is conventionally performed using scatter plots showing initial rates of proton flux versus intracellular pH. We present justification for determining proton flux over a wide range of pHi, by digitally smoothing a pHi trace and then directly taking the first derivative versus time of smoothed data. Compared to conventional least-squares analysis of initial rates, the derivative method generates much more information per experiment. Compared to other methods which fit pHi traces to a defined equation prior to rate calculation, the new method does not require that the pHi trace be well fit by any given mathematical function. The derivative technique is illustrated in an analysis of Na+/H+ exchange in Caco-2 cells. Intracellular pH is measured fluorometrically in cells loaded with BCECF (2',7'-bis[2-carboxyethyl]-5-(and-6)carboxyfluorescein). To validate the analysis of Na+/H+ exchange over an extended time range, we demonstrate that cellular acidification with NH4Cl does not change steady state Na+ content. We find that proton flux rates analyzed by the derivative method are equivalent to initial rates measured by least-squares analysis.     

Novel localization of a Na+/H+ exchanger in a late endosomal compartment of yeast. Implications for vacuole biogenesis

Na+/H+ exchangers catalyze the electrically silent countertransport of Na+ and H+, controlling the transmembrane movement of salt, water, and acid-base equivalents, and are therefore critical for Na+ tolerance, cell volume control, and pH regulation. In contrast to numerous well studied plasma membrane isoforms (NHE1-4), much less is known about intracellular Na+/H+ exchangers, and thus far no vertebrate isoform has been shown to have an exclusively endosomal distribution. In this context, we show that the yeast NHE homologue, Nhx1 (Nass, R., Cunningham, K. W., and Rao, R. (1997) J. Biol. Chem. 272, 26145-26152), localizes uniquely to prevacuolar compartments, equivalent to late endosomes of animal cells. In living yeast, we show that these compartments closely abut the vacuolar membrane in a striking bipolar distribution, suggesting that vacuole biogenesis occurs at distinct sites. Nhx1 is the founding member of a newly emergent cluster of exchanger homologues, from yeasts, worms, and humans that may share a common intracellular localization. By compartmentalizing Na+, intracellular exchangers play an important role in halotolerance; furthermore, we hypothesize that salt and water movement into vesicles may regulate vesicle volume and pH and thus contribute to vacuole biogenesis.