The SLC9A-C Mammalian Na+/H+ Exchanger Family: Molecules, Mechanisms, and Physiology.

Na+/H+ exchangers play pivotal roles in the control of cell and tissue pH by mediating the electroneutral exchange of Na+ and H+ across cellular membranes. They belong to an ancient family of highly evolutionarily conserved proteins, and they play essential physiological roles in all phyla. In this review, we focus on the mammalian Na+/H+ exchangers (NHEs), the solute carrier (SLC) 9 family. This family of electroneutral transporters constitutes three branches: SLC9A, -B, and -C. Within these, each isoform exhibits distinct tissue expression profiles, regulation, and physiological roles. Some of these transporters are highly studied, with hundreds of original articles, and some are still only rudimentarily understood. In this review, we present and discuss the pioneering original work as well as the current state-of-the-art research on mammalian NHEs. We aim to provide the reader with a comprehensive view of core knowledge and recent insights into each family member, from gene organization over protein structure and regulation to physiological and pathophysiological roles. Particular attention is given to the integrated physiology of NHEs in the main organ systems. We provide several novel analyses and useful overviews, and we pinpoint main remaining enigmas, which we hope will inspire novel research on these highly versatile proteins.

Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD.

The Na+/H+ antiporter, which regulates intracellular pH in virtually all cells, is one of the best examples of a mitogen- and oncogene-activated membrane target whose activity rapidly changes on stimulation. The activating mechanism is unknown. A Na+/H+ antiporter complementary DNA fragment was expressed in Escherichia coli as a beta-galactosidase fusion protein, and a specific antibody to the fusion protein was prepared. Use of this antibody revealed that the Na+/H+ antiporter is a 110-kilodalton glycoprotein that is phosphorylated in growing cells. Mitogenic activation of resting hamster fibroblasts and A431 human epidermoid cells with epidermal growth factor, thrombin, phorbol esters, or serum, stimulated phosphorylation of the Na+/H+ antiporter with a time course similar to that of the rise in intracellular pH.

Nucleotide sequence of the Chinese hamster Na+/H+ exchanger NHE1.

We have cloned and sequenced the cDNA coding for the Chinese hamster Na+/H+ antiporter expressed in the CCL39 fibroblast cell line. This cDNA contains an open reading frame encoding a protein of 822 amino acids which is homologous to the human NHE1 isoform. Sequence comparison studies show that the first cytoplasmic loop of the transporter is completely different between the two species indicating that the conservation of this domain is not required for the function of the antiporter.

Protective effects of HOE642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion.

OBJECTIVE: The aim was to characterise the new compound HOE642 as a selective and cardioprotective Na+/H+ exchange inhibitor in various models. METHODS: The effect of HOE642 was tested in the osmotically activated Na+/H+ exchange of rabbit erythrocytes and in propionate induced swelling of human thrombocytes. Recovery of pH after an NH4Cl prepulse and effects on other ion transport systems by patch clamp technique were investigated in rat cardiomyocytes. NHE subtype specifity of the compound was determined by 22Na+ uptake inhibition in a fibroblast cell line separately expressing subtype isoforms 1-3. Protective effects of HOE642 in cardiac ischaemia and reperfusion by ligation of coronary artery were investigated in isolated working rat hearts and in anaesthetised rats. RESULTS: HOE642 concentration dependently inhibited the amiloride sensitive sodium influx in rabbit erythrocytes, reduced the swelling of human platelets induced by intracellular acidification, and delayed pH recovery in rat cardiomyocytes. In the isolated working rat heart subjected to ischaemia and reperfusion HOE642 dose dependently reduced the incidence and the duration of reperfusion arrhythmias. It also reduced the the release of lactate dehydrogenase and creatine kinase, and preserved the tissue content of glycogen, ATP, and creatine phosphate. In anaesthetised rats undergoing coronary artery ligation intravenous and oral pretreatment with HOE642 caused a dose dependent reduction or a complete prevention of ventricular premature beats, ventricular tachycardia, and ventricular fibrillation. The compound was well tolerated and neutral to circulatory variables. Other cardiovascular agents tested in this model were not, or were only partly, effective at doses showing marked cardiodepressive effects. CONCLUSIONS: HOE642 is a very selective NHE subtype 1 inhibitor showing cardioprotective and antiarrhythmic effects in ischaemic and reperfused hearts. Further development of well tolerated compounds like HOE642 could lead to a new therapeutic approach in clinical indications related to cardiac ischaemia and reperfusion.

Structure, function, and regulation of vertebrate Na+/H+ exchangers.

The Na+/H+ exchangers are vital transmembrane transporters that participate in the regulation of intracellular pH and volume as well as transepithelial ion transport. Several isoforms of these antiporters belonging to the same gene family have been cloned, and they display tissue-specific expression. All these transporters are highly regulated via various stimuli, which can modulate their activity and expression level. Impairments in regulation of the antiporters may contribute to certain pathologic situations. Biochemical techniques and the tools of molecular genetics now provide the means to analyze the different aspects of these transporters at a molecular level.

A point mutation of the Na+/H+ exchanger gene (NHE1) and amplification of the mutated allele confer amiloride resistance upon chronic acidosis.

The diuretic drug amiloride and its 5-amino substitute N5-methyl-N5-propylamiloride (MPA) are potent inhibitors of the growth factor-activatable Na+/H+ exchanger isoform 1 (NHE1). This inhibitor competes with Na+, presumably by interacting with the ion-transport site of the NHE molecule. As an approach to identify this site, we previously reported the use of a specific H(+)-killing selection technique for isolating amiloride-resistant variants of Chinese hamster lung fibroblasts. After long-term selection, two variants, AR40 and AR300, 100- and 1000-fold, respectively, resistant to MPA, were isolated. By comparing NHE1 cDNA sequences of parental and two variant cell lines, we show that the 1000-fold resistance to MPA results from two sequential genetic events. (i) In one AR40 allele a point mutation, Phe-167--> Leu, occurs in the middle of the fourth putative transmembrane segment of NHE1. Producing this mutant protein from human NHE1 cDNA by site-directed mutagenesis increased the Ki for MPA by 30-fold, as seen in AR300 cells. (ii) An approximately 10-fold amplification of the mutated allele, which contributes to the acquired MPA resistance, accounts for the Vmax increase. Mutating a close residue, Phe-165--> Tyr, increased by 40-fold the Ki for amiloride and reduced Na+ transport rate 3- to 4-fold, indicating that we have identified a critical domain of the NHE molecule that controls amiloride binding and Na+ transport. Interestingly, the epithelial amiloride-resistant NHE isoforms that occurred naturally possess some of the amino acid substitutions described here.

Second-site revertants of a low-sodium-affinity mutant of the Na+/H+ exchanger reveal the participation of TM4 into a highly constrained sodium-binding site.

On the basis of intracellular acidifications in the presence of 30 microM cariporide, we selected a fibroblast cell line termed CR5, expressing a mutated Na(+)/H(+) exchanger NHE-1 with a low affinity for cariporide (87 +/- 11.6 microM) and extracellular sodium (248 +/- 63.7 mM). This mutated exchanger displays a Phe162Ser substitution in its fourth transmembrane segment. Using intracellular acidifications in the presence of 3 mM external sodium on the CR5 fibroblasts, we isolated two revertants which exhibited a complete recovery for sodium affinity but were still resistant to cariporide. Sequencing the cDNAs encoding these revertants revealed the presence of two mutations situated at a distant location from Phe162 in the same fourth transmembrane segment (Ile169Ser and Ile170Thr). Interestingly, introducing these two mutations in the wild-type cDNA did not result in a significant increase in affinity for sodium. Furthermore, all the mutants characterized in this study display an unchanged affinity for lithium, another transported cation. These data suggest that the mutation resulting in the low sodium affinity and the two mutations responsible for the reversion of this phenotype affect the binding of sodium itself instead of the conformational changes triggering substrate translocation. Taken together, these results allow us to propose that optimal sodium binding by the Na(+)/H(+) exchangers requires the geometrical integrity of a highly constrained sodium coordination site.

The Na+/H+ exchanger NHE-1 possesses N- and O-linked glycosylation restricted to the first N-terminal extracellular domain.

The ubiquitously-expressed human Na+H+ exchanger (NHE-1) contains three consensus sites (Asn-X-Ser/Thr) for N-linked glycosylation at asparagines 75, 370, and 410. The first extracellular loop is rich in serine and threonine residues which may contain O-linked carbohydrate. In order to determine unambiguously the sites of glycosylation and their role in biosynthesis and cation transport, site-directed mutagenesis at the individual potential N-glycosylation sites (Asn to Asp) was performed and all possible double and triple mutants were constructed. The mutated DNAs were expressed in PS120 hamster fibroblasts lacking endogenous exchanger, and the transfected cells were selected by their ability to survive acute intracellular acidification. All constructs produced functional exchangers that had transport rates and pharmacological profiles that were similar to that of wild-type. Immunoblot analysis of the expressed proteins with and without N-glycosidase F treatment showed that only the first N-glycosylation site (Asn 75) is utilized. In addition, treatment of NHE-1 with neuraminidase and O-glycosidase demonstrated that NHE-1 also contains O-linked oligosaccharide. Two forms of NHE-1 was consistently observed, a mature form with a molecular mass of 110,000 Da which contains N-linked and O-linked oligosaccharide and is expressed at the cell surface, and a lower molecular mass form (85,000 Da) present in the endoplasmic reticulum which only contains N-linked high-mannose oligosaccharide. NHE-3, an apically-expressed epithelial isoform which does not possess the N75 N-linked putative glycosylation site and any extracellular loops enriched in serine and threonine residues, does not exhibit any detectable glycosylation.

Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties.

A fibroblast mutant cell line devoid of Na+/H+ exchange was used to stably express cDNAs encoding the NHE1, NHE2, and NHE3 Na+/H+ antiporters. Pharmacological studies using amiloride and two of its 5-N-substituted derivatives, 5-N-dimethyl amiloride and 5-N-(methyl-propyl)amiloride (MPA), demonstrate that the NHE1 isoform is the ubiquitously expressed amiloride-sensitive Na+/H+ antiporter (Ki of 0.08 microM for MPA), whereas the NHE2 and NHE3 isoforms exhibit a lower affinity for these inhibitors (Ki of 0.5 microM and 10 microM, respectively, for MPA) and are therefore likely to be members of the epithelial Na+/H+ exchanger's family. In addition, we have used this system to test a new Na+/H+ exchanger inhibitor possessing anti-ischemic properties on myocardial cells [(3-methylsulphonyl-4-piperidinobenzoyl) guanidine methanesulphonate]. This compound inhibits competitively NHE1 (Ki of 0.16 microM) with a much greater affinity than NHE2 and NHE3 (Ki of 5 microM and 650 microM, respectively) and therefore appears to be much more discriminative between these two classes of antiporter isoforms than the amiloride-related molecules. These results suggest an explanation for the observed difference of physiological effects between amiloride and HOE694, and identify this new inhibitor as a useful tool for studies of Na+/H+ exchange.

Random mutagenesis reveals a novel site involved in inhibitor interaction within the fourth transmembrane segment of the Na+/H+ exchanger-1.

We constructed and expressed human Na+/H+ exchanger (NHE-1 isoform) cDNAs randomly mutagenized within the sequence encoding the transmembrane region of the exchanger. Using acute intracellular acidifications in the presence of the NHE-1 inhibitor amiloride (300 microM), we selected a clone expressing a NHE-1 protein exhibiting a 3.3-fold increase in K(i) for amiloride (10 microM instead of 3 microM). Sequencing its cDNA revealed one point mutation resulting in a Gly174Ser substitution near the carboxy-terminal end of the putative fourth transmembrane domain of NHE-1. The introduction of this mutation into the wild-type NHE-1 cDNA and its expression reproduced the features of the mutant. Site-directed Gly174Ala and Gly174Asp substitutions resulted, respectively, in no change and in an approximately 4-fold decrease in the amiloride affinity. An additional mutation (Leu163Phe) in transmembrane segment four has previously been shown to result in a decreased sensitivity to amiloride and its derivatives. The Leu163Phe/Gly174Ser double mutant possesses a strongly reduced affinity for various inhibitors (17 microM for amiloride, 2 microM for MPA, and 20 microM for HOE694) and also a decreased affinity (28 mM instead of 14 mM) for sodium. Although distant in the transmembrane segment, Leu163 and Gly174 residues are both not hydrogen-bonded, being one helix turn from proline residues, and are therefore located in highly flexible regions of the protein. This flexibility and the availability of free carbonyls may play an important role in the interaction with the inhibitors and transported cations.

Na/H exchange activities in NHE1-transfected OK-cells: cell polarity and regulation.

The human fibroblast, "amiloride-sensitive" Na/H exchanger (NHE1) was transfected into opossum kidney cells (OK cells) (OK/NHE1 cells). Northern blot analysis confirmed that the NHE1 message is expressed in OK/NHE1 cells. In immunoblot analysis, an anti-human NHE1 antibody labelled a membrane protein only present in OK/NHE1 cells. In contrast to the parental cell line containing only an apically located, "amiloride-resistant" Na/H exchange activity, OK/NHE1 cells contain apically and basolaterally located Na/H exchange activities, the apical activity being "amiloride resistant" and the basolateral being "amiloride sensitive". Parathyroid hormone (PTH) inhibited apical transport activity (OK and OK/NHE1 cells) but had no effect on basolateral transport activity (OK/NHE1 cells). Pharmacological activation of protein kinase A (forskolin) decreased both apical and basolateral Na/H exchange activity. Incubation with phorbol ester (exogenous activation of protein kinase C) reduced apical Na/H exchange activity (OK and OK/NHE1 cells) but had only a moderate, inhibitory effect on basolateral Na/H exchange activity (OK/NHE1 cells). These results indicate that transfection of OK cells with human fibroblast NHE1 cDNA encoding an "amiloride-sensitive" form of the Na/H exchanger results in expression of basolaterally located "NHE1-related" transport activity. Regulatory control of intracellular Na/H exchange activities (apically versus basolaterally located) and intercellular Na/H exchange activities (NHE1-related) differs. This may relate to cell-specific properties as well as to exchanger-specific properties.

Structure of the 5'-flanking regulatory region and gene for the human growth factor-activatable Na/H exchanger NHE-1.

The amiloride-sensitive, growth factor-activatable Na/H exchanger (NHE-1) is a ubiquitous mammalian protein that is involved in the regulation of intracellular pH and cell volume. We have determined the intron/exon boundaries and the transcription initiation sites and have characterized a portion of the 5'-flanking regulatory region of the human NHE-1 gene. The Na/H exchanger gene spans approximately 70 kilobases. The coding region is divided into 12 exons and 11 introns, one of which is 41.5 kilobases in length. The first exon contains the entire 5'-noncoding region, which is 786 bases long, and 352 bases of the coding sequence. Primer extension identified two discrete start sites for RNA polymerase. 1377 bases of the 5'-regulatory region were sequenced. The promoter/enhancer region is characterized by a TATA box, four GC boxes, two CAAT boxes, five CACCC boxes, three Ap-1 sites, a cyclic AMP response element, and four partial glucocorticoid response elements. Promoter activities of a 313- and a 1441-base pair fragment containing the TATA box were demonstrated by their ability to direct chloramphenicol acetyltransferase expression when transiently expressed in fibroblasts.

CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts.

To study the potential influence of cystic fibrosis conductance regulator (CFTR) on intracellular pH regulation during apoptosis induction, we used PS120 Chinese hamster lung fibroblasts devoid of the Na(+)/H(+) exchanger (NHE1 isoform) transfected with constructs, allowing the expression of CFTR and/or NHE1. Kinetics of lovastatin-induced apoptosis were measured by orcein staining, double staining with Hoechst-33258, propidium iodide, DNA fragmentation, and annexin V labeling. In PS120 control cells, the percentage of apoptotic cells after 40 h of lovastatin treatment was 23 +/- 3%, whereas in PS120 CFTR-transfected cells, this percentage was 40 +/- 4%. In PS120 NHE1 cells, the transfection with CFTR did not modify the percentage of apoptotic cells after 40 h (control: 19 +/- 3%, n = 8; CFTR: 17 +/- 1%, n = 8), indicating that blocking intracellular acidification by overexpressing the Na(+)/H(+) exchanger inhibited the enhancement of apoptosis induced by CFTR. In all cell lines, the initial pH values were identical (pH = 7.46 +/- 0.04, n = 9), and treatment with lovastatin led to intracellular acidification. However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 +/- 0.02, n = 10) than in PS120 cells (pH = 7.15 +/- 0.03, n = 10). To further investigate the origin of this increased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitable Cl(-)/HCO exchanger was studied. 8-Bromoadenosine 3',5'-cyclic monophosphate incubation resulted in Cl(-)/HCO exchanger activation in PS120 CFTR-transfected cells but had no effect on PS120 cells. Together, our results suggest that CFTR can enhance apoptosis in Chinese hamster lung fibroblasts, probably due to the modulation of the Cl(-)/HCO exchanger, resulting in a more efficient intracellular acidification.

Exploration of the pore structure of a peptide-gated Na+ channel.

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.

Na+/H+ and CI-/HCO3-antiporters of bovine pigmented ciliary epithelial cells.

Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor. Published data have indicated that both paired Na+/H+ and Cl-/HCO3- antiporters and the Na+-K+-2Cl- symporter are involved in net uptake. The molecular identities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated 22Na+ uptake was inhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCECF-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antiporter. Removing external Cl- also triggered an alkalinization, which was Na+-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Application of hypotonicity followed by return to isotonicity triggered a regulatory volume increase, which was pharmacologically similar to the uptake mechanisms described for intact rabbit ciliary epithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from the human ciliary body detected expression of the AE2 Cl-/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine PE cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.