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A human Na+/H+ antiporter sharing evolutionary origins with bacterial NhaA may be a candidate gene for essential hypertension
Abstract
Phylogenetic analysis of the cation/proton antiporter superfamily has uncovered a previously unknown clade of genes in metazoan genomes, including two previously uncharacterized human isoforms, NHA1 and NHA2, found in tandem on human chromosome 4. The NHA (sodium hydrogen antiporter) family members share significant sequence similarity with Escherichia coli NhaA, including a conserved double aspartate motif in predicted transmembrane 5. We show that HsNHA2 (Homo sapiens NHA2) resides on the plasma membrane and, in polarized MDCK cells, localizes to the apical domain. Analysis of mouse tissues indicates that NHA2 is ubiquitous. When expressed in the yeast Saccharomyces cerevisiae lacking endogenous cation/proton antiporters and pumps, HsNHA2 can confer tolerance to Li⁺ and Na⁺ ions but not to K⁺. HsNHA2 transformants accumulated less Li⁺ than the salt-sensitive host; however, mutagenic replacement of the conserved aspartates abolished all observed phenotypes. Functional complementation by HsNHA2 was insensitive to amiloride, a characteristic inhibitor of plasma membrane sodium hydrogen exchanger isoforms, but was inhibited by phloretin. These are hallmarks of sodium–lithium countertransport activity, a highly heritable trait correlating with hypertension. Our findings raise the possibility that NHA genes may contribute to sodium–lithium countertransport activity and salt homeostasis in humans.
• sodium–lithium countertransport
• yeast expression
• red blood cell
• pancreas
... The SLC9B2 gene is located in tandem with the SLC9B1 gene on human Chromosome 4 and is comprised of 13 exons. In contrast to SLC9B1, the SLC9B2 gene exhibits near ubiquitous expression, including expression in testis and sperm, in both mouse and human [6,10,80,81]. In mature mouse sperm, NHA2 has been reported to reside in the membrane of the principal piece. ...
... NHA2 is also reported to localize to mitochondria in both mouse osteoclasts and rat kidney cells [80,84]. Additionally, NHA2 was reported to localize to the plasma membrane of mouse kidney cells, mouse osteoclasts, and MDCK cells (canine kidney cells) [81,83,85,86]. ...
... Physiologically, NHA2 has been shown to be important for osteoclast function, insulin secretion, and nephron function where it has been implicated in playing a significant role in maintaining salt balance to support normotension [81][82][83]87]. NHA2 was reported to localize to the distal convoluted tubules of mouse kidney cells, and subcellularly found to localize to endosomes in mpkDCT4 cells, a mouse distal convoluted tubule epithelial cell line, although NHA2 was found to not influence endosomal pH homeostasis [87]. ...
Na+/H+ exchangers (NHEs) are known to be important regulators of pH in multiple intracellular compartments of eukaryotic cells. Sperm function is especially dependent on changes in pH and thus it has been postulated that NHEs play important roles in regulating the intracellular pH of these cells. For example, in order to achieve fertilization, mature sperm must maintain a basal pH in the male reproductive tract and then alkalize in response to specific signals in the female reproductive tract during the capacitation process. Eight NHE isoforms are expressed in mammalian testis/sperm: NHE1, NHE3, NHE5, NHE8, NHA1, NHA2, NHE10, and NHE11. These NHE isoforms are expressed at varying times during spermatogenesis and localize to different subcellular structures in developing and mature sperm where they contribute to multiple aspects of sperm physiology and male fertility including proper sperm development/morphogenesis, motility, capacitation, and the acrosome reaction. Previous work has provided evidence for NHE3, NHE8, NHA1, NHA2, and NHE10 being critical for male fertility in mice and NHE10 has recently been shown to be essential for male fertility in humans. In this article we review what is known about each NHE isoform expressed in mammalian sperm and discuss the physiological significance of each NHE isoform with respect to male fertility.
... NHA2, also known as SLC9B2, is the most recently identified mammalian NHE. [7][8][9] Based on chromosomal localization, expression pattern and transport characteristics, NHA2 was proposed to be the long-sought sodium/lithium countertransporter. [8] Sodium/lithium countertransporter activity is a highly heritable trait associated with diabetes mellitus and arterial hypertension in humans. ...
... [7][8][9] Based on chromosomal localization, expression pattern and transport characteristics, NHA2 was proposed to be the long-sought sodium/lithium countertransporter. [8] Sodium/lithium countertransporter activity is a highly heritable trait associated with diabetes mellitus and arterial hypertension in humans. [10,11] At the outset of our studies, the physiological role and molecular function of NHA2 was unknown and the pharmacology of NHA2 unexplored. ...
... [9] Protein and mRNA expression studies revealed that NHA2 is ubiquitously expressed in all organs but confined to specialized cell types. [7][8][9] Of all cell types examined, osteoclasts (bone degrading cells) exhibited by far the highest NHA2. Intriguingly, NHA2 was found to be one of the most prominently upregulated proteins during receptor-activator of the NF-κB ligand (RANKL)-induced osteoclast differentiation, and knock-down of NHA2 in an osteoclast cell line greatly Fig. 1. pH of endocytic and secretory organelles. ...
NHA2, also known as SLC9B2, is an orphan intracellular Na+/H+ exchanger (NHE) that has been associated with arterial hypertension and diabetes mellitus in humans. The objective of this NCCR TransCure project was to define the physiological and molecular function of NHA2, to develop a high resolution kinetic transport assay for NHA2 and to identify specific and potent compounds targeting NHA2. In this review, we summarize the results of this highly interdisciplinary and interfaculty effort, led by the groups of Proffs. Jean-Louis Reymond, Christoph von Ballmoos and Daniel Fuster.
... Moreover, the NHA2 gene is located in a human chromosomal region (4q24) which has been associated with hypertension in numerous linkage studies. 6 So far, particular physiological roles of NHA2 have been studied in a range of organisms and tissues. Single RNAi knockdowns of NHA2 or its homolog NHA1 in Drosophila melanogaster reduced survival, and their combination was lethal, suggesting their essentiality for life. ...
... 11,30 Pharmacologically, NHA2 is resistant to amiloride, a typical inhibitor of members of the NHE subfamily, but it is a phloretin-sensitive transport system. 6,30 So far, several functional and mutagenesis studies of HsNHA2 have been done upon its expression in the model yeast S. cerevisiae, in a strain that lacked the three Na + transporters-the Na + /H + antiporter Nha1 and Na + -ATPases Ena from the plasma membrane and the endosomal Na + /H + antiporter Nhx1. 4,6,23,31 This strain is highly sensitive to all alkali-metal-cations salts and the expression of HsNHA2 improved the growth of these cells in the presence of sodium or lithium in a pH dependent manner. ...
... 6,30 So far, several functional and mutagenesis studies of HsNHA2 have been done upon its expression in the model yeast S. cerevisiae, in a strain that lacked the three Na + transporters-the Na + /H + antiporter Nha1 and Na + -ATPases Ena from the plasma membrane and the endosomal Na + /H + antiporter Nhx1. 4,6,23,31 This strain is highly sensitive to all alkali-metal-cations salts and the expression of HsNHA2 improved the growth of these cells in the presence of sodium or lithium in a pH dependent manner. 4,6 Examination of the growth of yeast cells expressing mutated variants of HsNHA2 in the presence of salts was also used to identify some residues that are important for the activity of HsNHA2. ...
The human Na⁺/H⁺ antiporter NHA2 (SLC9B2) transports Na⁺ or Li⁺ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N‐terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt‐sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2‐specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50–70 residues of the N‐terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N‐terminal part of the protein appears to allosterically auto‐inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity.
... The orthologues of the SLC9B subfamily exist in most of metazoan genomes, including nematodes, flies, puffer fish, mice and humans. NHA1 is specifically expressed in testis, while NHA2 has been found to be fairly ubiquitously expressed (Ye et al., 2006;Xiang et al., 2007;Battaglino et al., 2008;Fuster et al., 2008). There is scant information on the physiological function of NHA1, and its role in human disease has not been explored. ...
... Compared to NHA1, NHA2 has received much more attention since its initial discovery (Xiang et al., 2007;Battaglino et al., 2008;Fuster et al., 2008). Based on genomic localization, tissue distribution pattern and inhibitor characteristics (tolerance to amiloride but sensitivity to phloretin), NHA2 was proposed as a possible candidate for the elusive sodium-lithium countertransporter (SLC) (Xiang et al., 2007). ...
... Compared to NHA1, NHA2 has received much more attention since its initial discovery (Xiang et al., 2007;Battaglino et al., 2008;Fuster et al., 2008). Based on genomic localization, tissue distribution pattern and inhibitor characteristics (tolerance to amiloride but sensitivity to phloretin), NHA2 was proposed as a possible candidate for the elusive sodium-lithium countertransporter (SLC) (Xiang et al., 2007). An increased activity of this countertransporter in erythrocytes and fibroblasts is a heritable trait that has been linked to the pathogenesis of arterial hypertension and diabetes mellitus (Canessa et al., 1980;Mangili et al., 1988;Laurenzi et al., 1997;Strazzullo et al., 1998;Vaccaro et al., 2005). ...
The SLC9 gene family encodes Na + /H + exchangers (NHEs), a group of membrane transport proteins critically involved in the regulation of cytoplasmic and organellar pH, cell volume, as well as systemic acid-base and volume homeostasis. NHEs of the SLC9A subfamily (NHE 1-9) are well-known for their roles in human physiology and disease. Much less is known about the two members of the SLC9B subfamily, NHA1 and NHA2, which share higher similarity to prokaryotic NHEs than the SLC9A paralogs. NHA2 (also known as SLC9B2) is ubiquitously expressed and has recently been shown to participate in renal blood pressure and electrolyte regulation, insulin secretion and systemic glucose homeostasis. In addition, NHA2 has been proposed to contribute to the pathogenesis of polycystic kidney disease, the most common inherited kidney disease in humans. NHA1 (also known as SLC9B1) is mainly expressed in testis and is important for sperm motility and thus male fertility, but has not been associated with human disease thus far. In this review, we present a summary of the structure, function and regulation of expression of the SLC9B subfamily members, focusing primarily on the better-studied SLC9B paralog, NHA2. Furthermore, we will review the potential of the SLC9B subfamily as drug targets.
... NHE1 to NHE9 (solute carrier family 9 members A1-9) belong to the CPA1 clade, and are well known for their roles in human physiology, such as Na + reabsorption in the kidney and in acid-base homeostasis 2,5-7 . By contrast, the mammalian CPA2 clade members NHA1 and NHA2 (SLC9 family 9 members B1 and B2) were more recently identified 4,8,9 and share a closer evolutionary relationship to bacterial Na + /H + antiporters 9 (Fig. 1a). Based on tissue expression, genome location and phloretin sensitivity, human NHA2 was proposed to be the candidate gene for the Na + (Li + ) countertransport activity associated with the development of essential hypertension and diabetes in humans 9-12 . ...
... The truncated NHA2 ΔN sequence shares 97% sequence identity to human NHA2 ΔN (Supplementary Fig. 2). It has been shown previously that only functional human NHA2 can rescue growth in the salt-sensitive S. cerevisiae AB11c strain 9,32 , which lacks the main Na + -and K + -extrusion systems. We confirmed that bison NHA2 ΔN was expressed in the AB11c strain and complemented growth under Fig. 1 | the cryo-eM structure of NHA2 reveals a domain-swapped homodimer. ...
... 1a,b and 3a,b). By contrast, poor S. cerevisiae AB11c growth was apparent under Na + -or Li + -salt stress conditions for either non-induced cells or the double ion-binding aspartate mutant of bison NHA2 ΔN Asp277Cys-Asp278Cys, previously shown to abolish human NHA2 activity 9 (Extended Data Fig. 1a,b and Supplementary Figs. 1a,b and 3b). ...
The Na+/H+ exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na+/Li+ exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na+/H+ exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity. NHA2 exchanges sodium ions for protons across cell membranes, and its activity is linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Drew et al. report the cryo-EM structure of NHA2 in detergent and nanodiscs.
... The CPA1 subgroup includes the electroneutral NHE family of Na + (K + )/H + exchangers represented by the well-characterized plasma membrane transporter NHE1 (Donowitz et al. 2013;Fuster & Alexander, 2014;Kondapalli et al. 2014). More recently, a new clade of CPA2 genes was discovered in animals, sharing ancestry with electrogenic bacterial NapA and NhaA antiporters (Xiang et al. 2007;Fuster et al. 2008). Of the two human CPA2 gene products, expression of NHA2 has ubiquitous tissue distribution, whereas the closely related NHA1 isoform is restricted to testis (Xiang et al. 2007;Fuster et al. 2008;Chen et al. 2016). ...
... More recently, a new clade of CPA2 genes was discovered in animals, sharing ancestry with electrogenic bacterial NapA and NhaA antiporters (Xiang et al. 2007;Fuster et al. 2008). Of the two human CPA2 gene products, expression of NHA2 has ubiquitous tissue distribution, whereas the closely related NHA1 isoform is restricted to testis (Xiang et al. 2007;Fuster et al. 2008;Chen et al. 2016). Despite the multiplicity of genes encoding Na + /H + exchangers in mammals, emerging evidence indicates that CPA1 and CPA2 subtypes have distinct, non-redundant transport roles based on differences in chemiosmotic coupling, inhibitor sensitivity and pH activation (Kondapalli et al. 2012). ...
... Human NHA2 has been implicated as a marker of essential hypertension, with potential roles in kidney diseases relating to salt and water balance (Xiang et al. 2007;Schushan et al. 2010;Kondapalli et al. 2017). NHA2 expression in kidney is confined to the distal nephron and renal collecting duct, regions that play critical roles in salt, pH and volume homeostasis (Fuster et al. 2008). ...
Key points
Significant and selective up‐regulation of the Na⁺/H⁺ exchanger NHA2 (SLC9B2) was observed in cysts of patients with autosomal dominant polycystic kidney disease.
Using the MDCK cell model of cystogenesis, it was found that NHA2 increases cyst size. Silencing or pharmacological inhibition of NHA2 inhibits cyst formation in vitro.
Polycystin‐1 represses NHA2 expression via Ca²⁺/NFAT signalling whereas the dominant negative membrane‐anchored C‐terminal fragment (PC1‐MAT) increased NHA2 levels.
Drugs (caffeine, theophylline) and hormones (vasopressin, aldosterone) known to exacerbate cysts elicit NHA2 expression.
Taken together, the findings reveal NHA2 as a potential new player in salt and water homeostasis in the kidney and in the pathogenesis of polycystic kidney disease.
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 encoding polycystin‐1 (PC1) and polycystin‐2 (PC2), respectively. The molecular pathways linking polycystins to cyst development in ADPKD are still unclear. Intracystic fluid secretion via ion transporters and channels plays a crucial role in cyst expansion in ADPKD. Unexpectedly, we observed significant and selective up‐regulation of NHA2, a member of the SLC9B family of Na⁺/H⁺ exchangers, that correlated with cyst size and disease severity in ADPKD patients. Using three‐dimensional cultures of MDCK cells to model cystogenesis in vitro, we showed that ectopic expression of NHA2 is causal to increased cyst size. Induction of PC1 in MDCK cells inhibited NHA2 expression with concordant inhibition of Ca²⁺ influx through store‐dependent and ‐independent pathways, whereas reciprocal activation of Ca²⁺ influx by the dominant negative membrane‐anchored C‐terminal tail fragment of PC1 elevated NHA2. We showed that NHA2 is a target of Ca²⁺/NFAT signalling and is transcriptionally induced by methylxanthine drugs such as caffeine and theophylline, which are contraindicated in ADPKD patients. Finally, we observed robust induction of NHA2 by vasopressin, which is physiologically consistent with increased levels of circulating vasopressin and up‐regulation of vasopressin V2 receptors in ADPKD. Our findings have mechanistic implications on the emerging use of vasopressin V2 receptor antagonists such as tolvaptan as safe and effective therapy for polycystic kidney disease and reveal a potential new regulator of transepithelial salt and water transport in the kidney.
... The CPA1 subgroup includes the electroneutral NHE family of Na + (K + )/H + exchangers represented by the well-characterized plasma membrane transporter NHE1 (11)(12)(13). More recently, a new clade of CPA2 genes was discovered in animals, sharing ancestry with electrogenic bacterial NapA and NhaA antiporters (14,15). Of the two human CPA2 genes, NHA2 has ubiquitous tissue distribution, whereas the closely related NHA1 isoform is restricted to testis (14)(15)(16). ...
... More recently, a new clade of CPA2 genes was discovered in animals, sharing ancestry with electrogenic bacterial NapA and NhaA antiporters (14,15). Of the two human CPA2 genes, NHA2 has ubiquitous tissue distribution, whereas the closely related NHA1 isoform is restricted to testis (14)(15)(16). Despite the multiplicity of genes encoding Na + /H + exchangers in mammals, emerging evidence indicates that CPA1 and CPA2 subtypes have distinct, non-redundant transport roles based on differences in chemiosmotic coupling, inhibitor sensitivity and pH activation (17). ...
... Human NHA2 has been implicated as a marker of essential hypertension, with potential roles in kidney diseases relating to salt and water balance (14,18,19). NHA2 expression in kidney is confined to the distal nephron and renal collecting duct, regions that play critical roles in salt, pH and volume homeostasis (15). ...
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The molecular pathways linking polycystins to cyst development in ADPKD are still unclear. Intracystic fluid secretion via ion transporters and channels plays a crucial role in cyst expansion in ADPKD. Unexpectedly, we observed significant and selective up-regulation of NHA2, a member of the SLC9B family of Na+/H+ exchangers that correlated with cyst size and disease severity in ADPKD patients. Using three-dimensional cultures of MDCK cells to model cystogenesis in vitro, we show that ectopic expression of NHA2 is causal to increased cyst size. Induction of PC1 in MDCK cells inhibited NHA2 expression with concordant inhibition of Ca2+ influx through store-dependent and independent pathways, whereas reciprocal activation of Ca2+ influx by a dominant negative, membrane-anchored C-terminal tail fragment of PC1 elevated NHA2. We show that NHA2 is a target of Ca2+/NFAT signaling and is transcriptionally induced by methylxanthine drugs such as caffeine and theophylline, which are contraindicated in ADPKD patients. Finally, we observe robust induction of NHA2 by vasopressin, which is physiologically consistent with increased levels of circulating vasopressin and up-regulation of vasopressin V2 receptors in ADPKD. Our findings have mechanistic implications on the emerging use of vasopressin V2 receptor antagonists such as tolvaptan as safe and effective therapy for PKD and reveal a potential new regulator of transepithelial salt and water transport in the kidney.
... NHA2 is a recently identified sodium proton (Na + /H + ) antiporter belonging to a group of cation proton antiporters, CPA2, whose eukaryotic members have been poorly characterized [1]. Gene linkage analysis implicated NHA2 in essential hypertension, and heterologous expression in yeast provided the first evidence for a role in sodium homeostasis [29]. In kidney-derived MDCK cells, NHA2 was found to mediate phloretin-sensitive sodium lithium counter-transport (SLC) activity, an established marker for hypertension [14]. ...
... Anesthetized C57BL/6 mice were fixed by perfusion with 4 % periodate-lysineparaformaldehyde (PLP), and tissue sections were prepared and probed with the antibodies at the following dilutions: (a) anti-NHA2 antibody (1:100 dilution) raised in rabbit against a 15-aa peptide of HsNHA2 [29], (b) anti-AQP2 raised in chicken (gift from Dr. James Wade, Univ. of Maryland) at a dilution of 1:300, (c) anti-NCC antibody (Feldan, Qubec, Canada) was used at 1:100 dilution, (d) anti-α-NaKATPase antibody was used as previously described [24], (e) antibody Alexa Fluor 594-phalloidin (Molecular probes, Invitrogen) was used at 1:200 dilution to label actin, and (f) V-ATPase B1/2 (Santa Cruz Biotechnology, Inc.) at 1:50 dilution. ...
... We sought to establish the distribution of NHA2 to specific regions of the nephron (Fig. 1a) using a previously characterized polyclonal antibody [29]. Double-labeling of mouse kidney sections with the F-actin marker phalloidin, abundant in the brush border cells of the proximal tubule [22], showed a clear separation of NHA2 and phalloidin signals (Fig. 1b), consistent with the absence of NHA2 mRNA from proximal tubules in RT-PCR studies of microdissected rat tubules [11]. ...
Increased renal reabsorption of sodium is a significant risk factor in hypertension. An established clinical marker for essential hypertension is elevated sodium lithium countertransport (SLC) activity. NHA2 is a newly identified Na⁺(Li⁺)/H⁺ antiporter with potential genetic links to hypertension, which has been shown to mediate SLC activity and H⁺-coupled Na⁺(Li⁺) efflux in kidney-derived MDCK cells. To evaluate a putative role in sodium homeostasis, we determined the effect of dietary salt on NHA2. In murine kidney sections, NHA2 localized apically to distal convoluted (both DCT1 and 2) and connecting tubules, partially overlapping in distribution with V-ATPase, AQP2, and NCC1 transporters. Mice fed a diet high in sodium chloride showed elevated transcripts and expression of NHA2 protein. We propose a model in which NHA2 plays a dual role in salt reabsorption or secretion, depending on the coupling ion (sodium or protons). The identified novel regulation of Na⁺/H⁺ antiporter in the kidney suggests new roles in salt homeostasis and disease.
... A mitochondrial inner membrane Na + /H + exchanger (NHA2 or NHEDC2; designated as SLC9B2), was originally identified in yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), with the human homologue showing ubiquitous expression, particularly in tissues with high mitochondrial content, including the kidney distal convoluted tubule [6,7]. SLC9B2 has been identified as a contributor to sodium-lithium counter-transport activity (SLC), and may serve as a candidate gene for essential hypertension in human populations, particularly in urban black populations [7,8]. ...
... A mitochondrial inner membrane Na + /H + exchanger (NHA2 or NHEDC2; designated as SLC9B2), was originally identified in yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), with the human homologue showing ubiquitous expression, particularly in tissues with high mitochondrial content, including the kidney distal convoluted tubule [6,7]. SLC9B2 has been identified as a contributor to sodium-lithium counter-transport activity (SLC), and may serve as a candidate gene for essential hypertension in human populations, particularly in urban black populations [7,8]. Moreover, genetic linkage studies of SLC activity in baboons have reported association with chromosome 5, the homologue to human chromosome 4 [9]. ...
... Human SLC9B2 has also been shown to reverse the Na + /H + exchangernull phenotype when expressed in Na + /H + exchanger deficient yeast [10], which supports a major role for SLC9B2 as a sodium/hydrogen exchanger. A second gene (NHA1 or NHEDC1; designated as SLC9B1) has been located in tandem with SLC9B2 on human chromosome 4 which is specifically expressed in testis in mammals [7,11]. This is in contrast to Drosophila, for which two SLC9B-like genes (designated as NHA1 and NHA2) are widely expressed in epithelial tissues and play crucial roles in organismal ion homeostasis and as Na + /H + exchangers [12]. ...
SLC9B genes and proteins are members of the sodium/lithium hydrogen antiporter family which function as solute exchangers within cellular membranes of mammalian tissues. SLC9B2 and SLC9B1 amino acid sequences and structures and SLC9B-like gene locations were examined using bioinformatic data from several vertebrate genome projects. Vertebrate SLC9B2 sequences shared 56-98% identity as compared with ~50% identities with mammalian SLC9B1 sequences. Sequence alignments, key amino acid residues and conserved predicted transmembrane structures were also studied. Mammalian SLC9B2 and SLC9B1 genes usually contained 11 or 12 coding exons with differential tissue expression patterns: SLC9B2, broad tissue distribution; and SLC9B1, being testis specific. Transcription factor binding sites and CpG islands within the human SLC9B2 and SLC9B1 gene promoters were identified. Phylogenetic analyses suggested that SLC9B1 originated in an ancestral marsupial genome from a SLC9B2 gene duplication event.
... As the main transporters for eliminating the cytosolic protons, six isoforms of NHEs: NHE1 [31][32][33], NHE5 [34], NHE8 [35], sNHE [36,37], NHA1 [38], and NHA2 [39] had been reported to be expressed in sperm. However, which NHE is the determinant modulator for the activation of KSper and CatSper in murine sperm remained ambiguous. ...
... Therefore, we speculated that NHE1 activity did not account for the activation of pH-sensitive ion channels. sNHE and NHAs expressed in the principal piece of sperm were also candidates for mediating the activation of pH-dependent ion channels [36,39]. Given that NHAs were insensitive to amiloride [41], we supposed that KSper and CatSper were functionally coupled to sNHE. ...
Sperm-specific K+ ion channel (KSper) and Ca2+ ion channel (CatSper), whose elimination causes male infertility in mice, determine the membrane potential and Ca2+ influx, respectively. KSper and CatSper can be activated by cytosolic alkalization, which occurs during sperm going through the alkaline environment of the female reproductive tract. However, which intracellular pH (pHi) regulator functionally couples to the activation of KSper/CatSper remains obscure. Although Na+/H+ exchangers (NHEs) have been implicated to mediate pHi in sperm, there is a lack of direct evidence confirming the functional coupling between NHEs and KSper/CatSper. Here, 5-(N, N-dimethyl)-amiloride (DMA), an NHEs inhibitor that firstly proved not to affect KSper/CatSper directly, was chosen to examine NHEs function on KSper/CatSper in mouse sperm. The results of patch clamping recordings showed that, when extracellular pH was at the physiological level of 7.4, DMA application caused KSper inhibition and the depolarization of membrane potential when pipette solutions were not pH-buffered. In contrast, these effects were minimized when pipette solutions were pH-buffered, indicating that they solely resulted from pHi acidification caused by NHEs inhibition. Similarly, DMA treatment reduced CatSper current and intracellular Ca2+, effects also dependent on the buffer capacity of pH in pipette solutions. The impairment of sperm motility was also observed after DMA incubation. These results manifested that NHEs activity is coupled to the activation of KSper/CatSper under physiological conditions.
... Human NHA2 Antiport Activity Is Likely Electroneutral. In recent years, human Na + /H + exchangers NHA1 and NHA2 have been described, with the latter proposed to have a role in hypertension and insulin secretion (21)(22)(23). Compared with the human cation exchangers NHE1-9, NHA1 and NHA2 have higher sequence similarity to the bacterial homologues, especially NapA (21% protein sequence identity). ...
... Consistent with this idea, NHA2 is found to localize to late endosomes, where it further colocalizes with the vacuolar ATPase (V-ATPase) (25), which establishes a low pH on the inside of endosomes. Because of the two conserved aspartates and the similarity to the bacterial homologues, NHA2 was thought to be electrogenic, but experiments in whole cells have not supported this assumption (2,(22)(23)(24)26). We therefore aimed to clarify the energetics of human NHA2 in an isolated system using purified components. ...
Significance
Cells express transporters that strictly exchange protons for sodium ions to regulate many fundamental processes, such as intracellular pH and cell volume. The bacterial Na ⁺ /H ⁺ exchangers typically use the energy stored in membrane potentials, whereas the mammalian transporters do not. The energetic difference stems from the ability of the bacterial proteins to transport multiple protons in exchange for one sodium ion. The mechanism for how this is achieved, however, has remained elusive. Here, using purified components in synthetic membranes, we have compared the energetics and kinetics of the bacterial exchanger NapA to the human exchanger NHA2, which has been linked to insulin secretion. Remarkably, the judicial placement of a transmembrane embedded lysine is the key for harnessing a membrane potential.
... NHA2, also known as SLC9B2 or NHEDC2, is a recently cloned, poorly characterized NHE isoform [3]. Previous studies suggested that NHA2 is the correlate of the long sought sodium/lithium countertransporter that was linked to the pathogenesis of diabetes mellitus and essential hypertension in humans [2,4,5]. ...
... While NHA2 is ubiquitously expressed on tissue level, it is mainly confined to specialized cells within individual organs, e.g. osteoclasts in the bone or distal tubules of the kidney [4,6,7]. We recently reported that NHA2 is present in human and rodent β-cells of the endocrine pancreas [8]. ...
We previously demonstrated that the sodium/hydrogen exchanger NHA2, also known as NHEDC2 or SLC9B2, is critical for insulin secretion by β–cells. To gain more insights into the role of NHA2 on systemic glucose homeostasis, we studied the impact of loss of NHA2 during the physiological aging process and in the setting of diet-induced obesity. While glucose tolerance was normal at 2 months of age, NHA2 KO mice displayed a significant glucose intolerance at 5 and 12 months of age, respectively. An obesogenic high fat diet further exacerbated the glucose intolerance of NHA2 KO mice. Insulin levels remained similar in NHA2 KO and WT mice during aging and high fat diet, but fasting insulin/glucose ratios were significantly lower in NHA2 KO mice. Peripheral insulin sensitivity, measured by insulin tolerance tests and hyperinsulinemic euglycemic clamps, was unaffected by loss of NHA2 during aging and high fat diet. High fat diet diminished insulin secretion capacity in both WT and NHA2 KO islets and reduced expression of NHA2 in WT islets. In contrast, aging was characterized by a gradual increase of NHA2 expression in islets, paralleled by an increasing difference in insulin secretion between WT and NHA2 KO islets. In summary, our results demonstrate that loss of the sodium/hydrogen exchanger NHA2 exacerbates obesity- and aging-induced glucose intolerance in mice. Furthermore, our data reveal a close link between NHA2 expression and insulin secretion capacity in islets.
... More recently discovered are the sodium/hydrogen antiporters (NHAs), a subbranch of the CPA2 family, which is much less well understood (3,4). Originally studied in bacteria, yeast, and plants (5,6), this subbranch in humans includes two NHA genes in tandem: NHA1 (SLC9B1), which is testis-specific, and NHA2 (SLC9B2), which is ubiquitous (7,8). ...
... In the pancreas, NHA2 is necessary for insulin secretion but localizes not to insulin-containing vesicles, but rather to transferrin-positive endocytic vesicles (4,10). NHA2 also has been linked to hypertension (8). Thus, NHAs are multifunctional proteins expressed in a wide range of subcellular domains; however, a mechanistic understanding of the roles of NHAs in animals is lacking in comparison with their exhaustively studied NHE relatives, and a simple animal model is clearly needed. ...
Significance
Cation/proton antiporters (CPAs) are essential to life. The sodium/proton exchanger (NHE) branch of the CPA family has been studied exhaustively and is an important drug target; however, much less is known about the recently discovered NHA branch, represented by two genes in both humans and flies. Here we show that sodium/hydrogen antiporter (NHA) function is essential to life, and that both NHAs protect against salt stress. Their transport mechanisms are radically different, however, suggesting that function cannot be inferred from structural similarity. Although NHA2 was found to be a Na ⁺ /H ⁺ exchanger as expected, NHA1 was seen to act as an electroneutral H ⁺ /Cl ⁻ cotransporter. This is an important finding for future studies of these transporters.
... We newly identified NhaA as a dry-resistance factor by screening a Tn-insertion mutant library of E. coli DH5α. The NhaA transporter is reported to be important for regulating and maintaining the pH in and around bacteria [36], and is extremely well conserved from bacteria to eukaryotes [37]. Although the role of NhaA in drying is not fully understood, it is likely responsible for controlling the leakage of ions from bacterial cells whilst maintaining bacterial structures [38,39]. ...
Healthcare-associated infections have become a major health issue worldwide. One route of transmission of pathogenic bacteria is through contact with "high-touch" dry surfaces, such as handrails. Regular cleaning of surfaces with disinfectant chemicals is insufficient against pathogenic bacteria and alternative control methods are therefore required. We previously showed that warming to human-skin temperature affected the survival of pathogenic bacteria on dry surfaces, but humidity was not considered in that study. Here, we investigated environmental factors that affect the number of live bacteria on dry surfaces in hospitals by principal component analysis of previously-collected data (n = 576, for CFU counts), and experimentally verified the effect of warming to human-skin temperature on the survival of pathogenic bacteria on dry surfaces under humidity control. The results revealed that PCA divided hospital dry surfaces into four groups (Group 1~4) and hospital dry surfaces at low temperature and low humidity (Group 3) had much higher bacterial counts as compared to the others (Group 1 and 4) (p
... Because SLC9A1 is absent from sperm, SLC9B1, SLC9B2, and SLC9C1/2 are the remaining candidates for Na + /H + exchange. However, human SLC9B2 and sea urchin SLC9C1 are insensitive to amiloride 31,75,76 , and amiloride sensitivity of SLC9B1 is not known. ...
The reaction of CO2 with H2O to form bicarbonate (HCO3⁻) and H⁺ controls sperm motility and fertilization via HCO3⁻-stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3⁻ in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pHi is set by amiloride-sensitive Na⁺/H⁺ exchange. The sperm-specific putative Na⁺/H⁺ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO3⁻ transport are not detected, and may be present at copy numbers < 10 molecules/sperm cell. Instead, HCO3⁻ is produced by diffusion of CO2 into cells and readjustment of the CO2/HCO3⁻/H⁺ equilibrium. The proton channel Hv1 may serve as a unidirectional valve that blunts the acidification ensuing from HCO3⁻ synthesis. This work provides a new framework for the study of male infertility.
... Within the CHX clade, plant and fungal transporters may be found, such as the CHX genes from A. thaliana and the KHA genes from S. cerevisiae [20]. A group of related genes in animals has been identified and named as NHA, on the basis of their similarity to fungal NHA and bacterial nhaA genes [40]. The prototype of the CPA2 family, and the CPA superfamily in general, is the bacterial EcNhaA, an electrogenic transporter with a H + :Na + stoichiometry of 2:1 [41]. ...
Na+/H+ exchangers are essential for Na+ and pH homeostasis in all organisms. Human Na+/H+ exchangers are of high medical interest, and insights into their structure and function are aided by the investigation of prokaryotic homologues. Most prokaryotic Na+/H+ exchangers belong to either the Cation/Proton Antiporter (CPA) superfamily, the Ion Transport (IT) superfamily, or the Na+-translocating Mrp transporter superfamily. Several structures have been solved so far for CPA and Mrp members, but none for the IT members. NhaA from E. coli has served as the prototype of Na+/H+ exchangers due to the high amount of structural and functional data available. Recent structures from other CPA exchangers, together with diverse functional information, have allowed elucidation of some common working principles shared by Na+/H+ exchangers from different families, such as the type of residues involved in the substrate binding and even a simple mechanism sufficient to explain the pH regulation in the CPA and IT superfamilies. Here, we review several aspects of prokaryotic Na+/H+ exchanger structure and function, discussing the similarities and differences between different transporters, with a focus on the CPA and IT exchangers. We also discuss the proposed transport mechanisms for Na+/H+ exchangers that explain their highly pH-regulated activity profile.
... Overexpression of the Gal4 transcription factor [60] S. cerevisiae AB11c ena1-4∆nhx1∆nha1∆ Deletion of endogenous cation/proton antiporters and pumps [61] S. cerevisiae MSY6210 MAT α leu2-3,112 ura3-52 his3200 trp1-901lys2-801suc2-9 smf1::HIS3,smf2::KANR Deletion of Mg 2+ transporters [62] S. cerevisiae MSY6211 MAT a leu2-3,112 ura3-52 his3200 trp1-901 ade2-101 suc2-9 smf3::LEU2 Deletion of Mg 2+ transporters [62] P. pastoris GS115 his4 ...
For more than 20 years, yeast has been a widely used system for the expression of human membrane transporters. Among them, more than 400 are members of the largest transporter family, the SLC superfamily. SLCs play critical roles in maintaining cellular homeostasis by transporting nutrients, ions, and waste products. Based on their involvement in drug absorption and in several human diseases, they are considered emerging therapeutic targets. Despite their critical role in human health, a large part of SLCs’ is ‘orphans’ for substrate specificity or function. Moreover, very few data are available concerning their 3D structure. On the basis of the human health benefits of filling these knowledge gaps, an understanding of protein expression in systems that allow functional production of these proteins is essential. Among the 500 known yeast species, S. cerevisiae and P. pastoris represent those most employed for this purpose. This review aims to provide a comprehensive state-of-the-art on the attempts of human SLC expression performed by exploiting yeast. The collected data will hopefully be useful for guiding new attempts in SLCs expression with the aim to reveal new fundamental data that could lead to potential effects on human health.
... They contribute to cellular and organellar pH and volume regulation and transepithelial Na + transport (Hayashi et al., 2002). In regard to slc9B genes, which are rarely studied than NHEs, were reported to display differential tissue expression patterns: slc9B2, the higher levels and wider distribution patterns of expression have been associated with the Na + /H + exchanger, sodium-lithium counter transport, cation/proton antiporter and salt homeostasis roles across tissue organelle lipid bilayers (Xiang et al., 2007); and slc9B1, only expressed in testis but no specific role for the unique testis-specific slc9B1 expression has been identified in mammalian (Ye et al., 2006). ...
The solute carrier family 9 (slc9) genes, especially slc9a isoform coding proteins contribute to electroneutral countertransport of H⁺ for Na⁺ across the plasmalemmal and organellar membranes, intracellular pH and cellular volume regulation as well as the electrolyte, acid-base, and fluid volume homeostasis at the systemic level. These functional properties determine a potential basis for organisms to challenge stressful conditions. However, these well-done researches have been reported more in mammals. Thus, in this study, a total of eleven slc9 genes were identified from the latest version genome of L. waleckii, a cyprinid fish that could tolerate extremely alkaline environments (pH 9.6). The evolutionary footprint of slc9 genes was uncovered via the analysis of copy numbers, gene structure, motif composition, chromosome location and phylogenetic relationship. More importantly, there were two SNPs located on 5’ UTR and three non-synonymous mutations in the coding region of the slc9a3.2 gene by comparing freshwater with alkaline water populations attached to resequencing technology. Slc9a3.2 gene was a statistically significant low expression in gill tissue with extremely alkaline pressure. Generally, slc9 gene family in L. waleckii was highly conserved. Several important SNPs with high Fst values were identified where non-synonymous mutations occurred between freshwater and alkaline water populations, and they may play an important role in specific functional differentiation. Slc9 genes had clear tissue expression preferences and were involved in abiotic stress response, indicating their roles in physiological function and strong self-regulating capacity. Our insight into the genetic variations that take place in the individual genes under extreme conditions could provide a feasible example for studying specific molecular mechanisms based on genomic data with increasing environmental stress.
... The functions of the SLC9B and SLC9C isoforms are less well characterized. SLC9B isoforms are broadly expressed and have been implicated in hypertension (Xiang et al., 2007;Chintapalli et al., 2015;Kondapalli et al., 2017), insulin secretion (Deisl et al., 2013;Deisl et al., 2016) and sperm motility and fertility (Chen et al., 2016). By contrast, expression of the SLC9C isoforms is largely confined to testis where they are also essential for sperm function (Wang et al., 2003;Quill et al., 2006;Wang et al., 2007;Windler et al., 2018;Cavarocchi et al., 2021). ...
Endomembrane alkali cation (Na+, K+)/proton (H+) exchangers (eNHEs) are increasingly associated with neurological disorders. These eNHEs play integral roles in regulating the luminal pH, processing, and trafficking of cargo along the secretory (Golgi and post-Golgi vesicles) and endocytic (early, recycling, and late endosomes) pathways, essential regulatory processes vital for neuronal development and plasticity. Given the complex morphology and compartmentalization of multipolar neurons, the contribution of eNHEs in maintaining optimal pH homeostasis and cargo trafficking is especially significant during periods of structural and functional development and remodeling. While the importance of eNHEs has been demonstrated in a variety of non-neuronal cell types, their involvement in neuronal function is less well understood. In this review, we will discuss their emerging roles in excitatory synaptic function, particularly as it pertains to cellular learning and remodeling. We will also explore their connections to neurodevelopmental conditions, including intellectual disability, autism, and attention deficit hyperactivity disorders.
... We suggest that other ubiquitous transporters play housekeeping roles. spiSLC9A1, for example, could play a role in homeostatic pH regulation on the basolateral cell membrane, and spiSLC9B1-2 might participate in organismal ion homeostasis on the mitochondrial inner membrane, as reported in vertebrates [19,[90][91][92][93]. Interestingly, spiSLC9A1 was the only transporter affected by seawater acidification after 1 year of exposure (Fig. 8). ...
Background
Reef-building corals regularly experience changes in intra- and extracellular H ⁺ concentrations ([H ⁺ ]) due to physiological and environmental processes. Stringent control of [H ⁺ ] is required to maintain the homeostatic acid-base balance in coral cells and is achieved through the regulation of intracellular pH (pH i ). This task is especially challenging for reef-building corals that share an endosymbiotic relationship with photosynthetic dinoflagellates (family Symbiodinaceae), which significantly affect the pH i of coral cells. Despite their importance, the pH regulatory proteins involved in the homeostatic acid-base balance have been scarcely investigated in corals. Here, we report in the coral Stylophora pistillata a full characterization of the genomic structure, domain topology and phylogeny of three major H ⁺ transporter families that are known to play a role in the intracellular pH regulation of animal cells; we investigated their tissue-specific expression patterns and assessed the effect of seawater acidification on their expression levels.
Results
We identified members of the Na ⁺ /H ⁺ exchanger (SLC9), vacuolar-type electrogenic H ⁺ -ATP hydrolase (V-ATPase) and voltage-gated proton channel (H v CN) families in the genome and transcriptome of S. pistillata . In addition, we identified a novel member of the H v CN gene family in the cnidarian subclass Hexacorallia that has not been previously described in any species. We also identified key residues that contribute to H ⁺ transporter substrate specificity, protein function and regulation. Last, we demonstrated that some of these proteins have different tissue expression patterns, and most are unaffected by exposure to seawater acidification.
Conclusions
In this study, we provide the first characterization of H ⁺ transporters that might contribute to the homeostatic acid-base balance in coral cells. This work will enrich the knowledge of the basic aspects of coral biology and has important implications for our understanding of how corals regulate their intracellular environment.
... For the other ubiquitous transporters, we suggest they play housekeeping roles. spiSLC9A1, for example, could play a role in homeostatic pH regulation, on the basolateral cell membrane, and spiSLC9B1-2 might participate to organismal ion homeostasis, on the mitochondrial inner membrane, as reported in vertebrates [19,[86][87][88][89] Moreover, we assessed the tissue-speci city of the H + transporters gene families in the coral S. pistillata and we observed that their expression was not restricted to only one speci c tissue (oral or aboral) as reported for some members of the HCO 3 − gene family [20]. However, we observed higher or lower expression pro les in the oral or aboral tissues. ...
Background: Reef-building corals regularly experience changes in intra and extracellular H⁺ concentration ([H⁺]) due to physiological and environmental processes. Stringent control of [H⁺] is required for the maintenance of homeostatic acid-base balance in coral cells and is achieved through the regulation of intracellular pH (pHi). This task is especially challenging for reef-building corals that share an endosymbiotic relationship with photosynthetic dinoflagellates (family Symbiodinaceae), which exert a significant effect on the pHi of coral cells. Despite their importance, the pH regulatory proteins involved in the homeostatic acid-base balance have been scarcely investigated in corals. Here, we reported the full characterisation in terms of genomic structure, domain topology and phylogeny of three majors H⁺ transporter families implicated in pHi regulation; we investigated their tissue-specific expression and we assessed the effect of seawater acidification on their level of expression.
Results: We identified members of the Na⁺/H+ exchanger (SLC9), vacuolar-type electrogenic H⁺-ATP hydrolases (V-ATPase) and voltage-gated proton channels (HvCN) families in the genome and transcriptome of S. pistillata. In addition, we identified a novel member of the HvCN gene family in the cnidarian subclass Hexacorallia, which has never been described in any species to date. We also reported key residues that participate to the H⁺ transporters substrate specificity, protein function and regulation. Lastly, we demonstrated that some of these have different tissue expression patterns and are mostly unaffected by exposure to seawater acidification.
Conclusions: In this study, we provide the first characterization of the H+ transporters genes that contribute to homeostatic acid-base balance in coral cells. This work will enrich knowledge about basic aspects of coral biology, bearing important implications for our understanding of how corals regulate their intracellular environment.
... NHA1 and NHA2 (also known as SLC9B1 and SLC9B2) constitute the SLC9B family, but their function remains poorly defined. Based on chromosomal localization, transport characteristics and inhibitor sensitivity, NHA2 was proposed to be the long sought mediator of Na + /Li + counter-transport (SLC) 3 . SLC activity is a highly heritable trait, associated with abnormalities in Na + homeostasis, diabetes mellitus and arterial hypertension in humans [4][5][6][7][8][9] . ...
NHA2 is a sodium/proton exchanger associated with arterial hypertension in humans, but
the role of NHA2 in kidney function and blood pressure homeostasis is currently
unknown. Here we show that NHA2 localizes almost exclusively to distal convoluted
tubules in the kidney. NHA2 knock-out mice displayed reduced blood pressure,
normocalcemic hypocalciuria and an attenuated response to the thiazide diuretic
hydrochlorothiazide. Phosphorylation of the thiazide-sensitive sodium/chloride
cotransporter NCC and its upstream activating kinase Ste20/SPS1-related proline/alanine
rich kinase (SPAK), as well as the abundance of with no lysine kinase 4 (WNK4), were
significantly reduced in the kidneys of NHA2 knock-out mice. In vitro experiments
recapitulated these findings and revealed increased WNK4 ubiquitylation and enhanced
proteasomal WNK4 degradation upon loss of NHA2. The effect of NHA2 on WNK4
stability was dependent from the ubiquitylation pathway protein Kelch-like 3 (KLHL3) .
More specifically, loss of NHA2 selectively attenuated KLHL3 phosphorylation and
blunted protein kinase A- and protein kinase C-mediated decrease of WNK4 degradation.
Phenotype analysis of NHA2/NCC double knock-out mice supported the notion that
NHA2 affects blood pressure homeostasis by a kidney-specific and NCC-dependent
mechanism. Thus, our data show that NHA2 as a critical component of the WNK4-NCC
pathway and is a novel regulator of blood pressure homeostasis in the kidney.
... SLC9B2 is under-expressed in the TCR cell line and encodes for NHA2 protein that belongs to the sodium hydrogen antiporter family. 51 Aside from being involved in cell pH and sodium regulation, it has also been reported that a downregulation of NHA2 protein could affect endocytosis 52 and therefore could limit T-DM1 internalization. Further studies are required to determine whether these SLCs are involved in T-DM1 resistance mechanisms. ...
The development of targeted therapies has drastically improved the outcome of patients with different types of cancer. T‐DM1 (trastuzumab‐emtansine) is an antibody‐drug conjugate used for the treatment of HER2‐positive breast cancer combining the FDA approved mAb (monoclonal antibody) trastuzumab and the microtubule cytotoxic agent DM1 (emtansine). Despite clinical successes achieved by targeted therapies, a large number of patients develop resistance during treatment. To explore mechanisms of resistance to T‐DM1, the MDA‐MB‐361 HER2‐positive breast cancer cell line was exposed in vitro to T‐DM1 in the absence or presence of ciclosporin A. Previously reported mechanisms of resistance such as trastuzumab‐binding alterations, MDR1 upregulation, and SLC46A3 downregulation were not observed in these models. Despite a decrease in HER2 expression at the cell surface, both resistant cell lines remained sensitive to HER2 targeted therapies such as mAbs and tyrosine kinase inhibitors. In addition, sensitivity to DNA damaging agents and topoisomerase inhibitors were unchanged. Conversely resistance to anti‐tubulin agents increased. Resistant cells displayed a decreased content of polymerized tubulin and a decreased content of βIII tubulin although the downregulation of βIII tubulin by siRNA in the parental cell line did not modified the sensitivity to T‐DM1. Both cell lines resistant to T‐DM1 also presented giant aneuploid cells. Several SLC (solute carrier) transporters were found to be differentially expressed in the resistant cells in comparison to parental cells. These results suggest that some characteristics such as increased baseline aneuploidy and altered intracellular drug trafficking might be involved in resistance to T‐DM1.
... Nevertheless, two NHAs are present in every eukaryotic genome (Brett et al. 2005) and one or both of them have been cloned and localized in mosquitoes [Anopheles gambiae Na + /H + Antiporter 1, AgNHA1 (Rheault et al. 2007)], [Homo sapiens Na + /H + Antiporter 2, HsNHA1 (Xiang et al. 2007) and fruit flies [Drosophila melanogaster Na + /H + Antiporters 1 and 2, DmNHAs 1 and 2 (Day et al. 2008)]. Xiang and associates rescued NHA-lacking yeast mutants by transfection of HsNHA2 (functionally equivalent to AgNHA1) and Day et al. rescued yeast mutants with the Drosophila CPA2 homologues (DmNHAs) CG31052 and CG10806 from high Na + and High K + , respectively. ...
The present research report describes Na⁺/H⁺ antiport by brush border membrane vesicles isolated from whole larvae of Aedes aegypti (AeBBMVw). Our hypothesis is that acid quenching of acridine orange by AeBBMVw is predominantly mediated by Na⁺/H⁺ antiport via the NHA1 component of the AeBBMVw in the absence of amino acids and ATP. AeNHA1 is a Na⁺/H⁺ antiporter that has been postulated to exchange Na⁺ and H⁺ across the apical plasma membrane in posterior midgut of A. aegypti larvae. Its principal function is to recycle the H⁺ and Na⁺ that are transported during amino acid uptake, e.g., phenylalanine. This uptake is mediated, in part, by a voltage-driven, Na⁺-coupled, nutrient amino acid transporter (AeNAT8). The voltage is generated by an H⁺ V-ATPase. All three components, V-ATPase, antiporter, and nutrient amino acid transporter (VAN), are present in brush border membrane vesicles isolated from whole larvae of A. aegypti. By omitting ATP and amino acids, Na⁺/H⁺ antiport was measured by fluorescence quenching of acridine orange (AO) caused by acidification of either the internal vesicle medium (Na⁺in > Na⁺out) or the external fluid-membrane interface (Na⁺in < Na⁺out). Vesicles with 100 micromolar Na⁺ inside and 10 micromolar Na⁺ outside or with 0.01 micromolar Na⁺ inside and 100 micromolar Na⁺ outside quenched fluorescence of AO by as much as 30%. Acidification did not occur in the absence of AeBBMVw. Preincubation of AeBBMVw with antibodies to NHA1 inhibit Na⁺/H⁺ antiport dependent fluorescence quenching, indicating that AeNHA1 has a significant role in Na⁺/H⁺ exchange.
... Alongside the mammalian transporters, it also includes other eukaryotic taxa from arthropoda, nematode and protists. The most studied member of this clade is HsNHA2 that mediates the electroneutral exchange of sodium and lithium ions with protons 40,41 . Previously, human NHAs were proposed to be related to fungal NHA and bacterial NhaA genes 1 . ...
... NHX and sodium-proton exchanger (NHE) transporters belong to the cation proton antiporter1 CPA1 family [7,8]. It appears that CPA1 family has evolved from ancestral sodium-proton antiporter (NhaP) genes in prokaryotes [8][9][10]. Their primary physiological functions are regulation of cytoplasmic pH, extrusion of H + generated during metabolism in exchange of Na + or K + ions into cytoplasm and vacuoles in plants and animals [11][12][13]. ...
Na + transporters play an important role during salt stress and development. The present study is aimed at genome-wide identification, in silico analysis of sodium-proton antiporter (NHX) and sodium-proton exchanger (NHE)-type transporters in Sorghum bicolor and their expression patterns under varied abiotic stress conditions. In Sorghum, seven NHX and nine NHE homologs were identified. Amiloride (a known inhibitor of Na + /H + exchanger activity) binding motif was noticed in both types of the transporters. Chromosome 2 was found to be a hotspot region with five sodium transporters. Phylogenetic analysis inferred six ortholog and three paralog groups. To gain an insight into functional divergence of SbNHX/NHE transporters, real-time gene expression was performed under salt, drought, heat, and cold stresses in embryo, root, stem, and leaf tissues. Expression patterns revealed that both SbNHXs and SbNHEs are responsive either to single or multiple abiotic stresses. The predicted protein-protein interaction networks revealed that only SbNHX7 is involved in the calcineurin B-like proteins (CBL)-CBL interacting protein kinases (CIPK) pathway. The study provides insights into the functional divergence of SbNHX/NHE transporter genes with tissue specific expressions in Sorghum under different abiotic stress conditions.
... NHX and sodium-proton exchanger (NHE) transporters belong to the cation proton antiporter1 CPA1 family [7,8]. It appears that CPA1 family has evolved from ancestral sodium-proton antiporter (NhaP) genes in prokaryotes [8][9][10]. Their primary physiological functions are regulation of cytoplasmic pH, extrusion of H + generated during metabolism in exchange of Na + or K + ions into cytoplasm and vacuoles in plants and animals [11][12][13]. ...
Na+ transporters play an important role during salt stress and development. The present
study is aimed at genome-wide identification, in silico analysis of sodium-proton antiporter (NHX) and sodium-proton exchanger (NHE)-type transporters in Sorghum bicolor and their expression patterns under varied abiotic stress conditions. In Sorghum, seven NHX and nine NHE homologs were identified. Amiloride (a known inhibitor of Na+/H+ exchanger activity) binding motif was noticed in both types of the transporters. Chromosome 2 was found to be a hotspot region with five sodium transporters. Phylogenetic analysis inferred six ortholog and three paralog groups. To gain an insight
into functional divergence of SbNHX/NHE transporters, real-time gene expression was performed under salt, drought, heat, and cold stresses in embryo, root, stem, and leaf tissues. Expression patterns revealed that both SbNHXs and SbNHEs are responsive either to single or multiple abiotic stresses. The predicted protein–protein interaction networks revealed that only SbNHX7 is involved in the calcineurin B-like proteins (CBL)- CBL interacting protein kinases (CIPK) pathway. The study provides insights into the functional divergence of SbNHX/NHE transporter genes with tissue specific expressions in Sorghum under different abiotic stress conditions.
... 2011)). Using ena1-4 nha1 nhx1 mutants, several mammalian Na C /H C exchangers have also been characterized (Montero-Lomelí and Okorokova Façanha 1999; Flegelova et al. 2006;Xiang et al. 2007). One very interesting study used the yeast model system to characterize mutations in the human NHE9 Na C /H C antiporter that have been associated with autism ( Kondapalli et al. 2013). ...
As the proper maintenance of intracellular potassium and sodium concentrations is vital for cell growth, all living organisms have developed a cohort of strategies to maintain proper monovalent cation homeostasis. In the model yeast Saccharomyces cerevisiae, potassium is accumulated to relatively high concentrations and is required for many aspects of cellular function, whereas high intracellular sodium/potassium ratios are detrimental to cell growth and survival. The fact that S. cerevisiae cells can grow in the presence of a broad range of concentrations of external potassium (10 μM-2.5 M) and sodium (up to 1.5 M) indicates the existence of robust mechanisms that have evolved to maintain intracellular concentrations of these cations within appropriate limits. In this review, current knowledge regarding potassium and sodium transporters and their regulation will be summarized. The cellular responses to high sodium and potassium and potassium starvation will also be discussed, as well as applications of this knowledge to diverse fields, including antifungal treatments, bioethanol production and human disease.
... These recently identified NHEs exhibit greater homology to prokaryotic NHEs, and their physiologic roles remain poorly characterized, 8 with NHA1 apparently restricted to testis 16 and NHA2 identified in bone 17 and postulated to serve as a potential Na + /Li + countertransporter. 18 Finally, the SLC9C family contains proteins encoded by Slc9c1, which is believed to be sperm specific and to play an important role in sperm motility, 19 and Slc9c2, about which little is known. ...
Intracellular pH (pHi) homeostasis is key to the functioning of vascular smooth muscle cells, including pulmonary artery smooth muscle cells (PASMCs). Sodium-hydrogen exchange (NHE) is an important contributor to pHi control in PASMCs. In this review, we examine the role of NHE in PASMC function, in both physiologic and pathologic conditions. In particular, we focus on the contribution of NHE to the PASMC response to hypoxia, considering both acute hypoxic pulmonary vasoconstriction and the development of pulmonary vascular remodeling and pulmonary hypertension in response to chronic hypoxia. Hypoxic pulmonary hypertension remains a disease with limited therapeutic options. Thus, this review explores past efforts at disrupting NHE signaling and discusses the therapeutic potential that such efforts may have in the field of pulmonary hypertension.
... The plant flavonoid phloretin was the first compound of this class to be discovered, and inhibits UTs with micromolar affinity (Chou and Knepper 1989). However, phloretin is a relatively non-selective inhibitor of a large number of structurally unrelated transport proteins (Wheeler and Hinkle 1981;Tsukaguchi et al. 1998;Wang et al. 2000;Fan et al. 2001;Xiang et al. 2007), and may modulate transport by directly affecting the physical properties of the lipid bilayer (Andersen et al. 1976;Cseh and Benz 1999). More recently, the search for novel diuretics has lead to the discovery of multiple classes of high affinity inhibitors of the mammalian UTs Anderson et al. 2012;Yao et al. 2012;Esteva-Font et al. 2013;Li et al. 2013;Liu et al. 2013), including compounds that are selective for UT-B over UT-A ) and vice versa . ...
Members of the urea transporter (UT) family mediate rapid, selective transport of urea down its concentration gradient. To date, crystal structures of two evolutionarily distant UTs have been solved. These structures reveal a common UT fold involving two structurally homologous domains that encircle a continuous membrane-spanning pore and indicate that UTs transport urea via a channel-like mechanism. Examination of the conserved architecture of the pore, combined with crystal structures of ligand-bound proteins, molecular dynamics simulations, and functional data on permeation and inhibition by a broad range of urea analogs and other small molecules, provides insight into the structural basis of urea permeation and selectivity.
Cell pH and Na⁺ homeostasis requires Na⁺/H⁺ antiporters. The crystal structure of NhaA, the main Escherichia coli Na⁺/H⁺ antiporter, revealed a unique NhaA structural fold shared by prokaryotic and eukaryotic membrane proteins. Out of the 12 NhaA transmembrane segments (TMs), TMs III–V and X–XII are topologically inverted repeats with unwound TMs IV and XI forming the X shape characterizing the NhaA fold. We show that intramolecular cross-linking under oxidizing conditions of a NhaA mutant with two Cys replacements across the crossing (D133C-T340C) inhibits antiporter activity and impairs NhaA-dependent cell growth in high-salts. The affinity purified D133C-T340C protein binds Li⁺ (the Na⁺ surrogate substrate of NhaA) under reducing conditions. The cross-linking traps the antiporter in an outward-facing conformation, blocking the antiport cycle. As many secondary transporters are found to share the NhaA fold, including some involved in human diseases, our data have importance for both basic and clinical research.
Hypertension and heart failure (HF) are the leading causes of death and disability worldwide. Both are complex multifactorial conditions with each at either ends of the cardiovascular continuum. Rare mutations resulting in monogenic forms of hypertension, hypotension, and cardiomyopathies highlight the importance of genetics in disease causation and the consequent implications for disease prediction and treatment. Accelerating advances in genomics over the last decade have led to an unparalleled leap in our understanding of the genetic architecture of both hypertension and heart failure. In this chapter, we describe the current state of the art in the genetics of both conditions, focusing on the biological pathways that are perturbed and opportunities for early detection and treatment.
Cell pH and Na⁺ homeostasis requires Na⁺/H⁺ antiporters. The crystal structure of NhaA, the main Escherichia coli Na⁺/H⁺ antiporter, revealed a unique NhaA structural fold shared by prokaryotic and eukaryotic membrane proteins. Out of the 12 NhaA transmembrane segments (TMs), TMs III–V and X–XII are topologically inverted repeats with unwound TMs IV and XI forming the X shape characterizing the NhaA fold.
We show that intramolecular cross-linking under oxidizing conditions of a NhaA mutant with two Cys replacements across the crossing (D133C-T340C) inhibits antiporter activity and impairs NhaA-dependent cell growth in high-salts. The affinity purified D133C-T340C protein binds Li⁺ (the Na⁺ surrogate substrate of NhaA) under reducing conditions. The cross-linking traps the antiporter in an outward-facing conformation, blocking the antiport cycle. As many secondary transporters are found to share the NhaA fold, including some involved in human diseases, our data have importance for both basic and clinical research.
Na+/H+ exchangers (NHEs) are known to be important regulators of pH in multiple intracellular compartments of eukaryotic cells. Sperm function is especially dependent on changes in pH and thus it has been postulated that NHEs play important roles in regulating the intracellular pH of these cells. For example, in order to achieve fertilization, mature sperm must maintain a basal pH in the male reproductive tract and then alkalize in response to specific signals in the female reproductive tract during the capacitation process. Eight NHE isoforms are expressed in mammalian testis/sperm: NHE1, NHE3, NHE5, NHE8, NHA1, NHA2, NHE10, and NHE11. These NHE isoforms are expressed at varying times during spermatogenesis and localize to different subcellular structures in developing and mature sperm where they contribute to multiple aspects of sperm physiology and male fertility including proper sperm development/morphogenesis, motility, capacitation, and the acrosome reaction. Previous work has provided evidence for NHE3, NHE8, NHA1, NHA2, and NHE10 being critical for male fertility in mice and NHE10 has recently been shown to be essential for male fertility in humans. In this article we review what is known about each NHE isoform expressed in mammalian sperm and discuss the physiological significance of each NHE isoform with respect to male fertility.
It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.
The Na+/H+ exchanger transporters (NHE) play an important role in various biologic processes including Na+ absorption, intracellular pH homeostasis, cell volume regulation, proliferation, and apoptosis. The wide expression pattern and cellular localization of NHEs make these proteins pivotal players in virtually all human tissues and organs. In addition, recent studies suggest that NHEs may be one of the primeval transport protein forms in the history of life. Among the different isoforms, the most well-characterized NHEs are the Na+/H+ exchanger isoform 1 (NHE1) and Na+/H+ exchanger isoform 3 (NHE3). However, Na+/H+ exchanger isoform 8 (NHE8) has been receiving attention based on its recent discoveries in the gastrointestinal tract. In this review, we will discuss what is known about the physiological function and potential role of NHE8 in the main organ systems, including useful overviews that could inspire new studies on this multifaceted protein.
The protonation state of soluble and membrane-associated macromolecules dictates their charge, conformation and functional activity. In addition, protons (H ⁺ , or their equivalents) partake in numerous metabolic reactions and serve as a source of electrochemical energy to drive the transmembrane transport of both organic and inorganic substrates. Stringent regulation of the intracellular pH is therefore paramount to homeostasis. While the regulation of the cytosolic pH has been studied extensively, our understanding of the determinants of the [H ⁺ ] of intracellular organelles has developed more slowly, limited by their small size and inaccessibility. Recently, however, targeting of molecular probes to the organellar lumen, together with advances in genomic, proteomic and electrophysiological techniques have led to the identification and characterization of unique pumps, channels and transporters responsible for the establishment and maintenance of intraorganellar pH. These developments and their implications to cellular function in health and disease are the subject of this review.
Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.
Cation/proton antiporters (CPAs), which include Na⁺/H⁺ exchangers, are evolutionarily ancient transporters present in most species from prokaryotes to higher eukaryotes. Many of them are expressed in epithelial cells or various organs, where they utilize the electrochemical gradient of one ion to transport another ion against its electrochemical gradient. In the intestinal and renal epithelia, NHEs are critical for vectorial transport of Na⁺, HCO3⁻ and water, and consequently for the systemic volume and acid–base homeostasis. They also contribute to nutrient absorption, cellular proliferation, migration, and apoptosis, and modulate extracellular milieu, e.g., to regulate the intestinal microbial microenvironment. Their dysregulation, or in some instances, mutations, contributes to the human disease pathogenesis and some of the well-characterized Na⁺/H⁺ exchangers have been considered as attractive targets for pharmacological inhibition. In this chapter, we provide an overview of the members of the CPA superfamily of cation/proton antiporters, with particular focus on their roles in epithelial cells, their expression patterns, intracellular localization, regulation, and function, primarily as determined by gene targeting studies.
Cation and protons perform a substantial role in all the organism and its homeostasis within the cells are maintained by the cation-proton antiporters (CPAs). CPA is the huge family of the membrane transporter protein throughout the plant and animal kingdom including microorganism. In human, any malfunctioning of these proteins may lead to severe diseases like hypertension, heart diseases etc and CPAs are recently proposed to be responsible for the virulent property of various pathogens including Vibrio cholerae, Yersinia pestis etc. Human Sodium-Proton exchangers (Na⁺/H⁺ exchangers, NHEs) are crucial in ion homeostasis whereas Ec-NhaA, Na + -H + Antiporters maintain a balance of Na+ and proton in E. coli, regulating pH and cell volume within the cell. These Sodium-Proton antiporters are found to be responsible for the virulence in various pathogens causing human diseases. Understanding of these CPAs may assist investigators to target such human diseases, that further may lead to establishing the effective path for therapeutics or drug designing against associated human disease. Here we have compiled all such information on CPAs and provide a systematic approach to unravel the mechanism and role of antiporter proteins in a wide range of organisms. Being involved throughout all the species, this review on cation-proton antiporters may attract the attention of many investigators and concerned researchers and will be provided with the recent detailed information on the role of CPA in human health.
Hypertension and heart failure are leading causes of death and disability worldwide. Both are complex multifactorial conditions with hypertension and heart failure at either ends of the cardiovascular continuum. Rare mutations resulting in monogenic forms of hypertension, hypotension and cardiomyopathies highlight the importance of genetics in disease causation and consequent implications for disease prediction and treatment. Advances in genomics have accelerated over the last decade leading to an unparalleled leap in our understanding of the genetic architecture of both hypertension and heart failure. In this chapter, we describe the current state of the art in the genetics of both conditions, hypertension and heart failure, focussing on biologic pathways that are perturbed and opportunities for early detection and treatment.
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.
The mammalian Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH. It removes a single intracellular proton in exchange for one extracellular sodium ion. It has a large 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments with several membrane-associated segments including intracellular and extracellular loops. Extracellular regions of this domain are believed to contribute to sodium coordination. Intracellular loops may coordinate protons and modulate the sensitivity to intracellular pH. In this study we characterized the structure and function of intracellular loop 5 (IL5) amino acids Gly431-Lys443. Mutation of eleven residues to alanine caused partial inhibition of transport; notably, mutation of residues R440A and I436A, demonstrated that these residues were critical for NHE1 function. The structure of a peptide of IL5 revealed that it is unstructured in DMSO, however in sodium dodecyl sulfate solution it possessed significant alpha helical character. A significant finding was that Lys438 was in close proximity with Trp434 residue. Overall our results show that IL5 is a critical intracellular loop, with a propensity to form an alpha helix, with many residues being critical for proton transport.
The Slc9a family of Na⁺/H⁺ exchangers (NHEs) plays a critical role in neutral sodium absorption in the mammalian intestine as well as other absorptive and secretory epithelia of digestive organs. These transport proteins mediate the electroneutral exchange of Na⁺ and H⁺ and are crucial in a variety of physiological processes, including the transepithelial Na⁺ and water absorption, fine tuning of intracellular pH, cell volume control and systemic electrolyte, and acid-base and fluid volume homeostasis. They also secondarily affect other cellular transport mechanisms as well as cell survival, motility, adhesion, and repair mechanisms. In this chapter, we review the role of the Na⁺/H⁺ exchange mechanism as it relates to the physiology of organs and cells involved in nutrient intake and absorption, and we describe physiological and molecular aspects of individual isoforms, including their structure, tissue-, and subcellular distribution, as well as their regulation by physiological stimuli at the transcriptional and posttranscriptional levels. Consequences of gene-targeted mutation of individual isoforms are discussed in the context of the physiology of digestive organs. Where available, we also provide a review of pathophysiological states related to aberrant expression and/or activity of NHEs within the confines of the digestive system.
The transmembranal Na⁺/H⁺ antiporters transport sodium (or several other monovalent cations) in exchange for H⁺ across lipid bilayers in all kingdoms of life. They are critical in pH homeostasis of the cytoplasm and/or organelles. A particularly notable example is the SLC9 gene family, which encodes Na⁺/H⁺ exchangers (NHEs) in many species from prokaryotes to eukaryotes. In humans, these proteins are associated with the pathophysiology of various diseases. Yet, the most extensively studied Na⁺/H⁺ antiporter is Ec-NhaA, the main Na⁺/H⁺ antiporter of Escherichia coli.
The Slc9a family of Na + /H + exchangers (NHE) plays a critical role in neutral sodium absorption in the mammalian intestine as well as other absorptive and secretory epithelia of digestive organs. These transport proteins mediate the electroneutral exchange of Na + and H + and are crucial in a variety of physiological processes, including the transepithelial Na + and water absorption, fine-tuning of intracellular pH, cell volume control and systemic electrolyte, and acid–base and fluid volume homeostasis. They also secondarily affect other cellular transport mechanisms as well as cell survival, motility, adhesion, and repair mechanisms. In this chapter we review the role of the Na + /H + exchange mechanism as it relates to the physiology of organs and cells involved in nutrient intake and absorption, and we describe physiological and molecular aspects of individual isoforms, including their structure, tissue, and subcellular distribution, as well as their regulation by physiological stimuli at the transcriptional and post-transcriptional levels. Consequences of gene-targeted mutation of individual isoforms are discussed in the context of the physiology of digestive organs. Where available, we also provide a review of pathophysiological states related to aberrant expression and/or activity of Na + /H + exchangers within the confines of the digestive system.
The mammalian Na+–H+ exchanger isoform 1 (NHE1) is a plasma membrane protein that regulates intracellular pH in the myocardium by removing one intracellular hydrogen ion in exchange for one extracellular sodium ion. While NHE1 regulates intracellular pH, it is also involved in the damage that occurs to the myocardium with ischemia and reperfusion. Additionally, NHE1 levels are elevated in cardiac diseases such as hypertrophy, and NHE1 inhibition can reduce ischemia–reperfusion damage and prevent heart hypertrophy in animal models. Recently, it has been demonstrated that elevation of NHE1 levels occurs in several kinds of hearts disease. Surprisingly, the effect of elevation of these levels is varied, sometimes having beneficial and sometimes detrimental effects.
Objective:
To quantify genetic overlap between migraine and ischemic stroke (IS) with respect to common genetic variation.
Methods:
We applied 4 different approaches to large-scale meta-analyses of genome-wide data on migraine (23,285 cases and 95,425 controls) and IS (12,389 cases and 62,004 controls). First, we queried known genome-wide significant loci for both disorders, looking for potential overlap of signals. We then analyzed the overall shared genetic load using polygenic scores and estimated the genetic correlation between disease subtypes using data derived from these models. We further interrogated genomic regions of shared risk using analysis of covariance patterns between the 2 phenotypes using cross-phenotype spatial mapping.
Results:
We found substantial genetic overlap between migraine and IS using all 4 approaches. Migraine without aura (MO) showed much stronger overlap with IS and its subtypes than migraine with aura (MA). The strongest overlap existed between MO and large artery stroke (LAS; p = 6.4 × 10(-28) for the LAS polygenic score in MO) and between MO and cardioembolic stroke (CE; p = 2.7 × 10(-20) for the CE score in MO).
Conclusions:
Our findings indicate shared genetic susceptibility to migraine and IS, with a particularly strong overlap between MO and both LAS and CE pointing towards shared mechanisms. Our observations on MA are consistent with a limited role of common genetic variants in this subtype.
The functions of Na+/H+exchangers (NHEs) during osteoclastic differentiation were investigated using the NHE inhibitor amiloride and a monoclonal antibody (MAb). Compared with sRANKL-stimulated control cells, amiloride decreased the number of large TRAP-positive osteoclast cells (OCs) with ≥ 10 nuclei and increased the number of small TRAP-positive OCs with ≤ 10 nuclei during sRANKL-dependent osteoclastic differentiation of RAW264.7 cells. NHE10 mRNA expression and OC differentiation markers were increased by sRANKL stimulation in dose- and time- dependent manners. NHEs 1-9 mRNA expression was not increased by sRANKL stimulation. Similar to amiloride, a rat anti-mouse NHE10 MAb (clone 6B11) decreased the number of large TRAP-positive OCs, but increased the number of small TRAP-positive OCs. These findings suggested that inhibition of NHEs by amiloride or an anti-NHE10 MAb prevented sRANKL-promoted cellular fusion. The anti-NHE10 MAb has the potential for use as an effective inhibitor of bone resorption for targeted bone disease therapy.
Aquaporins (AQPs) are a family of membrane water channels that basically function as regulators of intracellular and intercellular water flow. To date, thirteen aquaporins have been characterized. They are distributed wildly in specific cell types in multiple organs and tissues. Each AQP channel consists of six membrane-spanning alpha-helices that have a central water-transporting pore. Four AQP monomers assemble to form tetramers, which are the functional units in the membrane. Some of AQPs also transport urea, glycerol, ammonia, hydrogen peroxide, and gas molecules. AQP-mediated osmotic water transport across epithelial plasma membranes facilitates transcellular fluid transport and thus water reabsorption. AQP-mediated urea and glycerol transport is involved in energy metabolism and epidermal hydration. AQP-mediated CO2 and NH3 transport across membrane maintains intracellular acid-base homeostasis. AQPs are also involved in the pathophysiology of a wide range of human diseases (including water disbalance in kidney and brain, neuroinflammatory disease, obesity, and cancer). Further work is required to determine whether aquaporins are viable therapeutic targets or reliable diagnostic and prognostic biomarkers.
Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.
The control by Na+/H+ antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na+/H+ antiporters. Their activity is tightly controlled by pH. Here we present the crystal structure of pH-downregulated NhaA, the main antiporter of Escherichia coli and many enterobacteria. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. We propose that the binding of charged substrates causes an electric imbalance, inducing movements, that permit a rapid alternating-access mechanism. This ion-exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the entry to the cytoplasmic funnel.
In human red cells, Li is extruded against its own concentration gradient if the external medium contains Na as a dominant cation. This uphill net Li extrusion occurs in the presence of external Na but not K, Rb, Cs, choline, Mg, or Ca, is ouabain-insensitive, inhibited by phloretin, and does not require the presence of cellular ATP. Li influx into human red cells has a ouabain-sensitive and a ouabain-insensitive but phloretin-sensitive component. Ouabain-sensitive Li influx is competitively inhibited by external K and Na and probably involves the site on which the Na-K pump normally transports K into red cells. Ouabain does not inhibit Li efflux from red cells containing Li concentrations below 10 mM in the presence of high internal Na or K, whereas a ouabain-sensitive Li efflux can be measured in cells loaded to contain 140 mM Li in the presence of little or no internal Na or K. Ouabain-insensitive Li efflux is stimulated by external Na and not by K, Rb, Cs, choline, Mg, or Ca ions. Na-dependent Li efflux does not require the presence of cellular ATP and is inhibited by phloretin, furosemide, quinine, and quinidine. Experiments carried out in cells loaded in the presence of nystatin to contain either only K or only Na show that the ouabain-insensitive, phloretin-inhibited Li movements into or out of human red cells are stimulated by Na on the trans side and inhibited by Na on the cis side of the red cell membrane. The characteristics of the Na-dependent unidirectional Li fluxes and uphill Li extrusion are similar, suggesting that they are mediated by the same Na-Li countertransport system.
A Na+/H+ antiporter coded by the nhaA (ant) gene of Escherichia coli has been overproduced and purified. The amino-terminal sequence of the protein has been determined and shown to correlate with initiation at a GUG codon, 75 bases upstream from the previously suggested AUG initiation codon. The purified protein, when reconstituted into proteoliposomes, has Na+/H+ antiport activity. It can mediate sodium uptake when a transmembrane pH gradient is applied. Downhill sodium efflux is shown to be highly dependent on pH and is accelerated by a transmembrane pH gradient. An imposed membrane potential negative inside accelerates Na+ efflux at all pH values tested. These findings suggest that the antiporter is electrogenic both at acid and alkaline pH. The activation at alkaline pH values (2000-fold increase) is consistent with the proposed role of the antiporter in regulation of internal pH at the alkaline pH range.
In a newly formulated growth medium lacking Na+ and NH4+, Saccharomyces cerevisiae grew maximally at 5 microM K+. Cells grown under these conditions transported K+ with an apparent Km of 24 microM, whereas cells grown in customary high-K+ medium had a significantly higher Km (2 mM K+). The two types of transport also differed in carbonyl cyanide-m-chlorophenyl hydrazone sensitivity, response to ATP depletion, and temperature dependence. The results can be accounted for either by two transport systems or by one system operating in two different ways.
Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.
All living cells maintain an inwardly directed Na+ gradient and a constant intracellular pH. Na+/H+ antiporters have been assigned an essential role in these homeostatic mechanisms in all cells. In Escherichia coli, two Na+/H+ antiporter genes, nhaA and nhaB, have been cloned. Deletion of either one or both showed that NhaA is essential for adaptation to high salinity, for growth at alkaline pH in the presence of Na+ and for challenging Li+ toxicity. NhaB confers tolerance to low levels of Na+ and becomes essential when the activity of NhaA limits growth.
The adaptive response to Na+ is mediated by the positive regulator nhaR, which transduces the signal (intracellular Na+) to expression of the nhaA gene. We have identified Glu-134 of NhaR as part of the ‘Na+ sensor’ of NhaA.
In agreement with the role of NhaA in pH homeostasis, its Na+-dependent expression is enhanced at alkaline pH. Reconstitution of pure NhaA and NhaB in proteoliposomes demonstrates that, whereas both are electrogenic (the H+/Na+ stoichiometry of NhaA is 2), only NhaA is pH-dependent, increasing its activity 1000-fold between pH 7 and 8.5. Mutating all the histidines of NhaA shows that His-226 is part of the ‘pH sensor’ of NhaA.
The H+:Na+ exchange stoichiometry of NhaA, a sodium-proton antiporter coded by the nhaA gene of Escherichia coli, has been determined using purified NhaA protein reconstituted into sodium-loaded proteoliposomes. One approach involved measuring, in parallel experiments, the Na+ efflux and H+ influx from such proteoliposomes and calculating the stoichiometry from the ratio of these fluxes. A second approach was based on measuring the membrane potential generated by NhaA at various sodium gradients and assuming complete coupling and thermodynamic equilibrium between the membrane potential and the ion gradients. The results from both methods agree with a stoichiometry of 2 H+ exchanged for each Na+. This value is independent of pH between pH 7.2 and 8.1. These results support the suggestion that a change in the catalytic rate of NhaA rather than its stoichiometry is crucial for its role in regulation of intracellular pH in alkaline environments.
PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, G.R. (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca2+-transporting ATPase in the Golgi, a hitherto unusual location for a Ca2+ pump. As a starting point for structure-function analysis using a mutagenic approach, we used the strong and inducible heat shock promoter to direct high level expression of PMR1 from a multicopy plasmid. Yeast lysates were separated on sucrose density gradients, and fractions assayed for organellar markers. PMR1 is found in fractions containing the Golgi marker guanosine diphosphatase, and is associated with an ATP-dependent, protonophore-insensitive 45Ca2+ uptake activity. This activity is virtually abolished in the absence of the expression plasmid. Furthermore, replacement of the active site aspartate within the phosphorylation domain had the expected effect of abolishing Ca2+ transport activity entirely. Interestingly, the mutant enzymes (Asp-371 --> Glu and Asp-371 --> Asn) demonstrated proper targeting to the Golgi, unlike analogous mutations in the related yeast H+-ATPase. Detailed characterization of calcium transport by PMR1 showed that sensitivity to inhibitors (vanadate, thapsigargin, and cyclopiazonic acid) and affinity for substrates (MgATP and Ca2+) were different from the previously characterized sarco/endoplasmic reticulum and plasma membrane Ca2+-ATPases. PMR1 therefore represents a new and distinct P-type Ca2+-ATPase. Because close homologs of PMR1 have been cloned from rat and other organisms, we suggest that Ca2+-ATPases in the Golgi will form a discrete subgroup that are important for functioning of the secretory pathway.
The bacterial Na+ (Li+)/H+ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na+, Li+) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H+ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na+, Li+) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H+ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionally of plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase.
The effect of ligands for phospholipase C-coupled receptors and of protein kinase C (PKC) stimulation with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or 1,2-dioctanoyl-sn-glycerol on the activity of the basolateral organic anion transporter (OAT) in S2 segments of single, nonperfused rabbit proximal tubules (PT) was measured with the use of fluorescein and epifluorescence microscopy. The initial uptake rate (25 s, OAT activity) was measured in real time by using conditions similar to those found in vivo. Stimulation of PKC with PMA or 1,2-dioctanoyl-sn-glycerol led to an inhibition of OAT activity, which could be prevented by 10(-7) mol/l of the PKC-specific inhibitor bisindolylmaleimide. The alpha(1)-receptor agonist phenylephrine as well as the peptide hormone bradykinin induced a reversible decrease of OAT activity, which was prevented by bisindolylmaleimide. The observed effect was not due to a decrease in the concentration of the counterion alpha-ketoglutarate or to impaired alpha-ketoglutarate recycling, because it was unchanged in the continuous presence of alpha-ketoglutarate or methyl succinate. We conclude that physiological stimuli can inhibit the activity of OAT in rabbit PT via PKC. The effect is not mediated by alterations in counterion availability but by a direct action on the OAT.
The biochemical mechanisms involved in regulation of insulin secretion are not completely understood. The rat INS-1 cell line has been used to gain insight in this area because it secretes insulin in response to glucose concentrations in the physiological range. However, the magnitude of the response is far less than that seen in freshly isolated rat islets. In the current study, we have stably transfected INS-1 cells with a plasmid containing the human proinsulin gene. After antibiotic selection and clonal expansion, 67% of the resultant clones were found to be poorly responsive to glucose in terms of insulin secretion (< or =2-fold stimulation by 15 mmol/l compared with 3 mmol/l glucose), 17% of the clones were moderately responsive (2- to 5-fold stimulation), and 16% were strongly responsive (5- to 13-fold stimulation). The differences in responsiveness could not be ascribed to differences in insulin content. Detailed analysis of one of the strongly responsive lines (832/13) revealed that its potent response to glucose (average of 10-fold) was stable over 66 population doublings (approximately 7.5 months of tissue culture) with half-maximal stimulation at 6 mmol/l glucose. Furthermore, in the presence of 15 mmol/l glucose, insulin secretion was potentiated significantly by 100 pmol/l isobutylmethylxanthine (320%), 1 mmol/l oleate/palmitate (77%), and 50 nmol/l glucagon-like peptide 1 (60%), whereas carbachol had no effect. Glucose-stimulated insulin secretion was also potentiated by the sulfonylurea tolbutamide (threefold at 3 mmol/l glucose and 50% at 15 mmol/l glucose) and was abolished by diazoxide, which demonstrates the operation of the ATP-sensitive K+ channel (K(ATP)) in 832/13 cells. Moreover, when the K(ATP) channel was bypassed by incubation of cells in depolarizing K+ (35 mmol/l), insulin secretion was more effectively stimulated by glucose in 832/13 cells than in parental INS-1 cells, which demonstrates the presence of a K(ATP) channel-independent pathway of glucose sensing. We conclude that clonal selection of INS-1 cells allows isolation of cell lines that exhibit markedly enhanced and stable responsiveness to glucose and several of its known potentiators. These lines may be attractive new vehicles for studies of beta-cell function.
Spectacular morning glory blooms rely on a behind-the-scenes proton
exchanger.
Tet(L) and Tet(K) are specific antibiotic-resistance determinants. They catalyze efflux of a tetracycline(Tc)-divalent metal complex in exchange for protons, as do other Tet efflux proteins. These Tet proteins also catalyze Na+ and K+ exchange for protons. Each of the "cytoplasmic substrates", Na+, K+ and the Tc-metal ion complex, can also be exchanged for K+, a catalytic mode that accounts for the long-recognized K+ uptake capacity conferred by some Tet proteins. The multiple catalytic modes of Tet(L) and Tet(K) provide potential new avenues for development of inhibitors of these efflux systems as well as avenues for exploration of structure-function relationships. The multiple catalytic modes of Tet(L), which is chromosomally encoded in Bacillus subtilis, also correspond to diverse physiological roles, including roles in antibiotic-, Na+-, and alkali-resistance as well as K+ acquisition. The use of K+ as an external coupling ion may contribute not only to the organism's K+ uptake capacity but also to its ability to exclude Na+ and Tc at elevated pH values. Regulation of the chromosomal tetL gene by Tc has been proposed to involve a translational re-initiation mechanism that is novel for an antibiotic-resistance gene and increases Tet expression seven-fold. Other elements of tetL expression and its regulation are already evident, including gene amplification and use of multiple promoters. However, further studies are required to clarify the full panoply of regulatory mechanisms, and their integration to ensure different levels of tetL expression that are optimal for its different functions. It will also be of interest to investigate the implications of Tet(L) and Tet(K) multifunctionality on the emergence and persistence of these antibiotic-resistance genes.
Increased red blood cell sodium-lithium countertransport (SLC) activity and elevated intracellular calcium have been observed in hypertensive patients. The association of these ion transport abnormalities with each other and with another phenotype, insulin resistance, has been suggested. We investigated whether elevated SLC activity and increased lymphocyte cytosolic calcium (Ca(cyt)) occur in the same individuals and whether either is associated with hyperinsulinaemia. We measured SLC activity, lymphocyte Ca(cyt)and fasting insulin levels in hypertensive patients and normal subjects. Consistent with prior studies, SLC activity was significantly and positively correlated with fasting insulin levels (r = 0.45, P < 0.01). However, SLC activity and lymphocyte Ca(cyt) were significantly but inversely correlated (r = -0.42, P < 0.01) and lymphocyte Ca(cyt) was also inversely correlated with fasting insulin (r = -0.55, P < 0.001). When the study participants were instead separated into two groups based on fasting insulin levels, those above the median (15 microU/ml) had significantly higher SLC activity and significantly lower Ca(cyt). When separated by lymphocyte Ca(cyt) levels (above or below 120 nM) those patients with low lymphocyte Ca(cyt) had significantly higher SLC activity and significantly higher insulin levels. Multiple linear regression showed that fasting insulin was significantly predictive of SLC activity (P = 0.05) and Ca(cyt) (P < 0.01). Thus, elevated SLC activity and increased lymphocyte Ca(cyt) are separate and distinct ion transport phenotypes in hypertensive patients, linked through a relationship to hyperinsulinaemia that is direct with SLC activity and inverse with lymphocyte Ca(cyt).
Directed cell movement is a multi-step process requiring an initial spatial polarization that is established by asymmetric stimulation of Rho GTPases, phosphoinositides (PIs), and actin polymerization. We report that the Na-H exchanger isoform 1 (NHE1), a ubiquitously expressed plasma membrane ion exchanger, is necessary for establishing polarity in migrating fibroblasts. In fibroblasts, NHE1 is predominantly localized in lamellipodia, where it functions as a plasma membrane anchor for actin filaments by its direct binding of ezrin/radixin/moesin (ERM) proteins. Migration in a wounding assay was impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ERM binding and cytoskeletal anchoring or ion transport. Disrupting either function of NHE1 impaired polarity, as indicated by loss of directionality, mislocalization of the Golgi apparatus away from the orientation of the wound edge, and inhibition of PI signaling. Both functions of NHE1 were also required for remodeling of focal adhesions. Most notably, lack of ion transport inhibited de-adhesion, resulting in trailing edges that failed to retract. These findings indicate that by regulating asymmetric signals that establish polarity and by coordinating focal adhesion remodeling at the cell front and rear, cytoskeletal anchoring by NHE1 and its localized activity in lamellipodia act cooperatively to integrate cues for directed migration.
PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions.
MdfA is an Escherichia coli multidrug-resistance transporter. Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance against a diverse group of toxic compounds. In this article, we show that, in addition to its role in multidrug resistance, MdfA confers extreme alkaline pH resistance and allows the growth of transformed cells under conditions that are close to those used normally by alkaliphiles (up to pH 10) by maintaining a physiological internal pH. MdfA-deleted E. coli cells are sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by MdfA expressed from a plasmid. This activity of MdfA requires Na⁺ or K⁺. Fluorescence studies with inverted membrane vesicles demonstrate that MdfA catalyzes Na⁺- or K⁺-dependent proton transport, and experiments with reconstituted proteoliposomes confirm that MdfA is solely responsible for this phenomenon. Studies with multidrug resistance-defective MdfA mutants and competitive transport assays suggest that these activities of MdfA are related. Together, the results demonstrate that a single protein has an unprecedented capacity to turn E. coli from an obligatory neutrophile into an alkalitolerant bacterium, and they suggest a previously uncharacterized physiological role for MdfA in pH homeostasis.
• MdfA
• multidrug transport
• sodium proton antiporter
• alkaline pH tolerance
• Escherichia coli
The relationship between endosomal pH and function is well documented in viral entry, endosomal maturation, receptor recycling, and vesicle targeting within the endocytic pathway. However, specific molecular mechanisms that either sense or regulate luminal pH to mediate these processes have not been identified. Herein we describe the use of novel, compartment-specific pH indicators to demonstrate that yeast Nhx1, an endosomal member of the ubiquitous NHE family of Na+/H+ exchangers, regulates luminal and cytoplasmic pH to control vesicle trafficking out of the endosome. Loss of Nhx1 confers growth sensitivity to low pH stress, and concomitant acidification and trafficking defects, which can be alleviated by weak bases. Conversely, weak acids cause wild-type yeast to present nhx1Delta trafficking phenotypes. Finally, we report that Nhx1 transports K+ in addition to Na+, suggesting that a single mechanism may responsible for both pH and K+-dependent endosomal processes. This presents the newly defined family of eukaryotic endosomal NHE as novel targets for pharmacological inhibition to alleviate pathological states associated with organellar alkalinization.
More than 200 genes annotated as Na+/H+ hydrogen exchangers (NHEs) currently reside in bioinformation databases such as GenBank and Pfam. We performed detailed phylogenetic analyses of these NHEs in an effort to better understand their specific functions and physiological roles. This analysis initially required examining the entire monovalent cation proton antiporter (CPA) superfamily that includes the CPA1, CPA2, and NaT-DC families of transporters, each of which has a unique set of bacterial ancestors. We have concluded that there are nine human NHE (or SLC9A) paralogs as well as two previously unknown human CPA2 genes, which we have named HsNHA1 and HsNHA2. The eukaryotic NHE family is composed of five phylogenetically distinct clades that differ in subcellular location, drug sensitivity, cation selectivity, and sequence length. The major subgroups are plasma membrane (recycling and resident) and intracellular (endosomal/TGN, NHE8-like, and plant vacuolar). HsNHE1, the first cloned eukaryotic NHE gene, belongs to the resident plasma membrane clade. The latter is the most recent to emerge, being found exclusively in vertebrates. In contrast, the intracellular clades are ubiquitously distributed and are likely precursors to the plasma membrane NHE. Yeast endosomal ScNHX1 was the first intracellular NHE to be described and is closely related to HsNHE6, HsNHE7, and HsNHE9 in humans. Our results link the appearance of NHE on the plasma membrane of animal cells to the use of the Na+/K(+)-ATPase to generate the membrane potential. These novel observations have allowed us to use comparative biology to predict physiological roles for the nine human NHE paralogs and to propose appropriate model organisms in which to study the unique properties of each NHE subclass.
In hair cells of the inner ear, robust Ca2+/H+ exchange mediated by plasma-membrane Ca2+-ATPase would rapidly acidify mechanically sensitive hair bundles without efficient removal of H+. We found that, whereas the basolateral membrane of vestibular hair cells from the frog saccule extrudes H+ via an Na+-dependent mechanism, bundles rapidly remove H+ in the absence of Na+ and HCO3(-), even when the soma is acidified. K+ was fully effective and sufficient for H+ removal; in contrast, Rb+ failed to support pH recovery. Na+/H+-exchanger isoform 1 (NHE1) was present on hair-cell soma membranes and was likely responsible for Na+-dependent H+ extrusion. NHE6 and NHE9 are organellar isoforms that can appear transiently on plasma membranes and have been proposed to mediate K+/H+ exchange. We identified NHE6 in a subset of hair bundles; NHE9 was present in all bundles. Heterologous expression of these isoforms in yeast strains lacking endogenous exchangers conferred pH-dependent tolerance to high levels of KCl and NaCl. NHE9 preferred cations in the order K+, Na+ > Rb+, consistent with the relative efficacies of these ions in promoting pH recovery in hair bundles. Electroneutral K+/H+ exchange, which we propose is performed by NHE9 in hair bundles, exploits the high-K+ endolymph, responds only to pH imbalance across the bundle membrane, is unaffected by the +80 mV endocochlear potential, and uses mechanisms already present in the ear for K+ recycling. This mechanism allows the hair cell to remove H+ generated by Ca2+ pumping without ATP hydrolysis in the cell.
It is well established that activation of the Na-H exchanger NHE1 and increases in intracellular pH (pHi) are early and universal responses to mitogens and have permissive effects in promoting cell proliferation. Despite this
evidence, a specific role for NHE1 or pHi in cell cycle progression remains undetermined. We now show that NHE1 activity and pHi regulate the timing of G2/M entry and transition. Prior to G2/M entry there is a rapid and transient increase in NHE1 activity and pHi, but in fibroblasts expressing a mutant NHE1 that lacks ion translocation activity, this increase in pHi is attenuated, S phase is delayed, and G2/M transition is impaired. In the absence of ion translocation by NHE1, expression of cyclin B1 and the kinase activity of
Cdc2 are decreased and Wee1 kinase expression increases. Increasing pHi in the absence of NHE1 activity, however, is sufficient to restore Cdc2 activity and cyclin B1 expression and to promote
G2/M entry and transition. These data indicate that a transient increase in pHi induced by NHE1 promotes the timing of G2/M, and they suggest that increases in pHi at the completion of S phase may constitute a previously unrecognized checkpoint for progression to G2 and mitosis.
In patients with affective disorders who are being treated with lithium carbonate, lithium concentration is lower in RBCs than in plasma, suggesting an active transport of the ion from the cells (similar to Na+ efflux). Wide variations have been observed in the steady state in vivo distribution of Li+ ratio (defined as intracellular Li concentration/extracellular Li+ concentration), which is under genetic control to some degree.1-3The relationship of this ratio to psychiatric diagnosis, to short- and long-term response to lithium therapy, and to the side effects of Li+ administration has been studied. Preliminary evidence shows that patients with bipolar illness have higher Li+ ratios in vivo than normal controls.4 The finding that patients who respond to lithium therapy have higher Li+ ratios than nonresponders5-7 has not been confirmed by all investigators.8-9Side effects of Li+ therapy may be more highly correlated with intracellular Li + concentrations or the Li+
The primary abnormalities that contribute to the pathogenesis of human essential hypertension are unknown. The known genetic contribution to this disorder suggests the possible use of genetic linkage analysis to test whether specific candidate genes contribute to the pathogenesis of either essential hypertension or intermediate phenotypes. Among such phenotypes, elevated erythrocyte Na(+)-Li+ countertransport (SLC) is the best known, supporting major gene inheritance by pedigree analysis. Striking similarities between SLC and Na(+)-H+ exchange suggest that mutations at the Na(+)-H+ antiporter gene locus (APNH) might result in elevated SLC and contribute to the subsequent pathogenesis of hypertension. We have tested these hypotheses by genetic linkage analysis, with APNH as a candidate gene. By determining genotypes at APNH and flanking loci in pedigrees that support major gene segregation of elevated SLC, we have excluded linkage of APNH and the major SLC locus with a LOD score of -5.91, an odds ratio of almost 1,000,000:1 against linkage. In the analysis of 93 hypertensive sibling pairs, we have further demonstrated that APNH explains none of the variance in SLC in hypertensive individuals (r2 = 6 x 10(-7), p greater than 0.99). Finally, we have directly tested for linkage of APNH to genes predisposing toward hypertension by linkage in hypertensive sibling pairs. Mean allele sharing at APNH is not greater than expected from random assortment in hypertensive siblings (0.92 versus 1.0, p greater than 0.80), and the upper 95% confidence limit of this value (1.04) indicates that mutations at APNH rarely if ever contribute to the pathogenesis of hypertension in this population.(ABSTRACT TRUNCATED AT 250 WORDS)
Human essential hypertension is a family of diseases; one subtype has an increased maximum velocity for red blood cell lithium-sodium countertransport activity. To begin the localization of the gene or genes responsible for this phenotype, we examined the association of blood pressure, lithium-sodium countertransport, and two genetic markers previously associated with hypertension--the MN blood group antigen (chromosome 4) and the plasma protein haptoglobin (chromosome 18)--in a population-based sample of 592 young adults from Tecumseh, Mich., the site of an ongoing cardiovascular epidemiological investigation. Our results suggest that the relation between MN phenotype and systolic blood pressure is significantly different and oppositely directed in men and women. Analysis of data available from previous examinations revealed that similar blood pressure differences related to MN phenotype had been present at least a decade earlier in both men and women. There also was a significant relation between systolic blood pressure and haptoglobin phenotype for the combined group of men and women. In addition to having high systolic blood pressure, men with the MM phenotype had significantly elevated red blood cell lithium-sodium countertransport activity. In studies of brother-brother pairs, we found evidence for significant genetic linkage between the MN locus and red blood cell lithium-sodium countertransport activity.
Susceptibility to diabetic nephropathy may be related to a predisposition to arterial hypertension. We have studied the activity of sodium-lithium countertransport in red cells, a marker of risk for essential hypertension, in white European adults with insulin-dependent diabetes and diabetic nephropathy, a matched group of patients with diabetes without renal disease, and nondiabetic patients with renal disease. Measures of metabolic control and concentrations of plasma free insulin and growth hormone were similar in the two diabetic groups. The degree of impairment in renal function was similar in the diabetic and nondiabetic patients with renal disease. Body-mass index and plasma potassium concentrations were similar in all three groups. Diastolic blood pressure was elevated to a similar degree in the two groups with renal disease, as compared with that in the diabetic patients without renal disease. The rates of sodium-lithium countertransport in red cells were significantly higher in the diabetic patients with renal disease (mean +/- SD, 0.55 +/- 0.19 mmol of lithium per liter of red cells per hour) than in the diabetic patients without renal disease (0.33 +/- 0.16; P less than 0.005) and in the nondiabetic patients with renal disease (0.31 +/- 0.14; P less than 0.001). Predisposition to hypertension, as indicated by elevated sodium-lithium countertransport activity in red cells, may serve as a marker for the risk of renal disease in patients with insulin-dependent diabetes.
Red-cell lithium-sodium countertransport is increased in patients with essential hypertension. It has been proposed that sodium-hydrogen ion exchange in the brush border of the renal proximal tubules is analogous to red-cell countertransport. To investigate the rate of sodium reabsorption by the proximal renal tubules in hypertension, we measured lithium clearance (a measure of proximal tubular reabsorption of sodium), as well as red-cell countertransport, in 14 patients with untreated essential hypertension and in 31 controls. As a group, the hypertensive patients had a higher average (+/- SEM) rate of red-cell countertransport (0.378 +/- 0.030 mmol of lithium per liter of cells per hour, P less than 0.01) and a lower renal fractional lithium clearance (13.96 +/- 0.69 percent, P less than 0.01) than normotensive subjects (0.317 +/- 0.015 mmol of lithium per liter of cells per hour and 17.75 +/- 0.81 percent, respectively). Within the normotensive group, subjects with hypertension in at least one first-degree relative had significantly lower fractional lithium clearances than subjects with no hypertensive relatives (15.37 +/- 0.84 percent vs. 19.06 +/- 1.07 percent, P less than 0.05). We conclude that hypertensive patients have heightened proximal tubular reabsorption of sodium and that red-cell countertransport is a marker of the renal abnormality. Enhanced proximal tubular sodium reabsorption may precede the development of essential hypertension.
The intracellular pH in animal cells in generally maintained at a higher level than would be expected if H+ were passively distributed across the plasma membrane. In a wide variety of cells including sea urchin eggs, skeletal muscle, renal and intestinal epithelial cells, and neuroblastoma cells, plasma membrane Na+-H+ exchangers mediate the uphill extrusion of H+ coupled to, and thus energized by, the downhill entry of Na+. Plasma membrane vesicles isolated from the luminal (microvillus, brush border) surface of renal proximal tubular cells possess a Na+-H+ exchanger that seems to be representative of the Na+-H+ exchangers found in other tissues. For example, the renal microvillus membrane Na+-H+ exchanger, like other Na+-H+ exchangers, mediates electroneutral cation exchange, is sensitive to inhibition by the diuretic drug amiloride, and has affinity for Li+ in addition to Na+ and H+ (refs 5, 9). Here we have examined the effect of internal H+ on the activity of the Na+-H+ exchanger in renal microvillus membrane vesicles. Our results suggest that internal H+, independent of its role as a substrate for exchange with external independent of its role as a substrate for exchange with external independent of its role as a substrate for exchange with external Na+, has an important modifier role as an allosteric activator of the Na+-H+ exchanger. Allosteric behaviour with respect to internal H+ is a property that would enhance the ability of plasma membrane Na+-H+ exchangers to extrude intracellular acid loads and thereby contribute to the regulation of intracellular pH.
This paper describes experiments showing that one of the pathways of sodium transport across the red-cell membrane, sodium-lithium countertransport, is faster in patients with essential hypertension than in control subjects. This transport system accepts only sodium or lithium and is not inhibited by ouabain. The maximum rate of transport shows inherited differences. The mean maximum rate of sodium-lithium countertransport was found to be 0.55 +/- 0.02 (mean +/- S.E.M.) mmol (liter of red cells X hour)(-1) in a group of 36 patients with essential hypertension and 0.24 +/- 0.02 in 26 control subjects (P less than 0.001). The first-degree relatives of eight patients with essential hypertension and 10 control subjects had mean maximum rates of sodium-lithium countertransport of 0.54 +/- 0.05 and 0.23 +/- 0.02, respectively. Five patients with secondary hypertension had normal mean maximum rates of sodium-lithium countertransport. The relation between heritability of red-cell sodium-lithium countertransport and essential hypertension should be investigated further.
The importance of negatively charged residues in transmembrane helices of many cation-coupled transporters has been widely demonstrated. Four Asp residues were located in the putative transmembrane helices of the Escherichia coli Na+/H+ antiporter, NhaA. We replaced each of these Asp residues by Asn in plasmid encoded nhaA and expressed these constructs in an E. coli mutant defective in both nhA and nhaB. Substitution of Asp-65 or Asp-282 (in the extramembrane region) had no effect on supporting the host mutant growth in the high NaCl- or LiCl-containing medium, and these two mutants had normal Na+/H+ and Li+/H+ antiporter activities. In contrast, substitution of Asp-133, Asp-163 or Asp-164 was detrimental to survival of the host mutant and impaired both Na+/H+ and Li+/H+ antiporter activities. These three Asp residues, conserved in the nhaA homologs from different species and which are located closely in the 3rd and 4th putative transmembrane helices, appear to play important roles in cation binding and transport.
In this paper we report some results of our studies on patients with immunoglobulin (Ig)A nephropathy regarding (1) the familiar aggregation of erythrocyte sodium-lithium (Na,Li) countertransport; (2) the association of Na,Li countertransport with the presence of arterial hypertension and lipid abnormalities; (3) the correlation between Na,Li countertransport activity and renal functional reserve; and (4) the preliminary results of a longitudinal study. In 13 families of patients with IgA nephropathy, selected because both parents were available, we found a significant correlation between midparent and offspring Na,Li countertransport activity (Spearman's rank correlation = 0.65; P = 0.023), but no husband-wife relationship. In 49 patients, the activity of Na,Li countertransport was significantly higher in erythrocytes from 20 hypertensive patients than from either 29 normotensive patients or from 36 healthy age- and sex-matched normal subjects. Hyperlipidemic patients had an erythrocyte Na,Li countertransport activity significantly higher than normolipidemic patients and controls. In 17 patients a significant inverse correlation was found between the peak variation of creatinine clearance over baseline value after an oral protein load and the erythrocyte Na,Li countertransport activity (Spearman r = 0.54; P = 0.03). In a longitudinal study of 36 patients followed from 12 to 36 months, those showing a progression toward renal failure had an erythrocyte Na,Li countertransport activity higher than median value. The results of our studies show that in patients with IgA nephropathy a high erythrocyte Na,Li countertransport rate, genetically determined, is associated with the presence of arterial hypertension and lipid abnormalities, and perhaps with a less favorable disease outcome.
An elevated red blood cell (RBC) sodium-lithium countertransport (Na-Li CT) is associated with high blood pressure (BP) in cross-sectional investigations; however, its value as a predictor of future hypertension, and thus of cardiovascular risk, has not been defined. The present study evaluated the association between Na-Li CT and risk of future hypertension in a sample of 106 untreated normotensive middle-aged men participating in the Olivetti Prospective Heart Study in southern Italy. BP, anthropometric and metabolic variables, and RBC Na-Li CT were measured at baseline in 1987 and at a follow-up visit in 1994 through 1995. Na-Li CT was stable over time (r=0.85) and was significantly associated to systolic BP in both visits. Of the 106 initially normotensive participants, 14 were found to be hypertensive at the 8-year follow-up examination. Eleven of these 14 hypertensives were in the highest tertile of systolic BP at baseline, and 9 of 11 also had an elevated baseline Na-Li CT. In multiple logistic regression analysis, baseline BP, Na-Li CT, and age were all significant predictors of the risk of future hypertension. Individuals with baseline systolic BP in the highest tertile had a 60% risk of developing hypertension if their Na-Li CT was also high, whereas their risk was only 5% if Na-Li CT was in the two lowest tertiles (P=0.003). RBC Na-Li CT was a valuable predictor of subsequent hypertension in middle-aged men with a high-normal BP level for their age.
Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 micrograms/ml, respectively) and also increased K'[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+ activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+ exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.
Pmr1, a novel member of the family of P-type ATPases, localizes to the Golgi compartment in yeast where it provides Ca(2+) and Mn(2+) for a variety of normal secretory processes. We have previously characterized Ca(2+) transport in isolated Golgi vesicles, and described an expression system for the analysis of Pmr1 mutants in a yeast strain devoid of background Ca(2+) pump activity [Sorin, A., Rosas, G., and Rao, R. (1997) J. Biol. Chem. 272, 9895-9901]. Here we show, using recombinant bacterial fusions, that an N-terminal EF hand-like motif in Pmr1 binds Ca(2+). Increasing disruptions of this motif led to progressive loss of pump function; thus, the single point mutations D51A and D53A retained pump activity but with drastic reductions in the affinity for Ca(2+) transport, while the double mutant was largely unable to exit the endoplasmic reticulum. In-frame deletions of the Ca(2+)-binding motif resulted in complete loss of function. Interestingly, the single point mutations conferred differential affinities for transport of Ca(2+) and Mn(2+) ions. Further, the proteolytic stability of the catalytic ATP-binding domain is altered by the N-terminal mutations, suggesting an interaction between these two regions of polypeptide. These studies implicate the N-terminal domain of Pmr1 in the modulation of ion transport, and may help elucidate the role of N-terminal metal-binding sites of Cu(2+)-ATPases, defective in Wilson and Menkes disease.
The genes involved in the regulation of cellular sodium transport characteristics, which are correlated with some forms of essential hypertension, have not yet been identified. We are studying the genes and environmental factors that affect red blood cell sodium-lithium countertransport (SLC) activity and intracellular sodium (ICNa) concentration in 634 baboons that comprise 11 pedigrees of 2 and 3 generations each. To detect and locate possible quantitative trait loci (QTLs) that affect SLC activity and ICNa concentration, we performed a genome screen by using a maximum likelihood-based variance-components linkage analysis program (SOLAR). SLC and ICNa phenotypes as well as genotypes on 281 microsatellite loci were available for all pedigreed animals. Both SLC and ICNa traits were highly heritable (residual heritability 0.593+/-0.083 [P<0.0001] and 0.739+/-0.082 [P<0.0001], respectively). We obtained evidence that a possible QTL for SLC activity is located on the baboon homologue of human chromosome 4 between D4S2456 and D4S2365 with a maximum multipoint lod score of 9.3 (P<10(-)(10)) near D4S1645. This QTL accounts for approximately two thirds of the total additive genetic variation in SLC activity in baboons. Although ICNa concentration was highly heritable, we found no evidence for linkage to a QTL with use of this methodology. Thus, we have evidence that a gene located on the baboon homologue of human chromosome 4 (baboon chromosome 5) affects cell sodium transport in baboons.
Na+/H+ antiporters are membrane proteins that play a major role in pH and Na+ homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants. The emerging genomic sequence projects already have started to reveal that the Na+/H+ antiporters cluster in several families. Structure and function studies of a purified antiporter protein have as yet been conducted mainly with NhaA, the key Na+/H+ antiporter of Escherichia coli. This antiporter has been overexpressed, purified and reconstituted in a functional form in proteoliposomes. It has recently been crystallized in both 3D as well as 2D crystals. The NhaA 2D crystals were analyzed by cryoelectron microscopy and a density map at 4 Å resolution was obtained and a 3D map was reconstructed. NhaA is shown to exist in the 2D crystals as a dimer of monomers each composed of 12 transmembrane segments with an asymmetric helix packing. This is the first insight into the structure of a polytopic membrane protein. Many Na+/H+ antiporters are characterized by very dramatic sensitivity to pH, a property that corroborates their role in pH homeostasis. The molecular mechanism underlying this pH sensitivity has been studied in NhaA. Amino acid residues involved in the pH response have been identified. Conformational changes transducing the pH change into a change in activity were found in loop VIII–IX and at the N-terminus by probing trypsin digestion or binding of a specific monoclonal antibody respectively. Regulation by pH of the eukaryotic Na+/H+ antiporters involves an intricate signal transduction pathway (recently reviewed by Yun et al., Am. J. Physiol. 269 (1995) G1–G11). The transcription of NhaA has been shown to be regulated by a novel Na+-specific regulatory network. It is envisaged that interdisciplinary approaches combining structure, molecular and cell biology as well as genomics should be applied in the future to the study of this important group of transporters.
Elevated erythrocyte Na+- Li+ countertransport (SLC) rates are commonly found in essential hypertension. We have recently shown that human skin fibroblasts functionally express a phloretin-sensitive Na+-H+ exchange (NHE) which may also be similar to erythrocyte SLC because of amiloride-insensitivity.
We investigated whether elevations in fibroblast SLC parallel the known elevations in erythrocyte SLC and in cell NHE that characterize essential hypertension.
Higher fibroblast SLC rates were found among hypertensive patients (n = 23, median 48.8 nmol Li+/ mg(protein) per min) than in 19 normotensive individuals of similar age and sex (median 14.8 nmol Li+/mg(protein) per min, P= 0.0002). As expected, erythrocyte SLC was elevated in patients with hypertension (median 411 versus 329 micromol/l(cell) per h, P= 0.0273), but was not quantitatively related to fibroblast SLC. Finally, fibroblast NHE exchange activity was higher in essential hypertension (median Vmax 14.2 versus 7.6 mmol H+/l(cell) per min, P= 0.002), but was unrelated to fibroblast SLC.
These findings extend to human skin fibroblasts the notion of abnormal Li+ transport in essential hypertension, and appear to be in accordance with the hypothesis that fibroblast SLC may be independent of NHE. However, molecular studies will be required to understand whether distinct exchangers and/or regulation mechanisms underlie these dysregulations.
Little is known about genetic determinants explaining variation in the erythrocyte sodium-lithium countertransport (SLC), an intermediate phenotype of essential hypertension. We characterized the SLC in immortalized lymphoblasts and showed that its behavior is similar to that of erythrocyte SLC. We then performed association and linkage analyses of the SLC in immortalized lymphoblasts from 5 large pedigrees from the Center d'Etude du Polymorphisme Humain (CEPH) genomics repository. The results of these analyses showed that a number of genomic regions harboring genes involved in glutathione metabolism might explain variations in SLC activity. These findings support evidence that thiol groups play a central role in SLC activity.
An increased activity of sodium-lithium countertransport (SLC) is a common finding in patients who have essential hypertension. The evidence that a similar dysfunction is shared also by patients with type 1 diabetes and nephropathy has suggested the hypothesis that a predisposition to essential hypertension may be the factor that, along with hyperglycemia, underlies the development of diabetic nephropathy. Despite the initial enthusiasm surrounding the potential use of SLC activity as a marker for the early detection and treatment of individuals who are predisposed to hypertension and diabetic nephropathy, its use has been so far restricted to epidemiologic studies, as specificity and sensitivity of the test are still too low to justify any clinical use. The recent finding, however, that the measurement of kinetic parameters of SLC can significantly increase the power to discriminate among individuals with and without hypertension or diabetic nephropathy could be of help toward a future clinical use of the measurement of this membrane transport. A second major point relates to the possibility that SLC per se might be directly involved in the pathogenesis of essential hypertension and diabetic nephropathy. This case has never been fully tested, as the gene responsible for this membrane transport has been, until recently, unknown. The recent identification of an alternative splicing of the first isoform of Na-H exchange that mediates SLC activity should allow for a rapid comprehension of the role of this transport in the pathophysiology of essential hypertension and diabetic nephropathy.
Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae, the high K+ content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K+ from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K+/H+ antiporter. We prepared kha1 and tok1-kha1 deletion strains in the B31 and MAB 2d background. Both the strains contain the ena1-4 and nha1 deletions; that means they lack the main active sodium and potassium efflux systems. MAB 2d has additional trk1 and trk2 deletions, i.e. is impaired in active K+ uptake as well. We performed a large physiological study with these strains to specify the phenotype of kha1 deletion. In our experiments, no difference in K+ content or efflux was observed in strains lacking the KHA1 gene compared with control strains. Two main phenotype manifestations of the kha1 deletion were growth defect on high external pH and hygromycin sensitivity. The correlation between these phenotypes and the kha1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of green fluorescent protein (GFP)-tagged Kha1p showed that this antiporter is localized preferentially intracellularly (in contrast to the plasma membrane Na+/H+ antiporter Nha1p). Based on these findings, Kha1p is probably not localized in plasma membrane and does not mediate efflux of alkali metal cations from cells, but is important for the regulation of intracellular cation homeostasis and optimal pH control, similarly as the Nhx1p.
Increased erythrocyte sodium-lithium countertransport (SLC) has been observed in patients with essential hypertension. An analytic strategy for identification of genetic variation contributing to hypertension is the evaluation of appropriate intermediate phenotypes for hypertension, such as SLC. Thus, in this study, genome-wide linkage scans for SLC were performed in two independent samples of pedigrees from the Rochester Family Heart Study (RFHS).
Genome-wide linkage scans for SLC were performed in independent samples of 232 and 252 non-Hispanic white families from the RFHS. Multipoint variance-component linkage analysis was performed using MERLIN.
Chromosomes 8, 9, 10, 19, and 20 contained evidence for linkage (log of the odds [LOD] > or =2) in at least one sample of RFHS pedigrees. Consistent evidence of linkage for SLC between the two samples was observed on chromosome 10 (LOD = 2.02 at 64 cM in the first sample and LOD = 2.27 at 55 cM in the second sample).
Consistent evidence of linkage (LOD > or =2) for SLC was observed in two independent samples of non-Hispanic white families on chromosome 10. Concordance of linkage evidence between the two samples provides confidence that a region on chromosome 10 contains genetic variation influencing SLC, which may potentially influence susceptibility to hypertension.
The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H+ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na+/H+ but not K+/H+ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na+ and K+ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb+ and Li+. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na+/H+ antiporters, whereas other known eukaryotic Na+/H+ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na+/H+ antiporters.
Drug and multidrug resistance have greatly compromised the compounds that were once the mainstays of antibiotic therapy. This resistance often persists despite reductions in the use of antibiotics, indicating that the proteins encoded by antibiotic-resistance genes have alternative physiological roles that can foster such persistence in the absence of selective pressure by antibiotics. The recent observations that Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both confer alkali tolerance offer a striking case study in support of this hypothesis.
Sodium-lithium countertransport (SLC) is an ouabain-insensitive exchange of Na for Li found in the erythrocyte membrane of several mammalian species. Although increased SLC activity is presently the most consistent intermediate phenotype of essential hypertension and diabetic nephropathy in humans, the gene responsible for this membrane transport has not been identified. Because of functional similarities, SLC was suggested to represent an in vitro mode of operation of the Na-H exchanger (NHE). This hypothesis, however, has been long hampered by the total insensitivity of SLC to amiloride, which is an intrinsic inhibitor of the first isoform of NHE, the only NHE isoform detected in human erythrocytes. We describe here the identification in human reticulocytes and erythrocytes of an alternative splicing of NHE lacking the amiloride binding site. Transfection experiments with this spliced variant restore amiloride-insensitive, phloretin-sensitive SLC activity. Expression of both regular and spliced transcripts of NHE is increased in subjects with high SLC activity. Altogether, these findings, by extending to NHE the characteristics of inheritance and predictivity previously attributed to SLC, eventually restore the candidacy of NHE isoform 1 as a gene involved in the pathogenesis of essential hypertension and diabetic nephropathy.
There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.
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