All living cells are critically dependent on homeostatic mechanisms that regulate intracellular pH, Na+ content, and, as a result, cell volume. Correspondingly, Na+ and H+ are among the most prevalent ions in living cells and are essential in cell bioenergetics. In 1974, West and Mitchell discovered sodium proton antiport activity in bacterial cells and suggested that Na+/H+ antiporter proteins have primary roles in the homeostasis of these cations (West and Mitchell 1974). Since then, sodium proton antiporters have been identified in the cytoplasmic and organelle membranes of almost all cells, including those of plants, animals and microorganisms. Furthermore, increasing numbers of these antiporters are being identified as human drug targets.
The SLC9 gene family 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 crystal structure of downregulated Ec-NhaA, determined at acidic pH, has provided the first structural insights into the antiport mechanism and pH regulation of an Na+/H+ antiporter (Hunte et al. 2005). Thus far, there is no crystal structure of any of the human Na+/H+ antiporters. Nevertheless, the Ec-NhaA crystal structure has enabled the structural modeling of NHE1, NHE9, and NHA2, three human plasmalemmal proteins that are members of the SLC9 family involved in human pathophysiology. Moreover, developments in the field, including cellular and biophysical methods that enable ion levels and fluxes to be measured in intact cells as well as in knockout mice, have led to advances in the identification and characterization of the transporters. For a more detailed reference list, see Padan and Landau (2016).
Phylogenic Classification of NHE Na+/H+ Antiporters
The NHE Na+/H+ antiporters are ubiquitous ion transporters, present in all kingdoms of life. Currently, 13 evolutionarily conserved NHE isoforms are known in mammals. Most species possess multiple Na+/H+ antiporters, which may serve redundant and/or specific functions (Donowitz et al. 2013).
The NHE Na+/H+ exchangers are encoded by the solute carrier family 9 (SLC9) gene family (HUGO nomenclature, http://www.genenames.org) and are a subgroup of the eukaryotic and prokaryotic monovalent cation/proton antiporter 1 (CPA1) family (transport protein database http://www.tcdb.org). The primary structures of the encoded proteins vary considerably in both sequence identity (13%–68% amino acid identity) and size (48–99 kDa).
On the basis of phylogeny, the SLC9 gene family is divided into three subgroups (http://www.bioparadigms.org). The SLC9A subgroup encompasses plasmalemmal isoforms NHE1-5 (SLC9A1-5) and the predominantly intracellular isoforms NHE6-9 (SLC9A6-9). The SLC9B subgroup consists of NHA1 and NHA2. The SLC9C subgroup consists of a sperm-specific plasmalemmal NHE (SLC9C1), as well as SLC9C2, a putative NHE for which functional data are currently lacking (Donowitz et al. 2013).
NHE (SLC9As) Function and Distribution
NHE1 exists in the plasma membranes of almost all mammals and uses the chemical energy of the Na+ gradient maintained by the Na+/K+ ATPase across the plasma membrane for electroneutral counter transport of H+. It is thereby imperative for regulation of intracellular pH, salt concentration, and cell volume (Donowitz et al. 2013; Wakabayashi et al. 2013). It can operate reversibly. NHE1 can exchange Li+ for Na+, but other alkali cations are excluded. NHE1 is the most abundant antiporter in the heart. It has long been a drug target because it has been suggested to play a role in heart pathology, hypertrophy, cardiac ischemia, and hypertension. NHE1 is highly sensitive to the drug amiloride and to lipophilic amiloride derivatives including ethylisopropylamiloride (EIPA), benzoylguanidines HOE694, and capiroide.
In contrast to the ubiquitous expression of NHE1, NHE2–NHE5 show a restricted expression pattern. For example, NHE2–NHE4 are predominantly expressed in the epithelial cells of the kidney, small intestine, and stomach, and NHE5 is mainly expressed in the brain. NHE2 has been suggested to be involved in intestinal and renal Na+ absorption and possibly also in repair of epithelial damage. Most recently, NHE2 expression was shown to be localized in the pituitary, with functional implications. NHE3 is one of the most highly regulated transport proteins, present in the Na+ absorptive cells of mammalian small intestine, colon, gall bladder, renal proximal tubule, and thick and thin limbs of the loop of Henle, and is responsible for the majority of intestinal and renal Na+ absorption (Donowitz et al. 2013). NHE3, in contrast to NHE1, is highly resistant to amiloride and its derivatives. NHE4 is localized to the basolateral membrane of epithelial cells and its expression is highest in the stomach and is also found, although at lower levels, in the kidney medulla, hippocampus, zymogen granules of the pancreas, and salivary gland. NHE5 expression is highly restricted to the brain and sperm, although it is also observed in the spleen and skeletal muscle. NHE5 has high homology with NHE3 (50% amino acid identity) and shares similar pharmacological, regulatory, and cell localization properties. Despite being classified as plasmalemmal, NHE3 and NHE5 are also present in internal membranes, specifically in recycling endosomes, and they cycle between the two pools.
Very little is known about the biochemistry of intracellular endomembrane isoforms of NHE: NHE6, NHE7, and NHE9. These intracellular exchangers may serve a function in cation homeostasis and/or osmoregulation, and not in pH regulation as is the case for the plasmalemmal isoforms. NHE6, NHE7, and NHE9 are considered to be derived evolutionarily from the Saccharomyces cerevisiae Nhx1, the first member of the NHE family to be molecularly identified. In contrast to the transport of plasmalemmal NHEs, active transport by endosomal NHE is driven by the H+ gradient generated by the V-type H+-ATPase, resulting in cation (Na+ or K+) sequestration coupled to removal of protons from the compartmental lumen. Hence, the plasma membrane and endosomal subtypes have different ion selectivity, with the former being Na+ selective, whereas most intracellular isoforms transport both K+ and Na+ ions with similar Vmax and Km.
On the basis of phylogenetic analysis, NHE8 has been predicted to encode an intracellular NHE and shares about 25% homology with other members of the gene family. NHE8 is ubiquitously expressed in human tissues, with the highest expression in the skeletal muscle and kidney, and some expression in the intestine. Both NHE3 and NHE8 are developmentally regulated. NHE8 seems to be the major intestinal brush border NHE in neonates, and NHE3 is the predominant brush border NHE in adults.
NHEs in Human Physiology and Disease
NHE1-5 participate in the regulation of cytosolic and organellar pH, cation composition, and cell volume, thereby contributing to the creation of environments suitable for cell function and survival. A bewildering collection of physiological agonists and antagonists regulates the NHEs, in most cases via the C-terminal domain (Orlowski and Grinstein 2011). An interesting example is that many agents that stimulate NHE1 inhibit NHE3, and vice versa. NHE2 generally responds in a manner akin to NHE1, while the limited information available suggests that NHE5 shares regulatory properties with NHE3. The regulation can be targeted to changes in the proton set point, protein exchange activity, protein expression, redistribution in different membranes, and protein synthesis and stability. NHE6 and NHE9 are currently the only NHEs directly linked to human disease. Mutations in these genes cause neurological disease in humans. However, it is becoming increasingly apparent that members of SLC9 gene family contribute to the pathophysiology of multiple human diseases. Certain important functions in cell physiology are accomplished by cooperation of several NHEs (Orlowski and Grinstein 2011). This should be taken into account when seeking to identify specific NHEs as drug targets. Mutations, knockout mice, and use of NHE inhibitors are highly instructive in the study of the physiopathological roles of a transporter.
Loss of NHE1 in mice was compatible with embryogenesis but was associated with a lower rate of postnatal growth and higher mortality (Bell et al. 1999). In addition, knockout mice suffered from ataxia and epileptic seizures. This phenotype was associated with brain and other neural damage. NHE1 inhibitors afford significant protection against ischemia–reperfusion cardiac injuries in animal models and in patients undergoing coronary interventions, suggesting that these injuries are mediated by NHE1 (Wakabayashi et al. 2013). Overexpression of NHE1 also exaggerates damage following myocardial ischemia and reperfusion. Accordingly, pharmacologic inhibition of NHE1 during episodes of ischemia–reperfusion has been shown to mitigate cardiac and neural injuries both in vivo and in vitro in rodents and pigs. NHE1 is also implicated in the cardiac hypertrophy and heart failure resulted from postinfarction remodeling. Thus, NHE1 was suggested as a promising drug target, protecting the myocardium from various diseases. However, clinical trials in humans have not been successful so far, showing no overall benefit of NHE1 inhibition by cariporide in acute coronary syndromes and no benefits in patients with acute myocardial infarction. The failure to obtain practical benefit from NHE1 inhibitors in humans is largely due to a lack of inhibitor specificity (Wakabayashi et al. 2013). Hence, increased knowledge of the atomic structure of NHE1 may facilitate the development of improved inhibitors for clinical use. NHE1 was also implicated in pathogenesis of cancer probably related to its fundamental role in cell migration.
Linkage of NHE3 to human diseases is limited, although animal models suggest likely clinical implications in metabolic acidosis and alkaline urine and absorptive defects of both sodium and water in both intestinal and renal tubular epithelia. NHE3 is also implicated in Ca+2 homeostasis, providing the driving force for Ca+2 absorption from renal and intestinal epithelia. In addition, NHE3 has been found to play a role in intestinal inflammation and is a target for treatment of acute diarrhea.
NHE5 is of particular interest in the nervous system. Knockdown of NHE5, or expression of its dominant-negative E209 mutant, results in spontaneous spine outgrowth. It is therefore suggested that NHE5 controls dendritic spine growth via a pH-dependent negative feedback mechanism. Increased activity of NHE5 results in alkalinization of the dendritic spine and concomitant acidification of the synaptic cleft. This may serve as an autocrine feedback mechanism that regulates pH-sensitive proteins at the synapse. Hence, NHE5 may be a very important pharmacological target.
The family of intracellular exchangers, NHE6, NHE7, and NHE9, which reside on endosomal and recycling compartments and regulate luminal pH and control vesicular trafficking, have been implicated in a range of neuropsychiatric disorders (Milosavljevic et al. 2014). Mutations in the NHE6 or NHE9 genes cause neurological disease in humans (Gilfillan et al. 2008). These are currently the only NHEs directly linked to human disease. NHE6 is encoded by the X-chromosome both in mice and in humans. Nonsense and missense mutations, as well as deletions in the SLC9A6 gene, have been observed (Gilfillan et al. 2008; Garbern et al. 2010). These cause three phenotypes in humans: (a) X-linked Angelman syndrome, characterized by intellectual disability, microcephaly, epilepsy, ataxia, and behavioral abnormalities (Gilfillan et al. 2008), (b) an Angelman-like syndrome known as Christianson syndrome (Christianson et al. 1999), and (c) a syndrome presenting with corticobasal degeneration with severe intellectual disability and autistic behavior (Garbern et al. 2010). The abnormal accumulation of glutamate in the brain of patients with NHE6 mutations is indicative of an underlying problem in glutamate clearance from the synapse (Gilfillan et al. 2008). Glutamate is excitotoxic at high concentrations, and aberrant glutamate levels are associated with neurological abnormalities of epilepsy and cerebellar degeneration, both characteristic symptoms of Angelman-like syndrome associated with NHE6 mutations (Gilfillan et al. 2008). The bulk of glutamate released into the neuronal synapse is cleared by rapid uptake into astrocytes, the single largest population of cells in the brain. Interestingly, NHE6 knockout mice have no obvious phenotype, even at older ages (Stromme et al. 2011). Extensive behavioral testing revealed that certain histological abnormalities result in mild motor hyperactivity and deficits in motor coordination in the mutant mice. Studying the same knockout mouse model, Morrow and coworkers found that NHE6-deficient neurons exhibit over-acidified endosomes, leading to disruption of endosomal signaling and to neuronal endolysosomal storage disease with cell death and impairment of the wiring of neuronal circuits (Schwede et al. 2014).
Genetic approaches have identified NHE9 as a candidate gene in attention deficit hyperactivity disorder (ADHD), addiction, autism, and mental retardation (Morrow et al. 2008). However, it cannot be excluded that variants associated with autism or ADHD represent benign polymorphisms (Gilfillan et al. 2008). Remarkably, the functional consequences of human NHE9 missense mutations in astrocytes have recently been clarified (Kondapalli et al. 2013). Overexpression of wild-type NHE9 but not of NHE9 mutants in astrocytes caused endosomal alkalinization and enhanced transferrin and glutamate uptake, indicating that the three missense mutations found in human NHE9 are loss-of-function mutations. Kondapalli and colleagues (Kondapalli et al. 2013) have developed a neurobiological model that establishes a role for NHE9.
Topology and Structural Models of Human Na+/H+ Antiporters
Structural information is important for describing the translocation process and regulation of this important antiporter family. Thus far, there are no available structures for any of the SLC9 family members, except for structures of short segments of NHE1. Nevertheless, it is possible to conclude, on the basis of a large volume of biochemical data, that all NHEs are organized in a similar fashion: A very short N-terminus facing the cytoplasm followed by an N-terminal domain composed of 11–14 transmembrane segments (TMs) (∼450 residues) that carries out the Na+/H+ exchange, and an intracellular C-terminal domain (∼125–440 residues, depending on the isoform) that is involved in regulation of the exchange activity. The C-terminal domain is suggested to associate with many regulatory factors. All SLC9 members appear to exist as dimers, while transport function occurs in a monomer. The dimerization is believed to provide stability.
Experimental structural information on NHE1 has been obtained only for segments of the protein. Structures of single or two putative TMs of the membrane domain were determined using nuclear magnetic resonance (NMR) (PDB IDs 2MDF, 2L0E, 2KBV). While much of the required structural information is contained within the primary amino acid sequences of the TM segments, interactions between TM segments can only partially be studied with single segments. Additionally, in some cases, the interactions between helices and the presence or absence of inter-helical loops can affect the conformation of TM segments. The C-terminus of NHE1 extends into the cytoplasm and is the least conserved region among the NHE family members (ranging in length from ∼50–450 amino acids). Fractions of its soluble domain were analyzed by NMR (PDB ID 2E30) or by crystallography (PDB IDs 2BEC and 2YGG) and have paved the way to better understanding of NHE1 regulation.
As structure determination for eukaryotic proteins remains highly challenging, experiment-based and theoretical models have been employed. Of note, even the membrane topology of NHE1 and other antiporters is still debatable. A topology model of the translocation module of NHE1 has been suggested on the basis of hydropathy plots and Cys scanning accessibility tests (Wakabayashi et al. 2000, 2013). The constructed model, called herein the Wakabayashi model, shows 12 TMs (designated here I–XII) with Nin–Cin topology. TM X in the hydrophobicity plot has extracellularly accessible residues on both ends and therefore was proposed to be a reentrant loop rather than a TM. Two intracellular loops, TMs IV–V and TMs VIII–IX, were also suggested to be re-reentrant loops.
The Landau model is compatible with evolutionary conservation analyses of Na+/H+ Exchangers (Fig. 1). Namely, the evolutionarily conserved amino acids are located in strategic regions at the interfaces between the TM segments, whereas variable residues face the membrane lipids. The extra-membranal loops are also enriched in variable amino acids. The Landau model is also supported by observations of conserved charged residues in the core of the protein, at the extended chain region. For example, D238 is thought to be equivalent to D133 in Ec-NhaA, compensating for the helix-end dipole charges. D163 and D164, which, in Ec-NhaA, are essential and bind ligands, are replaced with N266 and D267 in NHE1. In addition, NHE1 contains conserved amino acid clusters facing extracellularly, which are important in pH regulation and inhibition of NHE. Moreover, the model is consistent with mutagenesis studies, showing that many essential residues are located at the core of the transmembrane domain (Fig. 2). Finally, the Landau model is also consistent with Gunnar von Heijne and coworkers’ positive-inside rule.
Na+/H+ antiporters contribute to the creation of environments suitable for cell function and survival. Because NHEs are critical for maintaining fundamental processes in cellular physiology, such as homeostasis of intracellular pH, salt concentration, and cell volume (Donowitz et al. 2013; Wakabayashi et al. 2013), a change in its activity is detrimental to the cell. Future progress toward gaining an understanding of the SLC9 gene family, including its structure–function relationships and regulatory mechanisms in health and in disease, is likely to include insights into the pathophysiology of multiple diseases.
- Christianson AL, Stevenson RE, van der Meyden CH, Pelser J, Theron FW, van Rensburg PL, et al. X linked severe mental retardation, craniofacial dysmorphology, epilepsy, ophthalmoplegia, and cerebellar atrophy in a large South African kindred is localised to Xq24-q27. J Med Genet. 1999;36:759–66.PubMedPubMedCentralCrossRefGoogle Scholar
- Gilfillan GD, Selmer KK, Roxrud I, Smith R, Kyllerman M, Eiklid K, et al. SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome. Am J Hum Genet. 2008;82:1003–10. doi:10.1016/j.ajhg.2008.01.013.PubMedPubMedCentralCrossRefGoogle Scholar