Na+ /H+ Exchanger in Tissues of Lower Vertebrates

Na⁺/H⁺ exchange is one of the major pathways of ion transport in cells of pro- and eukaryots and plays an important role in intracellular pH and cell volume regulation, in cell division, proliferation, as well as in epithelial transport processes. Since 1989, investigations on the molecular nature of this transporter have revealed six isoforms (NHE1–NHE6) in mammalian tissues. Most works on studies of properties of the Na/H antiporter and regulation of its activity have been carried out on mammalian tissues. This review summarizes results of studies on the Na⁺/H⁺ exchange in tissues of lower vertebrates. Of the greatest interest are investigations on the rainbow trout, whose erythrocytes were found to contain a Na⁺/H⁺ exchanger activated by catecholamines. This carrier in trout erythrocytes has been cloned and called beta-NHE ( ;NHE). Another exchanger isoform, atNHE, was isolated from the red blood cells of the giant salamander Amphiuma tridactulum. Isoforms of antiporter isolated from oocytes (XL-NHE) and renal cells of the clawed frog Xenopus laevis (XNHE) have also been described.     

Structural and functional analysis of the Na+/H+ exchanger

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.     

Physiological role and regulation of the Na+/H+ exchanger

In mammalian eukaryotic cells, the Na+/H+ exchanger is a family of membrane proteins that regulates ions fluxes across membranes. Plasma membrane isoforms of this protein extrude 1 intracellular proton in exchange for 1 extracellular sodium. The family of Na+/H+ exchangers (NHEs) consists of 9 known isoforms, NHE1-NHE9. The NHE1 isoform was the first discovered, is the best characterized, and exists on the plasma membrane of all mammalian cells. It contains an N-terminal 500 amino acid membrane domain that transports ions, plus a 315 amino acid C-terminal, the intracellular regulatory domain. The Na+/H+ exchanger is regulated by both post-translational modifications including protein kinase-mediated phosphorylation, plus by a number of regulatory-binding proteins including phosphatidylinositol-4,5-bisphosphate, calcineurin homologous protein, ezrin, radixin and moesin, calmodulin, carbonic anhydrase II, and tescalcin. The Na+/H+ exchanger is involved in a variety of complex physiological and pathological events that include regulation of intracellular pH, cell movement, heart disease, and cancer. This review summarizes recent advances in the understanding of the physiological role and regulation of this protein.     

Mammalian NHE2 Na(+)/H+ exchanger mediates efflux of potassium upon heterologous expression in yeast

Na(+)/H+exchangers form a broad family of transporters that mediate opposing fluxes of alkali metal cations and protons across cell membranes. They play multiple roles in different organisms (protection from toxic cations, regulation of cell volume or pH). Rat NHE2 exchanger was expressed in a Saccharomyces cerevisiae mutant strain lacking its own exporters of alkali metal cations. Though most of the overexpressed NHE2 remained entrapped in the secretory pathway, part of it reached the plasma membrane and mediated K+ efflux from the yeast. We demonstrate for the first time that a mammalian Na(+)/H+ exchanger transports alkali metal cations in yeast in the opposite direction than in mammalian cells, and that the substrate specificity of the rat NHE2 exchanger is limited only to potassium cations upon expression in yeast cells.     

Cell surface levels of organellar Na+/H+ exchanger isoform 6 are regulated by interaction with RACK1

In mammalian cells, four Na(+)/H(+) exchangers (NHE6 - NHE9) are localized to intracellular compartments. NHE6 and NHE9 are predominantly localized to sorting and recycling endosomes, NHE7 to the trans-Golgi network, and NHE8 to the mid-trans-Golgi stacks. The unique localization of NHEs may contribute to establishing organelle-specific pH values and ion homeostasis in cells. Mechanisms underlying the regulation and targeting of organellar NHEs are largely unknown. We identified an interaction between NHE9 and RACK1 (receptor for activated C kinase 1), a cytoplasmic scaffold protein, by yeast two-hybrid screening using the NHE9 C terminus as bait. The NHE9 C terminus is exposed to the cytoplasm, verifying that the interaction is topologically possible. The binding region was further delineated to the central region of the NHE9 C terminus. RACK1 also bound NHE6 and NHE7, but not NHE8, in vitro. Endogenous association between NHE6 and RACK1 was confirmed by co-immunoprecipitation and co-localization in HeLa cells. The luminal pH of the recycling endosome was elevated in RACK1 knockdown cells, accompanied by a decrease in the amount of NHE6 on the cell surface, although the total level of NHE6 was not significantly altered. These results indicate that RACK1 plays a role in regulating the distribution of NHE6 between endosomes and the plasma membrane and contributes to maintaining luminal pH of the endocytic recycling compartments.     

Structure and function of the NHE1 isoform of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cytoskeletal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.     

Na+/H+ Exchanger 1 Gene Expression in Tissues of Yellow Chicken

The Na(+)/H(+) exchanger 1 (NHE1) transmembrane protein regulates intracellular pH, cell survival, cell growth, cell differentiation and plays a critical role in the progression of some diseases, including the pathogenesis of J avian leukosis. The chicken is an ideal model to study the function of NHE1 because it has developed highly efficient Na(+)-absorptive mechanisms in its small and large intestines. To date, there has been no detailed expression analysis to determine NHE1 expression in various tissues of the chicken. We determined the mRNA and protein expression levels of avian NHE1 by real-time quantitative PCR and immunohistochemical analysis. NHE1 mRNA was detected in all chicken tissues examined. Protein expression levels varied widely among tissues and did not always correlate with mRNA expression. Determining the mRNA and protein of NHE1 expression patterns in chicken should help to delineate the NHE1 role in different tissues and its contribution to physiological and pathological processes. These data provide the basis for examining the distinct function of chicken NHE1 compared with its mammalian counterpart.     

Structural and functional characterization of transmembrane segment IX of the NHE1 isoform of the Na+/H+ exchanger

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339-363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 function. Of the active mutants, Glu(346) and Ser(351) were inhibited >70% by positively charged [2-(trimethylammonium)-ethyl]methanethiosulfonate but not by anionic [2-sulfonatoethyl]methanethiosulfonate, suggesting that they are pore lining and make up part of the cation conduction pathway. Both mutants also had decreased affinity for Na(+) and decreased activation by intracellular protons. The structure of a peptide representing amino acids 338-365 was determined by using high resolution NMR in dodecylphosphocholine micelles. The structure contained two helical regions (amino acids Met(340)-Ser(344) and Ile(353)-Ser(359)) kinked with a large bend angle around a pivot point at amino acid Ser(351). The results suggest that transmembrane IX is critical with pore-lining residues and a kink at the functionally important residue Ser(351).     

The Na+/H+ exchanger isoform 1

The Na+/H+ exchanger (NHE) isoform 1 is a ubiquitously expressed integral membrane protein which regulates intracellular pH in mammalian cells. Nine isoforms of the Na+/H+ exchanger have been identified. The isoform first discovered has two domains: an N-terminal membrane domain containing approximately 500 amino acids and a C-terminal regulatory domain containing approximately 315 amino acids. The exchanger, which resides in the plasma membrane, exchanges an intracellular proton for an extracellular sodium, thereby regulating intracellular pH. It is involved in cell growth and differentiation, cell migration, and regulation of sodium fluxes. The Na+/H+ exchanger plays an important role in myocardial damage during ischemia and reperfusion and has recently been implicated as a mediator of cardiac hypertrophy. Inhibitors of the Na+/H+ exchanger, which may prove useful in the clinical treatment of these conditions, are currently being developed and clinical trials are underway.     

Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na(+)) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na(+)/H(+) exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na(+)/H(+) exchange activity by Na(+)-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na(+)/H(+) exchange activity by >30%. Moreover, the sgk2-mediated increase in Na(+)/H(+) exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na(+) transport through NHE3 in the proximal tubule.     

Role of NHE1 in calcium signaling and cell proliferation in human CNS pericytes

The central nervous system (CNS) pericytes play an important role in brain microcirculation. Na(+)/H(+) exchanger isoform 1 (NHE1) has been suggested to regulate the proliferation of nonvascular cells through the regulation of intracellular pH, Na(+), and cell volume; however, the relationship between NHE1 and intracellular Ca(2+), an essential signal of cell growth, is still not known. The aim of the present study was to elucidate the role of NHE1 in Ca(2+) signaling and the proliferation of human CNS pericytes. The intracellular Ca(2+) concentration was measured by fura 2 in cultured human CNS pericytes. The cells showed spontaneous Ca(2+) oscillation under quasi-physiological ionic conditions. A decrease in extracellular pH or Na(+) evoked a transient Ca(2+) rise followed by Ca(2+) oscillation, whereas an increase in pH or Na(+) did not induce the Ca(2+) responses. The Ca(2+) oscillation was inhibited by an inhibitor of NHE in a dose-dependent manner and by knockdown of NHE1 by using RNA interference. The Ca(2+) oscillation was completely abolished by thapsigargin. The proliferation of pericytes was attenuated by inhibition of NHE1. These results demonstrate that NHE1 regulates Ca(2+) signaling via the modulation of Ca(2+) release from the endoplasmic reticulum, thus contributing to the regulation of proliferation in CNS pericytes.     

Mitogen-activated protein kinase-dependent activation of the Na+/H+ exchanger is mediated through phosphorylation of amino acids Ser770 and Ser771

We investigated regulation of the type 1 isoform of the Na(+)/H(+) exchanger by phosphorylation. Four specific groups of serine and threonine residues in the regulatory carboxyl-terminal tail were mutated to alanine residues: group 1, S693A; group 2, T718A and S723A/S726A/S729A; group 3, S766A/S770A/S771A; and group 4, T779A and S785A. The proteins were expressed in Na(+)/H(+) exchanger-deficient cells, and the activity was characterized. All of the mutants had proper expression, localization, and normal basal activity relative to wild type NHE1. Sustained intracellular acidosis was used to activate NHE1 via an ERK-dependent pathway that could be blocked with the MEK inhibitor U0126. Immunoprecipitation of (32)P-labeled Na(+)/H(+) exchanger from intact cells showed that sustained intracellular acidosis increased Na(+)/H(+) exchanger phosphorylation in vivo. This was blocked by U0126. The Na(+)/H(+) exchanger activity of mutants 1 and 2 was stimulated similar to wild type Na(+)/H(+) exchanger. Mutant 4 showed a partially reduced level of activation. However, mutant 3 was not stimulated by sustained intracellular acidosis, and loss of stimulation of activity correlated to a loss of sustained acidosis-mediated phosphorylation in vivo. Mutation of the individual amino acids within mutant 3, Ser(766), Ser(770), and Ser(771), showed that Ser(770) and Ser(771) are responsible for mediating increases in NHE1 activity through sustained acidosis. Both intact Ser(770) and Ser(771) were required for sustained acidosis-mediated activation of NHE1. Our results suggest that amino acids Ser(770) and Ser(771) mediate ERK-dependent activation of the Na(+)/H(+) exchanger in vivo.     

Analysis of the regulatory domain of yeast plasma membrane H+-ATPase by directed mutagenesis and intragenic suppression.

The yeast plasma membrane H+-ATPase is activated in vivo by glucose metabolism, and previous deletion analysis has shown the C-terminus of the enzyme to be involved in this regulation. Site-directed mutagenesis demonstrates that Arg909 and Thr912 at the C-terminus are important for the increase in Vmax of the ATPase induced by glucose. Other changes in kinetic parameters induced by glucose are largely independent of these amino acids. Arg909 and Thr912 form a potential phosphorylation site for calmodulin-dependent multiprotein kinase. A double mutation of Ser911 and Thr912 to Ala results in no cell growth in glucose medium and greatly reduced activation of the ATPase by glucose. Growth and activity are restored by a third mutation (Ala547----Val) at the catalytic domain, providing genetic evidence for domain interaction.     

CHP调节NHE1活性影响细胞生长和死亡

钠氢离子交换蛋白(NHE)定位于细胞膜,它的重要功能是调节细胞内pH值。钙调磷酸酶B同源蛋白(CHP)是NHE必要的活性调节亚单位。研究了NHE1结合CHP与否对细胞生长和死亡的影响。结果显示,CHP结合于NHE1细胞质调节区域之中靠近细胞膜部位,二者以疏水键结合而形成蛋白IV级结构。在细胞内pH5.4的非生理条件下,表达没有CHP结合能力的突变体NHE1-4R细胞只有表达野生型NHE细胞7.6%的最大摄取钠活性;在细胞内pH7.2的生理条件下,这个比例降至1.2%的摄取钠活性。与野生型NHE1比较,有血清时表达突变体NHE1-4R的细胞生长速度减慢;在血清饥饿时这些细胞因自身的胞浆酸性化而死亡数增加。实验结果证明,CHP是NHE1生理活性的必要调节因子,它能影响细胞生长和死亡。     

The yeast Na+/H+ exchanger Nhx1 is an N-linked glycoprotein. Topological implications

Nhx1, the endosomal Na(+)/H(+) exchanger of Saccharomyces cerevisiae represents the founding member of a newly emerging subfamily of intracellular Na(+)/H(+) exchangers. These proteins share significantly greater sequence homology to one another than to members of the mammalian Na(+)/H(+) exchanger (NHE) family encoding plasma membrane Na(+)/H(+) exchangers. Members of both subtypes are predicted to share a common organization, with an N-terminal transporter domain of transmembrane helices followed by a C-terminal hydrophilic tail. In the present study, we show that Nhx1 is an asparagine-linked glycoprotein and that the sites of glycosylation map to two residues within the C-terminal stretch of the polypeptide. This is the first evidence, to date, for glycosylation of the C-terminal region of any known NHE isoform. Importantly, the mapping of N-linked glycosylation to the C-terminal domain of Nhx1 is indicative of an unexpected membrane topology, particularly with regard to the orientation of the tail region. Although one recent study demonstrated that certain epitopes in the C-terminal domain of NHE3 were accessible from the exoplasmic side of the plasma membrane (Biemesderfer, D., DeGray, B., and Aronson, P. S. (1998) J. Biol. Chem. 273, 12391-12396), numerous other studies implicate a cytosolic disposition for the hydrophilic C-terminal tail of plasma membrane NHE isoforms. Our analysis of the glycosylation of Nhx1 is strongly indicative of residence of at least some portion of the hydrophilic tail domain within the endosomal lumen. These findings imply that the organization of the tail domain may be more complex than previously assumed.     

Regulation and characterization of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous protein present in all mammalian cell types that functions to remove one intracellular H+ for one extracellular Na+. Several isoforms of the protein exist, which are referred to as NHE1 to NHE6 (for Na+/H+ exchanger one through six). The NHE1 protein was the first isoform cloned and studied in a variety of systems. This review summarizes recent papers on this protein, particularly those that have examined regulation of the protein and its expression and activity.     

NHE基因家族的研究进展

Na⁺/H⁺交换泵(Na⁺/H⁺ exchanger, NHE)是存在于所有脊椎动物细胞中的重要跨膜蛋白,该蛋白质涉及细胞的多种功能,包括细胞内pH值调节、细胞体积的控制以及离子转运等.目前已克隆了五个亚型NHE的cDNA,它们构成了脊椎动物细胞离子转运泵的一个基因家族. 这五个亚型的表达水平及活性可受多种因素的调节.在肿瘤、高血压及糖尿病等疾病中,已发现NHE-1亚型的表达水平和活性显著增高.因此,研究NHE-1的转录及活性调节机制,将可能为这些疾病的诊治提供新的手段.     

Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin

In mammalian cells, nine conserved isoforms of the Na(+)/H(+) exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1-5 are localized to the plasma membrane, whereas NHE6-9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ～15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrin-dependent endocytosis of transferrin via pH regulation.