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.     

Membrane surface charge dictates the structure and function of the epithelial Na+/H+ exchanger

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 activity.     

Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

To assess the role of Ca²⁺in regulation of theNa⁺/H⁺exchanger (NHE1), we used CCL-39 fibroblasts overexpressing theNa⁺/Ca²⁺exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise andlong-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant withthe calmodulin (CaM)-binding domain deleted (amino acids 637-656),suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression ofNCX1 only slightly inhibited platelet-derived growth factor BB-inducedalkalinization and did not affect hyperosmolarity- or phorbol12-myristate 13-acetate-induced alkalinization. Downregulation ofprotein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely inNCX1-transfected cells, suggesting that the thrombin effect is mediatedexclusively via Ca²⁺ and PKC. Onthe other hand, deletion mutant study revealed that PKC-dependentregulation occurs through a small cytoplasmic segment (amino aids566-595). These data suggest that a mechanism involving directCa²⁺-CaM binding lasts for arelatively long period after agonist stimulation, despite apparentshort-lived Ca²⁺ mobilization, andfurther support our previous conclusion that Ca²⁺- and PKC-dependent mechanismsare mediated through distinct segments of the NHE1 cytoplasmic domain.

     

Structure and function of the human Na+/H+ exchanger isoform 1

Sodium proton exchangers (NHEs) constitute a large family of polytopic membrane protein transporters found in organisms across all domains of life. They are responsible for the exchange of protons for sodium ions. In archaea, bacteria, yeast and plants they provide increased salt tolerance by removing sodium in exchanger for extracellular protons. In humans they have a host of physiological functions, the most prominent of which is removal of intracellular protons in exchange for extracellular sodium. Human NHE is also involved in heart disease, cell growth and in cell differentiation. NHE’s physiological roles and the intriguing pathological consequences of their actions, make them a very important target of structural and functional studies. There are nine isoforms identified to date in humans. This review provides a brief overview of the human NHE’s physiological and pathological roles and cellular/tissue distribution, with special attention to the exemplar member NHE1. A summary of our knowledge to date of the structure and function of NHE1 is included focusing on a discussion of the recent discrepancies reported on the topology of NHE1. Finally we discuss a newly discovered relative of the NHE1 isoform, the Na+/Li+ exchanger, focusing on its predicted topology and its potential roles in disease.     

Shank2 associates with and regulates Na+/H+ exchanger 3

Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.     

Solution structure of the cytoplasmic region of Na+/H+ exchanger 1 complexed with essential cofactor calcineurin B homologous protein 1

Na+/H+ exchanger 1 (NHE1) regulates intracellular pH, Na+ content, and cell volume. Calcineurin B homologous protein 1 (CHP1) serves as an essential cofactor that facilitates NHE1 exchange activity under physiological conditions by direct binding to the cytoplasmic juxtamembrane region of NHE1. Here we describe the solution structure of the cytoplasmic juxtamembrane region of NHE1 complexed with CHP1. The region of NHE1 forms an amphipathic helix, which is induced by CHP1 binding, and CHP1 possesses a large hydrophobic cleft formed by EF-hand helices. The apolar side of the NHE1 helix participates in extensive hydrophobic interactions with the cleft of CHP1. We suggest that helix formation of the cytoplasmic region of NHE1 by CHP1 is a prerequisite for generating the active form of NHE1. The molecular recognition detailed in this study also provides novel insight into the target binding mechanism of EF-hand proteins.     

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.     

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.     

Functional analysis of acidic amino acids in the cytosolic tail of the Na+/H+ exchanger

The mammalian Na(+)/H(+) exchanger is a membrane protein with a C-terminal regulatory cytosolic domain and an N-terminal membrane domain. Na(+)/H(+) exchanger isoform 1 (NHE1) possesses a conserved amino acid sequence of seven consecutive acidic residues in the distal region of the cytosolic tail. We examined the structural and functional role of this acidic sequence. In human NHE1, varying mutations of the sequence (753)EEDEDDD(759) resulted in defective NHE1 activity. Mutation of the core acid sequence, (755)DED(757), or of the entire sequence caused a decrease in the activity of NHE1 in response to acute acid load. This was not due to changes in Na(+) affinity but rather due to decreased maximum velocity of the protein and delayed activation. Mutation of the target sequence did not affect the ability of the cytoplasmic domain to bind carbonic anhydrase II or tescalcin but did affect calmodulin binding. Mutation of the acidic domain also caused altered sensitivity to trypsin and changes in size of the protein in gel-filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results demonstrate that the acidic sequence is critical in maintaining proper conformation of the cytosolic domain, calmodulin binding, and in maintenance of Na(+)/H(+) exchanger activity.     

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.     

Na+-inhibitory sites of the Na+/H+ exchanger are Li+ substrate sites

Amiloride-inhibitable Li+ influx in dog red blood cells is mediated by the Na+/H+ exchanger, NHE. However, there are substantial differences between the properties of Li+ transport and Na+ transport through the NHE. Li+ influx is activated by cell shrinkage, and Na+ influx is not, as we reported previously (Dunham PB, Kelley SJ, and Logue PJ. Am J Physiol Cell Physiol 287: C336-C344, 2004). Li+ influx is a sigmoidal function of its concentration, and Na+ activation is linear at low Na+ concentrations. Li+ does not inhibit its own influx; in contrast, Na+ inhibits Na+ influx. Li+ prevents this inhibition by Na+. Na+ is a mixed or noncompetitive inhibitor of Li+ influx, implying that both a Na+ and a Li+ can be bound at the same time. In contrast, Li+ is a competitive inhibitor of Na+ influx, suggesting Li+ binding at one class of sites on the transporter. Because the properties of Li+ transport and Na+ transport are different, a simple explanation is that Na+ and Li+ are transported by separate sites. The similarities of the properties of Li+ transport and the inhibition of Na+ transport by Na+ suggest that Li+ is transported by the Na+-inhibitory sites.     

Dimerization is crucial for the function of the Na+/H+ exchanger NHE1

The Na(+)/H(+) exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H(+) concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na(+) or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.     

Dimeric structure of human Na+/H+ exchanger isoform 1 overproduced in Saccharomyces cerevisiae

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% alpha-helix, 23% beta-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1.     

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.     

Protein phosphatase regulation of Na+/H+ exchanger isoform I

We investigated regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) by dephosphorylation. Treatment of primary cultures of cardiomyocytes with the phosphatase inhibitor okadaic acid increased the rate of recovery from an acid load, suggesting that the okadaic acid sensitive PP1 may be involved in NHE1 regulation in vivo. We examined the ability of purified protein phosphatases PP1, PP2A, and PP2B to dephosphorylate the regulatory cytoplasmic tail. NHE1 was completely dephosphorylated by PP1, poorly dephosphorylated by PP2A, and not dephosphorylated by PP2B. Examination of NHE1 binding to PP1 or PP2B revealed that an association occurs between NHE1 and PP1 both in vitro and in vivo, but NHE1 did not associate with full-length PP2B. We expressed PP1 or inhibitor 2, a specific PP1 inhibitor, in cell lines to examine the effect of PP1 on NHE1 activity in vivo. Overexpression of PP1 causes a decrease in NHE1 activity but does not affect stimulation by thrombin. Cell lines expressing the specific PP1 inhibitor, inhibitor 2, had elevated proton efflux rates and could not be further stimulated by the Na(+)/H(+) exchanger agonist thrombin. The results suggest that PP1 is an important regulatory phosphatase of NHE1, that it can bind to and dephosphorylate the protein, and that it regulates NHE1 activity in vivo.     

The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.