The human Na,K-ATPase alpha 4 isoform is a ouabain-sensitive alpha isoform that is expressed in sperm

The Na,K-ATPase generates electrochemical gradients across the plasma membrane that are responsible for numerous cellular and physiological processes. The active Na,K-ATPase is minimally composed of an alpha and a beta subunit and families of isoforms for both subunits exist. Recent studies have identified a physiological role for the rat Na,K-ATPase alpha4 isoform in sperm motility. However, very little is known about the human Na,K-ATPase alpha4 isoform other than its genomic sequence and structure and its mRNA expression pattern. Here, the human alpha4 isoform of the Na,K-ATPase is cloned, expressed, and characterized. Full length cDNAs encoding the putative human alpha4 isoform of the Na,K-ATPase were identified from a number of ESTs and a protein product corresponding to this isoform was shown to be expressed from these cDNAs. The human Na,K-ATPase alpha4 isoform protein was found to be expressed in mature sperm in human testes sections and it is localized specifically to the principle piece of human sperm. In addition, the presence of the Na,K-ATPase alpha4 isoform is absent in immature testes however its expression appears coincident with sexual maturity. And finally, the human Na,K-ATPase alpha4 isoform was shown to be as sensitive to cardiac glycoside inhibition as the human Na,K-ATPase alpha1 isoform. Considering the important role of the rat Na,K-ATPase alpha4 isoform in rat sperm motility, the demonstration that the human alpha4 isoform is a sperm-specific protein localized to the flagellum suggests a role for the human Na,K-ATPase alpha4 isoform in human sperm physiology.     

Aquaporin-2 and Na+/H+ exchanger isoform 1 modulate the efficiency of renal cell migration

Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin β1 trafficking and the turnover of focal adhesions. The aim of this study was to investigate whether AQP2 also works in cooperation with Na⁺/H⁺ exchanger isoform 1 (NHE1), another well-known protein involved in the regulation of cell migration. Our results showed that the lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and areas of focal adhesions and more alkaline intracellular pH due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2, or its physically-associated calcium channel TRPV4, significantly reduced lamellipodia NHE1 activity. NHE1 blockage significantly reduced the rate of cell migration, the number of lamellipodia, and the assembly of F-actin only in AQP2-expressing cells. Our data suggest that AQP2 modulates the activity of NHE1 through its calcium channel partner TRPV4, thereby determining pH-dependent actin polymerization, providing mechanical stability to delineate lamellipodia structure and defining the efficiency of cell migration.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Recovery from hypoxia-induced internalization of cardiac Na+/H + exchanger 1 requires an adequate intracellular store of antioxidants

The heart is highly active metabolically but relatively underperfused and, therefore, vulnerable to ischemia. In addition to acidosis, a key component of ischemia is hypoxia that can modulate gene expression and protein function as part of an adaptive or even maladaptive response. Here, using cardiac-derived HL-1 cells, we investigate the effect of various hypoxic stimuli on the expression and activity of Na⁺/H ⁺ exchanger 1 (NHE1), a principal regulator of intracellular pH. Acute (10 min) anoxia produced a reversible decrease in the sarcolemmal NHE1 activity attributable to NHE1 internalization. Treatment with either 1% O ₂ or dimethyloxaloylglycine (DMOG; 1 mM) for 48-hr stabilized hypoxia-inducible factor 1 and reduced the sarcolemmal NHE1 activity by internalization, but without a change in total NHE1 immunoreactivity or message levels of the coding gene ( SLC9A1) determined in whole-cell lysates. Unlike the effect of DMOG, which was rapidly reversed on washout, reoxygenation after a prolonged period of hypoxia did not reverse the effects on NHE1, unless media were also supplemented with a membrane-permeant derivative of glutathione (GSH). Without a prior hypoxic episode, GSH supplementation had no effect on the NHE1 activity. Thus, posthypoxic NHE1 reinsertion can only take place if cells have a sufficient reservoir of a reducing agent. We propose that oxidative stress under prolonged hypoxia depletes intracellular GSH to an extent that curtails NHE1 reinsertion once the hypoxic stimulus is withdrawn. This effect may be cardioprotective, as rapid postischaemic restoration of the NHE1 activity is known to trigger reperfusion injury by producing an intracellular Na ⁺-overload, which is proarrhythmogenic.     

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.     

A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility

It has long been speculated that intracellular pH is a critical regulator of both invertebrate and vertebrate sperm motility, and sodium-hydrogen exchange has been suggested as a mediator of such pH(i) regulation in various instances. Two sodium-hydrogen exchangers (NHE1 and NHE5) are expressed in spermatozoa. However, elimination of the NHE1 gene fails to cause infertility, suggesting that normal sperm function is maintained in NHE1-null animals. Here, we used a functionally unbiased signal peptide trap screen to identify a novel sperm-specific NHE. The NHE contains 14 predicted transmembrane segments, including a potential voltage sensor and a consensus cyclic nucleotide-binding motif. Testis histology, sperm numbers and morphology were normal, but NHE-null males were completely infertile with severely diminished sperm motility. The addition of ammonium chloride, which elevates intracellular pH, partially rescued the motility and fertility defects. Surprisingly, cyclic AMP analogues almost completely rescued the motility and infertility phenotypes. The existence of this new sperm NHE provides an attractive contraceptive target, given its cell-specific expression and absolute requirement for fertility.     

The Na+/H+ Exchanger Present in Trout Erythrocyte Membranes is Not Responsible for the Amiloride-insensitive Na+/Li+ Exchange Activity

The protein responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na⁺/H⁺ exchanger could be responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li⁺/H⁺ exchange activity (mediated by the Na⁺/H⁺ exchanger βNHE) and the Na⁺/Li⁺ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O₂. We concluded that the DMA insensitive Na⁺/Li⁺ exchange activity originates from a different protein. To further examine these findings, we measured Li⁺ efflux in fibroblasts expressing the βNHE as the only Na⁺/H⁺ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification of fibroblasts expressing the Na⁺/H⁺ exchanger βNHE, induces a Na⁺ stimulated Li⁺ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA insensitive Na⁺/Li⁺ exchange activity. In these fibroblasts no significant DMA insensitive Na⁺/Li⁺ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na⁺/Li⁺ exchange activity is not mediated by the Na⁺/H⁺ exchanger (βNHE) present in these membranes. Received: 6 December 1996/Revised: 11 August 1997     

Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.     

NHE10, an osteoclast-specific member of the Na+/H+ exchanger family, regulates osteoclast differentiation and survival [corrected

Bone homeostasis is tightly regulated by the balanced actions of osteoblasts (OBs) and osteoclasts (OCs). We previously analyzed the gene expression profile of OC differentiation using a cDNA microarray, and identified a novel osteoclastogenic gene candidate, clone OCL-1-E7 [J. Rho, C.R. Altmann, N.D. Socci, L. Merkov, N. Kim, H. So, O. Lee, M. Takami, A.H. Brivanlou, Y. Choi, Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis, DNA Cell Biol. 21 (2002) 541-549]. In this study, we have isolated full-length cDNAs corresponding to this clone from mice and humans to determine the functional roles of this gene in osteoclastogenesis. The full-length cDNA of OCL-1-E7 encodes 12 membrane-spanning domains that are typical of isoforms of the Na⁺/H⁺ exchangers (NHEs), indicating that this clone is a novel member of the NHE family (hereafter referred to as NHE10). Here, we show that NHE10 is highly expressed in OCs in response to receptor activator of nuclear factor-κB ligand signaling and is required for OC differentiation and survival.     

Model for the allosteric regulation of the Na+/Ca2+ exchanger NCX

The Na⁺/Ca²⁺ exchanger provides a major Ca²⁺ extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca²⁺ concentrations. In Canis familiaris, Na⁺/Ca²⁺ exchanger (NCX) activity is regulated by the binding of Ca²⁺ to two cytosolic Ca²⁺‐binding domains, CBD1 and CBD2, such that Ca²⁺‐binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca²⁺‐regulation remains unclear. It was found previously that for NCX in the absence of Ca²⁺ the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca²⁺‐binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca²⁺‐binding to CBD1 inhibits Ca²⁺ exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca²⁺ modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca²⁺ regulation of the Na⁺/Ca²⁺ exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca²⁺‐binding to CBD1 controlling ion transport across the plasma membrane. Proteins 2016; 84:580–590. © 2016 Wiley Periodicals, Inc.     

Na+/H+ exchanger 1 inhibition contributes to K562 leukaemic cell differentiation

The effect of hypoxia on the differentiation of chronic myeloid leukaemic K562 cells were studied, as was the role of the NHE1 (Na+/H+ exchanger 1). Hypoxia induced differentiation of K562 cells as seen by modifications in their morphological features, up-regulation of C/EBPα (CCAAT/enhancer-binding protein α), and marked IL-8 (interleukin-8) release. Inhibition of NHE1 under hypoxia additionally enhanced the level of C/EBPα and further promoted leukaemic cells differentiation. Pharmacological inhibition of p38 MAPK (mitogen-activated protein kinase) also significantly suppressed C/EBPα expression under hypoxia conditions after NHE1 inhibition. These results indicate the enhancement of hypoxia-induced K562 differentiation by NHE1 inhibition, which may be due to up-regulation of C/EBPα via p38 MAPK signalling pathway, which suggests a possible therapeutic target of NHE1 under hypoxia microenvironment in the treatment of leukaemic diseases.     

Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger

We previously demonstrated that the progesterone‐ (P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na⁺ (Na⁺_o). Moreover, Na⁺_o depletion resulted in a decreased cytosolic pH (pH_i), suggesting involvement of a Na⁺‐dependent pH_i regulatory mechanism during the P‐initiated AR. We now report that the decreased pH_i resulting from Na⁺_o depletion is reversible and mediated by a Na⁺/H⁺ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pH_i, capacitated spermatozoa were incubated in Na⁺‐deficient, bicarbonate/CO₂‐buffered (0NaB) medium for 15–30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na⁺‐containing, bicarbonate/CO₂‐buffered (NaB) medium; Na⁺‐containing, Hepes‐buffered (NaH) medium; or maintained in the 0NaB medium. Included in the NaH medium was the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The steady‐state pH_i was then determined by spectrofluorometric measurement of bis(carboxyethyl)‐5(6)‐carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 μM) significantly (P < 0.05) inhibited the pH_i recovery produced by NaH medium. Moreover, the pH_i in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na⁺‐dependent, bicarbonate‐independent pH_i regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE‐1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P‐initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE‐dependent pH_i regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility. Mol. Reprod. Dev. 52:189–195, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Na+/H+逆向转运蛋白和植物耐盐性

Na^ /H^ 逆向转运蛋白对植物耐盐起着重要作用,它利用质膜H^ -ATPase或液泡膜H^ -ATPase及PPiase泵H^ 产生的驱动力把Na^ 排出细胞或在液泡中区隔化以消除Na^ 的毒害。主要讨论植物中Na^ /H^ 逆向转运蛋白研究在分子水平的最新进展。     

细菌Na+/H+逆向转运蛋白的研究进展

Na+/H+逆向转运蛋白在维持细胞内pH稳态、Na+离子动态平衡和调控细胞体积方面发挥着重要作用。目前,细菌中许多参与高盐或高碱性环境压力应答的Na+/H+逆向转运蛋白得到了鉴定和功能阐释。继续挖掘高效的Na+/H+逆向转运蛋白,深入探究Na+/H+逆向转运蛋白的分子机理,将为工业菌株或农作物的改良提供新的研究思路。本文以4种模式菌株为例,简要概述细菌Na+/H+逆向转运蛋白的种类和特征,同时对其结构和功能等方面也进行探讨。     

Combined blockade of the Na+ channel and the Na+/H+ exchanger virtually prevents ischemic Na+ overload in rat hearts

Blocking either the Na⁺ channel or the Na⁺/H⁺ exchanger (NHE) has been shown to reduce Na⁺ and Ca²⁺ overload during myocardial ischemia and reperfusion, respectively, and to improve post-ischemic contractile recovery. The effect of combined blockade of both Na⁺ influx routes on ionic homeostasis is unknown and was tested in this study. [Na⁺]_i, pH_i and energy-related phosphates were measured using simultaneous ²³Na- and ³¹P-NMR spectroscopy in isolated rat hearts. Eniporide (3 μM) and/or lidocaine (200 μM) were administered during 5 min prior to 40 min of global ischemia and 40 min of drug free reperfusion to block the NHE and the Na⁺ channel, respectively. Lidocaine reduced the rise in [Na⁺]_i during the first 10 min of ischemia, followed by a rise with a rate similar to the one found in untreated hearts. Eniporide reduced the ischemic Na⁺ influx during the entire ischemic period. Administration of both drugs resulted in a summation of the effects found in the lidocaine and eniporide groups. Contractile recovery and infarct size were significantly improved in hearts treated with both drugs, although not significantly different from hearts treated with either one of them.     

Na+ Sensitivity of ROMK1 K+ Channel: Role of the Na+/H+ Antiporter

To examine the extracellular Na⁺ sensitivity of a renal inwardly rectifying K⁺ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K⁺ channel, ROMK1 (Kir1.1). When extracellular Na⁺ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na⁺ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na⁺ removal from the bath solution. A blocker of Na⁺/H⁺ antiporters, amiloride, largely inhibited the Na⁺ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na⁺ removal. Na⁺ removal was found to induce a significant decrease in intracellular pH in the oocytes using H⁺-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na⁺/H⁺ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na⁺ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na⁺, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na⁺ sensitivity of ROMK1 channel via regulating intracellular pH. Received: 13 April 1999/Revised: 15 July 1999