Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser⁷⁸⁵ and Ser⁷⁸⁷, that were similar in location and context to two amino acids of the Arabidopsis Na⁺/H⁺ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.     

Prokaryotic Na+/H+ Exchangers—Transport Mechanism and Essential Residues

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.     

Na+/H+ Exchangers Involve in Regulating the pH-Sensitive Ion Channels in Mouse Sperm

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 (pH_i) regulator functionally couples to the activation of KSper/CatSper remains obscure. Although Na⁺/H⁺ exchangers (NHEs) have been implicated to mediate pH_i 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 pH_i 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.     

The Mechanism of Energy Coupling in H+/Na+-Pumping Membrane Pyrophosphatase—Possibilities and Probabilities

Membrane pyrophosphatases (mPPases) found in plant vacuoles and some prokaryotes and protists are ancient cation pumps that couple pyrophosphate hydrolysis with the H⁺ and/or Na⁺ transport out of the cytoplasm. Because this function is reversible, mPPases play a role in maintaining the level of cytoplasmic pyrophosphate, a known regulator of numerous metabolic reactions. mPPases arouse interest because they are among the simplest membrane transporters and have no homologs among known ion pumps. Detailed phylogenetic studies have revealed various subtypes of mPPases and suggested their roles in the evolution of the “sodium” and “proton” bioenergetics. This treatise focuses on the mechanistic aspects of the transport reaction, namely, the coupling step, the role of the chemically produced proton, subunit cooperation, and the relationship between the proton and sodium ion transport. The available data identify H⁺-PPases as the first non-oxidoreductase pump with a “direct-coupling” mechanism, i.e., the transported proton is produced in the coupled chemical reaction. They also support a “billiard” hypothesis, which unifies the H⁺ and Na⁺ transport mechanisms in mPPase and, probably, other transporters.     

HKT1;1 and HKT1;2 Na+ Transporters from Solanum galapagense Play Different Roles in the Plant Na+ Distribution under Salinity

Salt tolerance is a target trait in plant science and tomato breeding programs. Wild tomato accessions have been often explored for this purpose. Since shoot Na⁺/K⁺ is a key component of salt tolerance, RNAi-mediated knockdown isogenic lines obtained for Solanum galapagense alleles encoding both class I Na⁺ transporters HKT1;1 and HKT1;2 were used to investigate the silencing effects on the Na and K contents of the xylem sap, and source and sink organs of the scion, and their contribution to salt tolerance in all 16 rootstock/scion combinations of non-silenced and silenced lines, under two salinity treatments. The results show that SgHKT1;1 is operating differently from SgHKT1;2 regarding Na circulation in the tomato vascular system under salinity. A model was built to show that using silenced SgHKT1;1 line as rootstock would improve salt tolerance and fruit quality of varieties carrying the wild type SgHKT1;2 allele. Moreover, this increasing effect on both yield and fruit soluble solids content of silencing SgHKT1;1 could explain that a low expressing HKT1;1 variant was fixed in S. lycopersicum during domestication, and the paradox of increasing agronomic salt tolerance through silencing the HKT1;1 allele from S. galapagense, a salt adapted species.     

Deficiency of Thyroid Hormone Reduces Voltage-Gated Na+ Currents as Well as Expression of Na+/K+-ATPase in the Mouse Hippocampus

Mice lacking functional thyroid follicular cells, Pax8^−/− mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na⁺-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8^−/− mice should show deficits in the expression of Na⁺ currents and potentially also in the expression of Na⁺/K⁺-ATPases, which are necessary to maintain low intracellular Na⁺ levels. We thus compared Na⁺ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na⁺/K⁺-ATPase in wild type versus Pax8^−/− mice. Whereas the Na⁺ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na⁺ current density remained at a very low level in hippocampal neurons from Pax8^−/− mice. Pax8^−/− mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na⁺/K⁺-ATPase as well as decreased levels of the β2 isoform, with no changes in the α2 and β1 subunits.     

Suppressing Effect of Na+/Ca2+ Exchanger (NCX) Inhibitors on the Growth of Melanoma Cells

The role of calcium ion (Ca²⁺) signaling in tumorigenicity has received increasing attention in melanoma research. Previous Ca²⁺ signaling studies focused on Ca²⁺ entry routes, but rarely explored the role of Ca²⁺ extrusion. Functioning of the Na⁺/Ca²⁺ exchanger (NCX) on the plasma membrane is the major way of Ca²⁺ extrusion, but very few associations between NCX and melanoma have been reported. Here, we explored whether pharmacological modulation of the NCX could suppress melanoma and promise new therapeutic strategies. Methods included cell viability assay, Ca²⁺ imaging, immunoblotting, and cell death analysis. The NCX inhibitors SN-6 and YM-244769 were used to selectively block reverse operation of the NCX. Bepridil, KB-R7943, and CB-DMB blocked either reverse or forward NCX operation. We found that blocking the reverse NCX with SN-6 or YM-244769 (5–100 μM) did not affect melanoma cells or increase cytosolic Ca²⁺. Bepridil, KB-R7943, and CB-DMB all significantly suppressed melanoma cells with IC₅₀ values of 3–20 μM. Bepridil and KB-R7943 elevated intracellular Ca²⁺ level of melanoma. Bepridil-induced melanoma cell death came from cell cycle arrest and enhanced apoptosis, which were all attenuated by the Ca²⁺ chelator BAPTA-AM. As compared with melanoma, normal melanocytes had lower NCX1 expression and were less sensitive to the cytotoxicity of bepridil. In conclusion, blockade of the forward but not the reverse NCX leads to Ca²⁺-related cell death in melanoma and the NCX is a potential drug target for cancer therapy.     

Phytoglobin Expression Alters the Na+/K+ Balance and Antioxidant Responses in Soybean Plants Exposed to Na2SO4

Soybean (Glycine max) is an economically important crop which is very susceptible to salt stress. Tolerance to Na₂SO₄ stress was evaluated in soybean plants overexpressing or suppressing the phytoglobin GmPgb1. Salt stress depressed several gas exchange parameters, including the photosynthetic rate, caused leaf damage, and reduced the water content and dry weights. Lower expression of respiratory burst oxidase homologs (RBOHB and D), as well as enhanced antioxidant activity, resulting from GmPgb1 overexpression, limited ROS-induced damage in salt-stressed leaf tissue. The leaves also exhibited higher activities of the H₂O₂-quenching enzymes, catalase (CAT) and ascorbate peroxidase (APX), as well as enhanced levels of ascorbic acid. Relative to WT and GmPgb1-suppressing plants, overexpression of GmPgb1 attenuated the accumulation of foliar Na⁺ and exhibited a lower Na⁺/K⁺ ratio. These changes were attributed to the induction of the Na⁺ efflux transporter SALT OVERLY SENSITIVE 1 (SOS1) limiting Na⁺ intake and transport and the inward rectifying K⁺ channel POTASSIUM TRANSPORTER 1 (AKT1) required for the maintenance of the Na⁺/K⁺ balance.     

Higher Na+-Ca2+ Exchanger Function and Triggered Activity Contribute to Male Predisposition to Atrial Fibrillation

Male sex is one of the most important risk factors of atrial fibrillation (AF), with the incidence in men being almost double that in women. However, the reasons for this sex difference are unknown. Accordingly, in this study, we sought to determine whether there are sex differences in intracellular Ca²⁺ homeostasis in mouse atrial myocytes that might help explain male predisposition to AF. AF susceptibility was assessed in male (M) and female (F) mice (4–5 months old) using programmed electrical stimulation (EPS) protocols. Males were 50% more likely to develop AF. The Ca²⁺ transient amplitude was 28% higher in male atrial myocytes. Spontaneous systolic and diastolic Ca²⁺ releases, which are known sources of triggered activity, were significantly more frequent in males than females. The time to 90% decay of Ca²⁺ transient was faster in males. Males had 54% higher Na⁺-Ca²⁺ exchanger (NCX1) current density, and its expression was also more abundant. L-type Ca²⁺ current (I_CaL) was recorded with and without BAPTA, a Ca²⁺ chelator. I_CaL density was lower in males only in the absence of BAPTA, suggesting stronger Ca²⁺-dependent inactivation in males. Ca_V1.2 expression was similar between sexes. This study reports major sex differences in Ca²⁺ homeostasis in mouse atria, with larger Ca²⁺ transients and enhanced NCX1 function and expression in males resulting in more spontaneous Ca²⁺ releases. These sex differences may contribute to male susceptibility to AF by promoting triggered activity.     

The Rice Cation/H+ Exchanger Family Involved in Cd Tolerance and Transport

Cadmium (Cd), a heavy metal toxic to humans, easily accumulates in rice grains. Rice with unacceptable Cd content has become a serious food safety problem in many rice production regions due to contaminations by industrialization and inappropriate waste management. The development of rice varieties with low grain Cd content is seen as an economic and long-term solution of this problem. The cation/H⁺ exchanger (CAX) family has been shown to play important roles in Cd uptake, transport and accumulation in plants. Here, we report the characterization of the rice CAX family. The six rice CAX genes all have homologous genes in Arabidopsis thaliana. Phylogenetic analysis identified two subfamilies with three rice and three Arabidopsis thaliana genes in both of them. All rice CAX genes have trans-member structures. OsCAX1a and OsCAX1c were localized in the vacuolar while OsCAX4 were localized in the plasma membrane in rice cell. The consequences of qRT-PCR analysis showed that all the six genes strongly expressed in the leaves under the different Cd treatments. Their expression in roots increased in a Cd dose-dependent manner. GUS staining assay showed that all the six rice CAX genes strongly expressed in roots, whereas OsCAX1c and OsCAX4 also strongly expressed in rice leaves. The yeast (Saccharomyces cerevisiae) cells expressing OsCAX1a, OsCAX1c and OsCAX4 grew better than those expressing the vector control on SD-Gal medium containing CdCl₂. OsCAX1a and OsCAX1c enhanced while OsCAX4 reduced Cd accumulation in yeast. No auto-inhibition was found for all the rice CAX genes. Therefore, OsCAX1a, OsCAX1c and OsCAX4 are likely to involve in Cd uptake and translocation in rice, which need to be further validated.     

Peptide Modification Diminishes HLA Class II-restricted CD4+ T Cell Recognition of Prostate Cancer Cells

Prostate cancer poses an ongoing problem in the western world accounting for significant morbidity and mortality in the male population. Current therapy options are effective in treating most prostate cancer patients, but a significant number of patients progress beyond a manageable disease. For these patients, immunotherapy has emerged as a real option in the treatment of the late-stage metastatic disease. Unfortunately, even the most successful immunotherapy strategies have only led to a four-month increase in survival. One issue responsible for the shortcomings in cancer immunotherapy is the inability to stimulate helper CD4⁺ T cells via the HLA class II pathway to generate a potent antitumor response. Obstacles to proper HLA class II stimulation in prostate cancer vaccine design include the lack of detectable class II proteins in prostate tumors and the absence of defined class II specific prostate tumor antigens. Here, for the first time, we show that the insertion of a lysosomal thiol reductase (GILT) into prostate cancer cells directly enhances HLA class II antigen processing and results in increased CD4⁺ T cell activation by prostate cancer cells. We also show that GILT insertion does not alter the expression of prostate-specific membrane antigen (PSMA), an important target in prostate cancer vaccine strategies. Our study suggests that GILT expression enhances the presentation of the immunodominant PSMA₄₅₉ epitope via the HLA class II pathway. Biochemical analysis showed that the PSMA₄₅₉ peptide was cysteinylated under a normal physiologic concentration of cystine, and this cysteinylated form of PSMA₄₅₉ inhibited T cell activation. Taken together, these results suggest that GILT has the potential to increase HLA class II Ag presentation and CD4⁺ T cell recognition of prostate cancer cells, and GILT-expressing prostate cancer cells could be used in designing cell therapy and/or vaccines against prostate cancer.     

Regulation of Cytosolic pH: The Contributions of Plant Plasma Membrane H+-ATPases and Multiple Transporters

Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H⁺ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H⁺ transport activity, namely, H⁺-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H⁺ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H⁺-ATPases mediate H⁺ extrusion, whereas most members of other six proteins mediate H⁺ influx, thus contributing to cytosolic pH homeostasis by directly modulating H⁺ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H⁺-coupled substrate influx, whereas other intra-family members facilitate H⁺-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H⁺-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H⁺ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H⁺ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.     

Na+/K+- and Mg2+-ATPases and Their Interaction with AMPA,NMDA and D2 Dopamine Receptors in an Animal Model of Febrile Seizures

Febrile seizures (FS) are one of the most common seizure disorders in childhood which are classified into short and prolonged, depending on their duration. Short FS are usually considered as benign. However, epidemiological studies have shown an association between prolonged FS and temporal lobe epilepsy. The development of animal models of FS has been very useful to investigate the mechanisms and the consequences of FS. One of the most used, the “hair dryer model”, has revealed that prolonged FS may lead to temporal lobe epilepsy by altering neuronal function. Several pieces of evidence suggest that Na⁺/ K⁺-ATPase and Mg²⁺-ATPase may play a role in this epileptogenic process. In this work, we found that hyperthermia-induced seizures (HIS) significantly increased the activity of Na⁺/ K⁺-ATPase and Mg²⁺-ATPase five and twenty days after hyperthermic insult, respectively. These effects were diminished in response to AMPA, D₂ dopamine A₁ and A_2A receptors activation, respectively. Furthermore, HIS also significantly increased the protein level of the AMPA subunit GluR1. Altogether, the increased Na⁺/ K⁺-ATPase and Mg²⁺-ATPase agree well with the presence of protective mechanisms. However, the reduction in ATPase activities in the presence of NMDA and AMPA suggest an increased propensity for epileptic events in adults.     

Characterization in Inhibitory Effectiveness of Carbamazepine in Voltage-Gated Na+ and Erg-Mediated K+ Currents in a Mouse Neural Crest-Derived (Neuro-2a) Cell Line

Carbamazepine (CBZ, Tegretol^®) is an anticonvulsant used in the treatment of epilepsy and neuropathic pain; however, several unwanted effects of this drug have been noticed. Therefore, the regulatory actions of CBZ on ionic currents in electrically excitable cells need to be reappraised, although its efficacy in suppressing voltage-gated Na⁺ current (I_Na) has been disclosed. This study was undertaken to explore the modifications produced by CBZ on ionic currents (e.g., I_Na and erg-mediated K⁺ current [I_K(erg)]) measured from Neuro-2a (N2a) cells. In these cells, we found that this drug differentially suppressed the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of I_Na in a concentration-dependent manner with effective IC₅₀ of 56 and 18 μM, respectively. The overall current–voltage relationship of I_Na(T) with or without the addition of CBZ remained unchanged; however, the strength (i.e., ∆area) in the window component of I_Na (I_Na(W)) evoked by the short ascending ramp pulse (V_ramp) was overly lessened in the CBZ presence. Tefluthrin (Tef), a synthetic pyrethroid, known to stimulate I_Na, augmented the strength of the voltage-dependent hysteresis (Hys_(V)) of persistent I_Na (I_Na(P)) in response to the isosceles-triangular V_ramp; moreover, further application of CBZ attenuated Tef-mediated accentuation of I_Na(P)’s Hys_(V). With a two-step voltage protocol, the recovery of I_Na(T) inactivation seen in Neuro-2a cells became progressively slowed by adding CBZ; however, the cumulative inhibition of I_Na(T) evoked by pulse train stimulation was enhanced during exposure to this drug. Neuro-2a-cell exposure to CBZ (100 μM), the magnitude of erg-mediated K⁺ current measured throughout the entire voltage-clamp steps applied was mildly inhibited. The docking results regarding the interaction of CBZ and voltage-gate Na⁺ (Na_V) channel predicted the ability of CBZ to bind to some amino-acid residues in Na_V due to the existence of a hydrogen bond or hydrophobic contact. It is conceivable from the current investigations that the I_Na (I_Na(T), I_Na(L), I_Na(W), and I_Na(P)) residing in Neuro-2a cells are susceptible to being suppressed by CBZ, and that its block on I_Na(L) is larger than that on I_Na(T). Collectively, the magnitude and gating of Na_V channels produced by the CBZ presence might have an impact on its anticonvulsant and analgesic effects occurring in vivo.     

Albumin Stimulates Epithelial Na+ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na⁺ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na⁺ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na⁺ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na⁺ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na⁺ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na⁺ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na⁺ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway.     

Epigenomic Features and Potential Functions of K+ and Na+ Favorable DNA G-Quadruplexes in Rice

DNA G-quadruplexes (G4s) are non-canonical four-stranded DNA structures involved in various biological processes in eukaryotes. Molecularly crowded solutions and monovalent cations have been reported to stabilize in vitro and in vivo G4 formation. However, how K⁺ and Na⁺ affect G4 formation genome-wide is still unclear in plants. Here, we conducted BG4-DNA-IP-seq, DNA immunoprecipitation with anti-BG4 antibody coupled with sequencing, under K⁺ and Na⁺ + PEG conditions in vitro. We found that K⁺-specific IP-G4s had a longer peak size, more GC and PQS content, and distinct AT and GC skews compared to Na⁺-specific IP-G4s. Moreover, K⁺- and Na⁺-specific IP-G4s exhibited differential subgenomic enrichment and distinct putative functional motifs for the binding of certain trans-factors. More importantly, we found that K⁺-specific IP-G4s were more associated with active marks, such as active histone marks, and low DNA methylation levels, as compared to Na⁺-specific IP-G4s; thus, K⁺-specific IP-G4s in combination with active chromatin features facilitate the expression of overlapping genes. In addition, K⁺- and Na⁺-specific IP-G4 overlapping genes exhibited differential GO (gene ontology) terms, suggesting they may have distinct biological relevance in rice. Thus, our study, for the first time, explores the effects of K⁺ and Na⁺ on global G4 formation in vitro, thereby providing valuable resources for functional G4 studies in rice. It will provide certain G4 loci for the biotechnological engineering of rice in the future.