cGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

New functions and roles of the Na+-H+-exchanger NHE3.

The sodium/proton exchanger isoform 3 (NHE3) is expressed in the intestine and the kidney, where it contributes to hydrogen secretion and sodium (re)absorption. The roles of this transporter have been studied by the use of the respective knockout mice and by using pharmacological inhibitors. Whole-body NHE3 knockout mice suffer from a high mortality rate (with only ∼30% of mice surviving into adulthood), and based on the expression of NHE3 in both intestine and kidney, some conclusions that were originally derived were based on this rather complex phenotype. In the last decade, more refined models have been developed that added temporal and spatial control of NHE3 expression. For example, novel mouse models have been developed with a knockout of NHE3 in intestinal epithelial cells, tubule/collecting duct of the kidney, proximal tubule of the kidney, and thick ascending limb of the kidney. These refined models have significantly contributed to our understanding of the role of NHE3 in a tissue/cell type-specific manner. In addition, tenapanor was developed, which is a non-absorbable, intestine-specific NHE3 inhibitor. In rat and human studies, tenapanor lowered intestinal Pi uptake and was effective in lowering plasma Pi levels in patients on hemodialysis. Of note, diarrhea is seen as a side effect of tenapanor (with its indication for the treatment of constipation) and in intestine-specific NHE3 knockout mice; however, effects on plasma Pi were not supported by this mouse model which showed enhanced and not reduced intestinal Pi uptake. Further studies indicated that the gut microbiome in mice lacking intestinal NHE3 resembles an intestinal environment favoring the competitive advantage of inflammophilic over anti-inflammatory species, something similar seen in patients with inflammatory bowel disease. This review will highlight recent developments and summarize newly gained insight from these refined models.

Overexpression of transcription factor FaMYB63 enhances salt tolerance by directly binding to the SOS1 promoter in Arabidopsis thaliana.

Salinity is a pivotal abiotic stress factor with far-reaching consequences on global crop growth, yield, and quality and which includes strawberries. R2R3-MYB transcription factors encompass a range of roles in plant development and responses to abiotic stress. In this study, we identified that strawberry transcription factor FaMYB63 exhibited a significant upregulation in its expression under salt stress conditions. An analysis using yeast assay demonstrated that FaMYB63 exhibited the ability to activate transcriptional activity. Compared with those in the wild-type (WT) plants, the seed germination rate, root length, contents of chlorophyll and proline, and antioxidant activities (SOD, CAT, and POD) were significantly higher in FaMYB63-overexpressing Arabidopsis plants exposed to salt stress. Conversely, the levels of malondialdehyde (MDA) were considerably lower. Additionally, the FaMYB63-overexpressed Arabidopsis plants displayed a substantially improved capacity to scavenge active oxygen. Furthermore, the activation of stress-related genes by FaMYB63 bolstered the tolerance of transgenic Arabidopsis to salt stress. It was also established that FaMYB63 binds directly to the promoter of the salt overly sensitive gene SOS1, thereby activating its expression. These findings identified FaMYB63 as a possible and important regulator of salt stress tolerance in strawberries.

Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

Translatome and Transcriptome Analyses Reveal the Mechanism that Underlies the Enhancement of Salt Stress by the Small Peptide Ospep5 in Plants.

Salt stress significantly impedes plant growth and the crop yield. This study utilized de novo transcriptome assembly and ribosome profiling to explore mRNA translation's role in rice salt tolerance. We identified unrecognized translated open reading frames (ORFs), including 42 upstream transcripts and 86 unannotated transcripts. A noteworthy discovery was the role of a small ORF, Ospep5, in conferring salt tolerance. Overexpression of Ospep5 in plants increased salt tolerance, while its absence led to heightened sensitivity. This hypothesis was corroborated by the findings that exogenous application of the synthetic small peptide Ospep5 bolstered salt tolerance in both rice and Arabidopsis. We found that the mechanism underpinning the Ospep5-mediated salt tolerance involves the maintenance of intracellular Na+/K+ homeostasis, facilitated by upregulation of high-affinity potassium transporters (HKT) and Na+/H+ exchangers (SOS1). Furthermore, a comprehensive multiomics approach, particularly ribosome profiling, is instrumental in uncovering unannotated ORFs and elucidating their functions in plant stress responses.

The Hydrophilic C-terminus of Yeast Plasma-membrane Na+/H+ Antiporters Impacts Their Ability to Transport K.

Yeast plasma-membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkali-metal cations and protons in cells. They are predicted to consist of 13 transmembrane segments (TMSs) and a large hydrophilic C-terminal cytoplasmic part with seven conserved domains. The substrate specificity, specifically the ability to recognize and transport K+ cations in addition to Na+ and Li+, differs among homologs. In this work, we reveal that the composition of the C-terminus impacts the ability of antiporters to transport particular cations. In the osmotolerant yeast Zygosaccharomyces rouxii, the Sod2-22 antiporter only efficiently exports Na+ and Li+, but not K+. The introduction of a negative charge or removal of a positive charge in one of the C-terminal conserved regions (C3) enabled ZrSod2-22 to transport K+. The same mutations rescued the low level of activity and purely Li+ specificity of ZrSod2-22 with the A179T mutation in TMS6, suggesting a possible interaction between this TMS and the C-terminus. The truncation or replacement of the C-terminal part of ZrSod2-22 with the C-terminus of a K+-transporting Nha/Sod antiporter (Saccharomyces cerevisiae Nha1 or Z. rouxii Nha1) also resulted in an antiporter with the capacity to export K+. In addition, in ScNha1, the replacement of three positively charged arginine residues 539-541 in the C3 region with alanine caused its inability to provide cells with tolerance to Li+. All our results demonstrate that the physiological functions of yeast Nha/Sod antiporters, either in salt tolerance or in K+ homeostasis, depend on the composition of their C-terminal parts.

The signalling pathways, calcineurin B-like protein 5 (CBL5)-CBL-interacting protein kinase 8 (CIPK8)/CIPK24-salt overly sensitive 1 (SOS1), transduce salt signals in seed germination in Arabidopsis.

For plant growth under salt stress, sensing and transducing salt signals are central to cellular Na+ homoeostasis. The calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CIPK) complexes play critical roles in transducing salt signals in plants. Here, we show that CBL5, an ortholog of CBL4 and CBL10 in Arabidopsis, interacts with and recruits CIPK8/CIPK24 to the plasma membrane. Yeast cells coexpressing CBL5, CIPK8/CIPK24 and SOS1 demonstrated lesser Na+ accumulation and a better growth phenotype than the untransformed or SOS1 transgenic yeast cells under salinity. Overexpression of CBL5 improved the growth of the cipk8 or cipk24 single mutant but not the cipk8 cipk24 double mutant under salt stress, suggesting that CIPK8 and CIPK24 were the downstream targets of CBL5. Interestingly, seed germination in cbl5 was severely inhibited by NaCl, which was recovered by the overexpression of CBL5. Furthermore, CBL5 was mainly expressed in the cotyledons and hypocotyls, which are essential to seed germination. Na+ efflux activity in the hypocotyls of cbl5 was reduced relative to the wild-type under salt stress, enhancing Na+ accumulation. These findings indicate that CBL5 functions in seed germination and protects seeds and germinating seedlings from salt stress through the CBL5-CIPK8/CIPK24-SOS1 pathways.

NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosomes in hepatocellular carcinoma.

BACKGROUND: Small extracellular vesicles (sEVs) mediate intercellular communication that contributes to hepatocellular carcinoma (HCC) progression via multifaceted pathways. The success of cell entry determines the effect of sEV on recipient cells. Here, we aimed to delineate the mechanisms underlying the uptake of sEV in HCC. METHODS: Macropinocytosis was examined by the ability of cells to internalize dextran and sEV. Macropinocytosis was analyzed in Na(+)/H(+) exchanger 7 (NHE7)-knockdown and -overexpressing cells. The properties of cells were studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. The expression of NHE7 in patients' liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7. RESULTS: The data revealed that macropinocytosis controlled the internalization of sEVs and their oncogenic effect on recipient cells. It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells. Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) limited the entry of sEVs and compromised cell aggressiveness. Mechanistically, we delineated that high level of NHE7, a sodium-hydrogen exchanger, alkalized intracellular pH and acidized endosomal pH, leading to the maturation of macropinosomes. Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis. Clinically, NHE7 expression was upregulated and linked to dismal prognosis of HCC. CONCLUSIONS: This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells. Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.

Common as well as unique methylation-sensitive DNA regulatory elements in three mammalian SLC9C1 genes.

The SLC9C1 gene (which encodes the NHE10 protein) is essential for male fertility in both mice and humans, however the epigenetic mechanisms regulating its testis/sperm-specific gene expression have yet to be studied. Here we identify and characterize DNA regulatory elements of the SLC9C1 gene across three mammalian species: mouse, rat, and human. First, in silico analysis of these mammalian SLC9C1 genes identified a CpG island located upstream of the transcription start site in the same relative position in all three genes. Further analysis reveals that this CpG island behaves differently, with respect to gene regulatory activity, in the mouse SLC9C1 gene than it does in the rat and human SLC9C1 gene. The mouse SLC9C1 CpG island displays strong promoter activity by itself and seems to have a stronger gene regulatory effect than either the rat or human SLC9C1 CpG islands. While the function of the upstream SLC9C1 CpG island may be divergent across the three studied species, it appears that the promoters of these three mammalian SLC9C1 genes share similar DNA methylation-sensitive regulatory mechanisms. All three SLC9C1 promoter regions are differentially methylated in lung and testis, being more hypermethylated in lung relative to the testis, and DNA sequence alignments provide strong evidence of primary sequence conservation. Luciferase assays reveal that in vitro methylation of constructs containing different elements of the three SLC9C1 genes largely exhibit methylation-sensitive promoter activity (reduced promoter activity when methylated) in both HEK 293 and GC-1spg cells. In total, our data suggest that the DNA methylation-sensitive elements of the mouse, rat, and human SLC9C1 promoters are largely conserved, while the upstream SLC9C1 CpG island common to all three species seems to perform a different function in mouse than it does in rat and human. This work provides evidence that while homologous genes can all be regulated by DNA methylation-dependent epigenetic mechanisms, the location of the specific cis-regulatory elements responsible for this regulation can differ across species.

Genome-Wide Analysis of Cation/Proton Antiporter Family in Soybean (Glycine max) and Functional Analysis of GmCHX20a on Salt Response.

Monovalent cation proton antiporters (CPAs) play crucial roles in ion and pH homeostasis, which is essential for plant development and environmental adaptation, including salt tolerance. Here, 68 CPA genes were identified in soybean, phylogenetically dividing into 11 Na+/H+ exchangers (NHXs), 12 K+ efflux antiporters (KEAs), and 45 cation/H+ exchangers (CHXs). The GmCPA genes are unevenly distributed across the 20 chromosomes and might expand largely due to segmental duplication in soybean. The GmCPA family underwent purifying selection rather than neutral or positive selections. The cis-element analysis and the publicly available transcriptome data indicated that GmCPAs are involved in development and various environmental adaptations, especially for salt tolerance. Based on the RNA-seq data, twelve of the chosen GmCPA genes were confirmed for their differentially expression under salt or osmotic stresses using qRT-PCR. Among them, GmCHX20a was selected due to its high induction under salt stress for the exploration of its biological function on salt responses by ectopic expressing in Arabidopsis. The results suggest that the overexpression of GmCHX20a increases the sensitivity to salt stress by altering the redox system. Overall, this study provides comprehensive insights into the CPA family in soybean and has the potential to supply new candidate genes to develop salt-tolerant soybean varieties.

Structural basis for the activity regulation of Salt Overly Sensitive 1 in Arabidopsis salt tolerance.

The plasma membrane Na+/H+ exchanger Salt Overly Sensitive 1 (SOS1) is crucial for plant salt tolerance. Unlike typical sodium/proton exchangers, SOS1 contains a large cytoplasmic domain (CPD) that regulates Na+/H+ exchange activity. However, the underlying modulation mechanism remains unclear. Here we report the structures of SOS1 from Arabidopsis thaliana in two conformations, primarily differing in CPD flexibility. The CPD comprises an interfacial domain, a cyclic nucleotide-binding domain-like domain (CNBD-like domain) and an autoinhibition domain. Through yeast cell-based Na+ tolerance test, we reveal the regulatory role of the interfacial domain and the activation role of the CNBD-like domain. The CPD forms a negatively charged cavity that is connected to the ion binding site. The transport of Na+ may be coupled with the conformational change of CPD. These findings provide structural and functional insight into SOS1 activity regulation.

Structure and activation mechanism of the rice Salt Overly Sensitive 1 (SOS1) Na+/H+ antiporter.

Salinity is one of the most severe abiotic stresses that adversely affect plant growth and agricultural productivity. The plant Na+/H+ antiporter Salt Overly Sensitive 1 (SOS1) located in the plasma membrane extrudes excess Na+ out of cells in response to salt stress and confers salt tolerance. However, the molecular mechanism underlying SOS1 activation remains largely elusive. Here we elucidate two cryo-electron microscopy structures of rice (Oryza sativa) SOS1, a full-length protein in an auto-inhibited state and a truncated version in an active state. The SOS1 forms a dimeric architecture, with an NhaA-folded transmembrane domain portion in the membrane and an elongated cytosolic portion of multiple regulatory domains in the cytoplasm. The structural comparison shows that SOS1 adopts an elevator transport mechanism accompanied by a conformational transition of the highly conserved Pro148 in the unwound transmembrane helix 5 (TM5), switching from an occluded conformation in the auto-inhibited state to a conducting conformation in the active state. These findings allow us to propose an inhibition-release mechanism for SOS1 activation and elucidate how SOS1 controls Na+ homeostasis in response to salt stress.

Na+/H+ Exchangers (NHEs) in Mammalian Sperm: Essential Contributors to Male Fertility.

Na+/H+ exchangers (NHEs) are known to be important regulators of pH in multiple intracellular compartments of eukaryotic cells. Sperm function is especially dependent on changes in pH and thus it has been postulated that NHEs play important roles in regulating the intracellular pH of these cells. For example, in order to achieve fertilization, mature sperm must maintain a basal pH in the male reproductive tract and then alkalize in response to specific signals in the female reproductive tract during the capacitation process. Eight NHE isoforms are expressed in mammalian testis/sperm: NHE1, NHE3, NHE5, NHE8, NHA1, NHA2, NHE10, and NHE11. These NHE isoforms are expressed at varying times during spermatogenesis and localize to different subcellular structures in developing and mature sperm where they contribute to multiple aspects of sperm physiology and male fertility including proper sperm development/morphogenesis, motility, capacitation, and the acrosome reaction. Previous work has provided evidence for NHE3, NHE8, NHA1, NHA2, and NHE10 being critical for male fertility in mice and NHE10 has recently been shown to be essential for male fertility in humans. In this article we review what is known about each NHE isoform expressed in mammalian sperm and discuss the physiological significance of each NHE isoform with respect to male fertility.

Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal 494TRL496 PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a 494AAA496 substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg495His, or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495 changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

A Na+/H+ antiporter-encoding salt overly sensitive 1 gene, LpSOS1, involved in positively regulating the salt tolerance in Lilium pumilum.

Lilium pumilum has a strong salt tolerance. However, the molecular mechanism underlying its salt tolerance remains unexplored. Here, LpSOS1 was cloned from L. pumilum and found to be significantly enriched at high NaCl concentrations (100 mM). In tobacco epidermal cells, localization analysis showed that the LpSOS1 protein was primarily located in the plasma membrane. Overexpression of LpSOS1 resulted in up-regulation of salt stress tolerance in Arabidopsis, as indicated by reduced malondialdehyde levels and Na+/K+ ratio, and increased activity of antioxidant reductases (including superoxide dismutase, peroxidase, and catalase). Treatment with NaCl resulted in improved growth, as evidenced by increased biomass, root length, and lateral root growth, in both sos1 mutant (atsos1) and wild-type (WT) Arabidopsis plants that overexpressed LpSOS1, Under NaCl treatment,atsos1 and WT Arabidopsis plants overexpressing LpSOS1 exhibited better growth, with higher biomass, root length, and lateral root quantity, whereas in the absence of LpSOS1 overexpression, the plants of both lines were wilted and chlorotic and even died under salt stress. When exposed to salt stress, the expression of stress-related genes was notably upregulated in the LpSOS1 overexpression line of Arabidopsis as compared to the WT. Our findings indicate that LpSOS1 enhances salt tolerance in plants by regulating ion homeostasis, reducing Na+/K+ ratio, thereby protecting the plasma membrane from oxidative damage caused by salt stress, and enhancing the activity of antioxidant enzymes. Therefore, the increased salt tolerance conferred by LpSOS1 in plants makes it a potential bioresource for breeding salt-tolerant crops. Further investigation into the mechanisms underlying lily's resistance to salt stress would be advantageous and could serve as a foundation for future molecular improvements.

A salt stress-activated GSO1-SOS2-SOS1 module protects the Arabidopsis root stem cell niche by enhancing sodium ion extrusion.

Soil salinity impairs plant growth reducing crop productivity. Toxic accumulation of sodium ions is counteracted by the Salt Overly Sensitive (SOS) pathway for Na+ extrusion, comprising the Na+ transporter SOS1, the kinase SOS2, and SOS3 as one of several Calcineurin-B-like (CBL) Ca2 + sensors. Here, we report that the receptor-like kinase GSO1/SGN3 activates SOS2, independently of SOS3 binding, by physical interaction and phosphorylation at Thr16. Loss of GSO1 function renders plants salt sensitive and GSO1 is both sufficient and required for activating the SOS2-SOS1 module in yeast and in planta. Salt stress causes the accumulation of GSO1 in two specific and spatially defined areas of the root tip: in the endodermis section undergoing Casparian strip (CS) formation, where it reinforces the CIF-GSO1-SGN1 axis for CS barrier formation; and in the meristem, where it creates the GSO1-SOS2-SOS1 axis for Na+ detoxification. Thus, GSO1 simultaneously prevents Na+ both from diffusing into the vasculature, and from poisoning unprotected stem cells in the meristem. By protecting the meristem, receptor-like kinase-conferred activation of the SOS2-SOS1 module allows root growth to be maintained in adverse environments.

CycC1;1-WRKY75 complex-mediated transcriptional regulation of SOS1 controls salt stress tolerance in Arabidopsis.

SALT OVERLY SENSITIVE1 (SOS1) is a key component of plant salt tolerance. However, how SOS1 transcription is dynamically regulated in plant response to different salinity conditions remains elusive. Here, we report that C-type Cyclin1;1 (CycC1;1) negatively regulates salt tolerance by interfering with WRKY75-mediated transcriptional activation of SOS1 in Arabidopsis (Arabidopsis thaliana). Disruption of CycC1;1 promotes SOS1 expression and salt tolerance in Arabidopsis because CycC1;1 interferes with RNA polymerase II recruitment by occupying the SOS1 promoter. Enhanced salt tolerance of the cycc1;1 mutant was completely compromised by an SOS1 mutation. Moreover, CycC1;1 physically interacts with the transcription factor WRKY75, which can bind to the SOS1 promoter and activate SOS1 expression. In contrast to the cycc1;1 mutant, the wrky75 mutant has attenuated SOS1 expression and salt tolerance, whereas overexpression of SOS1 rescues the salt sensitivity of wrky75. Intriguingly, CycC1;1 inhibits WRKY75-mediated transcriptional activation of SOS1 via their interaction. Thus, increased SOS1 expression and salt tolerance in cycc1;1 were abolished by WRKY75 mutation. Our findings demonstrate that CycC1;1 forms a complex with WRKY75 to inactivate SOS1 transcription under low salinity conditions. By contrast, under high salinity conditions, SOS1 transcription and plant salt tolerance are activated at least partially by increased WRKY75 expression but decreased CycC1;1 expression.

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm.

Na+/H+ exchangers (NHEs) are a family of ion transporters that regulate the pH of various cell compartments across an array of cell types. In eukaryotes, NHEs are encoded by the SLC9 gene family comprising 13 genes. SLC9C2, which encodes the NHE11 protein, is the only one of the SLC9 genes that is essentially uncharacterized. Here, we show that SLC9C2 exhibits testis/sperm-restricted expression in rats and humans, akin to its paralog SLC9C1 (NHE10). Similar to NHE10, NHE11 is predicted to contain an NHE domain, a voltage sensing domain, and finally an intracellular cyclic nucleotide binding domain. An immunofluorescence analysis of testis sections reveals that NHE11 localizes with developing acrosomal granules in spermiogenic cells in both rat and human testes. Most interestingly, NHE11 localizes to the sperm head, likely the plasma membrane overlaying the acrosome, in mature sperm from rats and humans. Therefore, NHE11 is the only known NHE to localize to the acrosomal region of the head in mature sperm cells. The physiological role of NHE11 has yet to be demonstrated but its predicted functional domains and unique localization suggests that it could modulate intracellular pH of the sperm head in response to changes in membrane potential and cyclic nucleotide concentrations that are a result of sperm capacitation events. If NHE11 is shown to be important for male fertility, it will be an attractive target for male contraceptive drugs due to its exclusive testis/sperm-specific expression.

Inhibition of NHE1 transport activity and gene transcription in DRG neurons in oxaliplatin-induced painful peripheral neurotoxicity.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN), one of the major dose-limiting side effects of colorectal cancer treatment, is characterized by both acute and chronic syndromes. Acute exposure to low dose OHP on dorsal root ganglion (DRG) neurons is able to induce an increase in intracellular calcium and proton concentration, thus influencing ion channels activity and neuronal excitability. The Na+/H+ exchanger isoform-1 (NHE1) is a plasma membrane protein that plays a pivotal role in intracellular pH (pHi) homeostasis in many cell types, including nociceptors. Here we show that OHP has early effects on NHE1 activity in cultured mouse DRG neurons: the mean rate of pHi recovery was strongly reduced compared to vehicle-treated controls, reaching levels similar to those obtained in the presence of cariporide (Car), a specific NHE1 antagonist. The effect of OHP on NHE1 activity was sensitive to FK506, a specific calcineurin (CaN) inhibitor. Lastly, molecular analyses revealed transcriptional downregulation of NHE1 both in vitro, in mouse primary DRG neurons, and in vivo, in an OIPN rat model. Altogether, these data suggest that OHP-induced intracellular acidification of DRG neurons largely depends on CaN-mediated NHE1 inhibition, revealing new mechanisms that OHP could exert to alter neuronal excitability, and providing novel druggable targets.