Gill ionocyte remodeling mediates blood pH regulation in rockfish (Sebastes diploproa) exposed to environmentally relevant hypercapnia.

Marine fishes excrete excess H+ using basolateral Na+-K+-ATPase (NKA) and apical Na+/H+ exchanger 3 (NHE3) in gill ionocytes. However, the mechanisms that regulate H+ excretion during exposure to environmentally relevant hypercapnia (ERH) remain poorly understood. Here, we explored transcriptomic, proteomic, and cellular responses in gills of juvenile splitnose rockfish (Sebastes diploproa) exposed to 3 days of ERH conditions (pH ∼7.5, ∼1,600 µatm Pco2). Blood pH was fully regulated at ∼7.75 despite a lack of significant changes in gill 1) mRNAs coding for proteins involved in blood acid-base regulation, 2) total NKA and NHE3 protein abundance, and 3) ionocyte density. However, ERH-exposed rockfish demonstrated increased NKA and NHE3 abundance on the ionocyte plasma membrane coupled with wider apical membranes and greater extension of apical microvilli. The observed gill ionocyte remodeling is consistent with enhanced H+ excretion that maintains blood pH homeostasis during exposure to ERH and does not necessitate changes at the expression or translation levels. These mechanisms of phenotypic plasticity may allow fishes to regulate blood pH during environmentally relevant acid-base challenges and thus have important implications for both understanding how organisms respond to climate change and for selecting appropriate metrics to evaluate its impact on marine ecosystems.NEW & NOTEWORTHY Splitnose rockfish exposed to environmentally relevant hypercapnia utilize existing proteins (rather than generate additional machinery) to maintain homeostasis.

Potency and specificity of amiloride and its analogues on branchial sodium fluxes in freshwater trout and goldfish.

There is a consensus that electroneutral Na+/H+ exchangers (NHEs) are important in branchial Na+ uptake in freshwater fish. There is also widespread belief, based on mammalian data, that EIPA [5-(N-ethyl-N-isopropyl)-amiloride]], and HMA [5-(N,N-hexamethylene)-amiloride)] are more potent and specific in blocking Na+ uptake than amiloride. We evaluated this idea by testing the three drugs at 10-7 to 10-4 M, i.e. 0.1 to 100 µM in two model species, rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus), using 22Na+ to measure unidirectional Na+ influx and efflux rates. In both species, the potency order for inhibiting unidirectional Na+ influx was HMA > amiloride > EIPA (IC50 values in the 10-70 µM range), very different from in mammals. At 100 µM, all three drugs inhibited Na+ influx by >90% in both species, except for amiloride in goldfish (65%). However, at 60-100 µM, all three drugs also stimulated unidirectional Na+ efflux rates, indicating non-specific effects. In trout, HMA and EIPA caused significant increases (2.1- to 2.3-fold) in efflux rates, whereas in goldfish, significant efflux elevations were greater (3.1- to 7.2-fold) with all three drugs. We conclude that the inhibitory potency profile established in mammals does not apply to the NHEs in fish gills, that non-specific effects on Na+ efflux rates are a serious concern, and that EIPA and HMA offer no clear benefits in terms of potency or specificity. Considering its much lower cost, we recommend amiloride as the drug of choice for in vivo experiments on freshwater fishes.

Dissecting structure and function of the monovalent cation/H+ antiporters Mdm38 and Ylh47 in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Mdm38 and Ylh47 are homologs of the Ca2+/H+ antiporter Letm1, a candidate gene for seizures associated with Wolf-Hirschhorn syndrome in humans. Mdm38 is important for K+/H+ exchange across the inner mitochondrial membrane and contributes to membrane potential formation and mitochondrial protein translation. Ylh47 also localizes to the inner mitochondrial membrane. However, knowledge of the structures and detailed transport activities of Mdm38 and Ylh47 is limited. In this study, we conducted characterization of the ion transport activities and related structural properties of Mdm38 and Ylh47. Growth tests using Na+/H+ antiporter-deficient Escherichia coli strain TO114 showed that Mdm38 and Ylh47 had Na+ efflux activity. Measurement of transport activity across E. coli-inverted membranes showed that Mdm38 and Ylh47 had K+/H+, Na+/H+, and Li+/H+ antiport activity, but unlike Letm1, they lacked Ca2+/H+ antiport activity. Deletion of the ribosome-binding domain resulted in decreased Na+ efflux activity in Mdm38. Structural models of Mdm38 and Ylh47 identified a highly conserved glutamic acid in the pore-forming membrane-spanning region. Replacement of this glutamic acid with alanine, a non-polar amino acid, significantly impaired the ability of Mdm38 and Ylh47 to complement the salt sensitivity of E. coli TO114. These findings not only provide important insights into the structure and function of the Letm1-Mdm38-Ylh47 antiporter family but by revealing their distinctive properties also shed light on the physiological roles of these transporters in yeast and animals. IMPORTANCE: The inner membrane of mitochondria contains numerous ion transporters, including those facilitating H+ transport by the electron transport chain and ATP synthase to maintain membrane potential. Letm1 in the inner membrane of mitochondria in animals functions as a Ca2+/H+ antiporter. However, this study reveals that homologous antiporters in mitochondria of yeast, Mdm38 and Ylh47, do not transport Ca2+ but instead are selective for K+ and Na+. Additionally, the identification of conserved amino acids crucial for antiporter activity further expanded our understanding of the structure and function of the Letm1-Mdm38-Ylh47 antiporter family.

GGA1 interacts with the endosomal Na+/H+ exchanger NHE6 governing localization to the endosome compartment.

Mutations in the endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurological disorder. NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl terminus as bait, we identify Golgi-associated, gamma adaptin ear-containing, ADP-ribosylation factor (ARF) binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using: coimmunoprecipitation; overexpressed constructs in mammalian cells; and coimmunoprecipitation of endogenously expressed GGA1 and NHE6 from neuroblastoma cells, as well as from the mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7, and NHE9) and that there is significantly less interaction with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate the cytoplasmic tail of NHE6 interacts most strongly with GGA1. We demonstrate the colocalization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mislocalized in GGA1 KO cells, wherein we find less NHE6 in endosomes, but more NHE6 transport to lysosomes, and more Golgi retention of NHE6, with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mislocalization, and Golgi retention, we find the intraluminal pH in Golgi to be alkalinized in GGA1-null cells. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intracellular NHE to the endosome compartment.

The Total Denervation of the Ischemic Kidney Induces Differential Responses in Sodium Transporters' Expression in the Contralateral Kidney in Goldblatt Rats.

The Goldblatt model of hypertension (2K-1C) in rats is characterized by renal sympathetic nerve activity (rSNA). We investigated the effects of unilateral renal denervation of the clipped kidney (DNX) on sodium transporters of the unclipped kidneys and the cardiovascular, autonomic, and renal functions in 2K-1C and control (CTR) rats. The mean arterial pressure (MAP) and rSNA were evaluated in experimental groups. Kidney function and NHE3, NCC, ENaCß, and ENaCγ protein expressions were assessed. The glomerular filtration rate (GRF) and renal plasma flow were not changed by DNX, but the urinary (CTR: 0.0042 ± 0.001; 2K-1C: 0.014 ± 0.003; DNX: 0.005 ± 0.0013 mL/min/g renal tissue) and filtration fractions (CTR: 0.29 ± 0.02; 2K-1C: 0.51 ± 0.06; DNX: 0.28 ± 0.04 mL/min/g renal tissue) were normalized. The Na+/H+ exchanger (NHE3) was reduced in 2K-1C, and DNX normalized NHE3 (CTR: 100 ± 6; 2K-1C: 44 ± 14, DNX: 84 ± 13%). Conversely, the Na+/Cl- cotransporter (NCC) was increased in 2K-1C and was reduced by DNX (CTR: 94 ± 6; 2K-1C: 144 ± 8; DNX: 60 ± 15%). In conclusion, DNX in Goldblatt rats reduced blood pressure and proteinuria independently of GRF with a distinct regulation of NHE3 and NCC in unclipped kidneys.

A putative Na+/H+ antiporter BpSOS1 contributes to salt tolerance in birch.

White birch (Betula platyphylla Suk.) is an important pioneer tree which plays a critical role in maintaining ecosystem stability and forest regeneration. The growth of birch is dramatically inhibited by salt stress, especially the root inhibition. Salt Overly Sensitive 1 (SOS1) is the only extensively characterized Na+ efflux transporter in multiple plant species. The salt-hypersensitive mutant, sos1, display significant inhibition of root growth by NaCl. However, the role of SOS1 in birch responses to salt stress remains unclear. Here, we characterized a putative Na+/H+ antiporter BpSOS1 in birch and generated the loss-of-function mutants of the birch BpSOS1 by CRISPR/Cas9 approach. The bpsos1 mutant exhibit exceptional increased salt sensitivity which links to excessive Na+ accumulation in root, stem and old leaves. We observed a dramatic reduction of K+ contents in leaves of the bpsos1 mutant plants under salt stress. Furthermore, the Na+/K+ ratio of roots and leaves is significant higher in the bpsos1 mutants than the wild-type plants under salt stress. The ability of Na+ efflux in the root meristem zone is found to be impaired which might result the imbalance of Na+ and K+ in the bpsos1 mutants. Our findings indicate that the Na+/H+ exchanger BpSOS1 plays a critical role in birch salt tolerance by maintaining Na+ homeostasis and provide evidence for molecular breeding to improve salt tolerance in birch and other trees.

Genes for endosomal pH regulators NHE6 and NHE9 are dysregulated in the substantia nigra in Parkinson's disease.

Endosomal acid base balance functions as a master orchestrator within the cell, engaging with many cellular pathways to maintain homeostasis. Mutations in the endosomal pH regulator Na+/H+ exchanger NHE6 may disrupt this delicate balancing act and cause monogenic Parkinsonism. Here, gene expression studies in post-mortem substantia nigra of Parkinson's disease (PD) patients and normal controls were performed to investigate whether NHE6 represents a pathophysiological link between monogenic and sporadic PD. The substantia nigra in PD displayed down-regulation of NHE6, coincident with a loss of expression of several SNARE signalling pathway members, suggesting impaired membrane fusion and vesicle-recycling. Increased abundance of related NHE9 was also identified in the parkinsonian nigra that could reflect compensatory changes or be a consequence of neuronal dysfunction. The current model suggests the possibility that neurons expressing low levels of NHE6 are more susceptible to injury in PD, potentially directly contributing to the loss of nigral dopaminergic neurons and the genesis of the disease. These results have important implications for disease-modifying therapies as they suggest that endosomal pH correctors, including epigenetic modifiers that regulate NHE6 expression, may be beneficial for PD. Thus, aberrant endosomal acidification in the nigrostriatal pathway is a possible unifying pathomechanism in both monogenic and sporadic PD, with implications for understanding and treating this disorder. Replication of these observations in the post-mortem brains of Alzheimer's disease and frontotemporal dementia patients supports a model of conserved mechanisms underlying injury and death of neurons.

Proton-driven sodium secretion in a saline water animal.

Aquatic animals residing in saline habitats either allow extracellular sodium concentration to conform to environmental values or regulate sodium to lower levels. The latter strategy requires an energy-driven process to move sodium against a large concentration gradient to eliminate excess sodium that diffuses into the animal. Previous studies of invertebrate and vertebrate species indicate a sodium pump, Na+/K+ ATPase, powers sodium secretion. We provide the first functional evidence of a saline-water animal, Aedes taeniorhynchus mosquito larva, utilizing a proton pump to power this process. Vacuolar-type H+ ATPase (VHA) protein is highly expressed on the apical membrane of the posterior rectal cells, and in situ sodium flux across this epithelium increases significantly in larvae held in higher salinity and is sensitive to Bafilomycin A1, an inhibitor of VHA. We also report the first evidence of splice variants of the sodium/proton exchanger, NHE3, with both high and low molecular weight variants highly expressed on the apical membrane of the posterior rectal cells. Evidence of NHE3 function was indicated with in situ sodium transport significantly inhibited by a NHE3 antagonist, S3226. We propose that the outward proton pumping by VHA establishes a favourable electromotive gradient to drive sodium secretion via NHE3 thus producing a hyperosmotic, sodium-rich urine. This H+- driven Na+ secretion process is the primary mechanism of ion regulation in salt-tolerant culicine mosquito species and was first investigated over 80 years ago.

Drosophila Nhe2 overexpression induces autophagic cell death.

Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous manner. In cancer cells, autophagy is often up-regulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the Drosophila eye via overexpression of DNhe2, resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the DNhe2-induced cell death was caspase independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.

Identification and Validation of SLC9A2 as A Potential Tumor Suppressor in Colorectal Cancer: Integrating Bioinformatics Analysis with Experimental Confirmation.

OBJECTIVE: To uncover the mechanisms underlying the development of colorectal cancer (CRC), we applied bioinformatic analyses to identify key genes and experimentally validated their possible roles in CRC onset and progression. METHODS: We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis. RESULTS: We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway. CONCLUSION: Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.

The salt sensitivity of Drd4-null mice is associated with the upregulations of sodium transporters in kidneys.

To explore the mechanism of the hypertension in dopamine receptor-4 (Drd4) null mice, we determined the salt sensitivity and renal sodium transport proteins in Drd4-/- and Drd4+/+ mice with varied salt diets. On normal NaCl diet (NS), mean arterial pressures (MAP, telemetry) were higher in Drd4-/- than Drd4+/+; Low NaCl diet (LS) tended to decrease MAP in both strains; high NaCl diet (HS) elevated MAP with sodium excretion decreased and pressure-natriuresis curve shifted to right in Drd4-/- relative to Drd4+/+ mice. Drd4-/- mice exhibited increased renal sodium-hydrogen exchanger 3 (NHE3), sodium-potassium-2-chloride cotransporter (NKCC2), sodium-chloride cotransporter (NCC), and outer medullary α-epithelial sodium channel (αENaC) on NS, decreased NKCC2, NCC, αENaC, and αNa+-K+-ATPase on LS, and increased αENaC on HS. NKCC2, NCC, αENaC, and αNa+-K+-ATPase in plasma membrane were greater in Drd4-/- than in Drd4+/+ mice with HS. D4R was expressed in proximal and distal convoluted tubules, thick ascending limbs, and outer medullary collecting ducts and colocalized with NKCC2 and NCC. The phosphorylation of NKCC2 was enhanced but ubiquitination was reduced in the KO mice. There were no differences between the mouse strains in serum aldosterone concentrations and urinary dopamine excretions despite their changes with diets. The mRNA expressions of renal NHE3, NKCC2, NCC, and αENaC on NS were not altered in Drd4-/- mice. Thus, increased protein expressions of NHE3, NKCC2, NCC and αENaC are associated with hypertension in Drd4-/- mice; increased plasma membrane protein expression of NKCC2, NCC, αENaC, and αNa+-K+-ATPase may mediate the salt sensitivity of Drd4-/- mice.

Transporter function characterization via continuous-exchange cell-free synthesis and solid supported membrane-based electrophysiology.

Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as KM, IMAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.

Site-directed mutagenesis at the Glu78 in Ec-NhaA transporter impacting ion exchange: a biophysical study.

Na+/H+ antiporters facilitate the exchange of Na+ for H+ across the cytoplasmic membrane in prokaryotic and eukaryotic cells. These transporters are crucial to maintain the homeostasis of sodium ions, consequently pH, and volume of the cells. Therefore, sodium/proton antiporters are considered promising therapeutic targets in humans. The Na+/H+ antiporter in Escherichia coli (Ec-NhaA), a prototype of cation-proton antiporter (CPA) family, transports two protons and one sodium (or Li+) in opposite direction. Previous mutagenesis experiments on Ec-NhaA have proposed Asp164, Asp163, and Asp133 amino acids with the significant implication in functional and structural integrity and create site for ion-binding. However, the mechanism and the sites for the binding of the two protons remain unknown and controversial which could be critical for pH regulation. In this study, we have explored the role of Glu78 in the regulation of pH by Ec-NhaA. Although we have created various mutants, E78C has shown a considerable effect on the stoichiometry of NhaA and presented comparable phenotypes. The ITC experiment has shown the binding of ~ 5 protons in response to the transport of one lithium ion. The phenotype analysis on selective medium showed a significant expression compared to WT Ec-NhaA. This represents the importance of Glu78 in transporting the H+ across the membrane where a single mutation with Cys amino acid alters the number of H+ significantly maintaining the activity of the protein.

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.

The molecular sociology of NHERF1 PDZ proteins controlling renal hormone-regulated phosphate transport.

Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control extracellular phosphate levels by regulating renal NPT2A-mediated phosphate transport by a process requiring the PDZ scaffold protein NHERF1. NHERF1 possesses two PDZ domains, PDZ1 and PDZ2, with identical core-binding GYGF motifs explicitly recognizing distinct binding partners that play different and specific roles in hormone-regulated phosphate transport. The interaction of PDZ1 and the carboxy-terminal PDZ-binding motif of NPT2A (C-TRL) is required for basal phosphate transport. PDZ2 is a regulatory domain that scaffolds multiple biological targets, including kinases and phosphatases involved in FGF23 and PTH signaling. FGF23 and PTH trigger disassembly of the NHERF1-NPT2A complex through reversible hormone-stimulated phosphorylation with ensuing NPT2A sequestration, down-regulation, and cessation of phosphate absorption. In the absence of NHERF1-NPT2A interaction, inhibition of FGF23 or PTH signaling results in disordered phosphate homeostasis and phosphate wasting. Additional studies are crucial to elucidate how NHERF1 spatiotemporally coordinates cellular partners to regulate extracellular phosphate levels.

The crossing of two unwound transmembrane regions that is the hallmark of the NhaA structural fold is critical for antiporter activity.

Cell pH and Na+ homeostasis requires Na+/H+ antiporters. The crystal structure of NhaA, the main Escherichia coli Na+/H+ antiporter, revealed a unique NhaA structural fold shared by prokaryotic and eukaryotic membrane proteins. Out of the 12 NhaA transmembrane segments (TMs), TMs III-V and X-XII are topologically inverted repeats with unwound TMs IV and XI forming the X shape characterizing the NhaA fold. We show that intramolecular cross-linking under oxidizing conditions of a NhaA mutant with two Cys replacements across the crossing (D133C-T340C) inhibits antiporter activity and impairs NhaA-dependent cell growth in high-salts. The affinity purified D133C-T340C protein binds Li+ (the Na+ surrogate substrate of NhaA) under reducing conditions. The cross-linking traps the antiporter in an outward-facing conformation, blocking the antiport cycle. As many secondary transporters are found to share the NhaA fold, including some involved in human diseases, our data have importance for both basic and clinical research.

An Endosomal Acid-Regulatory Feedback System Rewires Cytosolic cAMP Metabolism and Drives Tumor Progression.

Although suppressed cAMP levels have been linked to cancer for nearly five decades, the molecular basis remains uncertain. Here, we identify endosomal pH as a novel regulator of cytosolic cAMP homeostasis and a promoter of transformed phenotypic traits in colorectal cancer. Combining experiments and computational analysis, we show that the Na+/H+ exchanger NHE9 contributes to proton leak and causes luminal alkalinization, which induces resting [Ca2+], and in consequence, represses cAMP levels, creating a feedback loop that echoes nutrient deprivation or hypoxia. Higher NHE9 expression in cancer epithelia is associated with a hybrid epithelial-mesenchymal (E/M) state, poor prognosis, tumor budding, and invasive growth in vitro and in vivo. These findings point to NHE9-mediated cAMP suppression as a pseudostarvation-induced invasion state and potential therapeutic vulnerability in colorectal cancer. Our observations lay the groundwork for future research into the complexities of endosome-driven metabolic reprogramming and phenotype switching and the biology of cancer progression. IMPLICATIONS: Endosomal pH regulator NHE9 actively controls cytosolic Ca2+ levels to downregulate the adenylate cyclase-cAMP system, enabling colorectal cancer cells to acquire hybrid E/M characteristics and promoting metastatic progression.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

STUDY QUESTION: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Intracellularly delivered human lactoferrin functions as an activator of Na+/H+ exchanger 7.

Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.