Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.

Imidazole-based sphingosine-1-phosphate transporter Spns2 inhibitors.

Sphingosine-1-phosphate (S1P) is a chemotactic lipid that influences immune cell positioning. S1P concentration gradients are necessary for proper egress of lymphocytes from the thymus and secondary lymphoid tissues. This trafficking is interdicted by S1P receptor modulators, and it is expected that S1P transporter (Spns2) inhibitors, by reshaping S1P concentration gradients, will do the same. We previously reported SLF1081851 as a prototype Spns2 inhibitor, which provided a scaffold to investigate the importance of the oxadiazole core and the terminal amine. In this report, we disclose a structure-activity relationship study by incorporating imidazole as both a linker and surrogate for a positive charge in SLF1081851. In vitro inhibition of Spns2-dependent S1P transport in HeLa cells identified 7b as an inhibitor with an IC50 of 1.4 ± 0.3 µM. The SAR studies reported herein indicate that imidazolium can be a substitute for the terminal amine in SLF1081851 and that Spns2 inhibition is highly dependent on the lipid alkyl tail length.

Aldosterone Regulates Pendrin and Epithelial Sodium Channel Activity through Intercalated Cell Mineralocorticoid Receptor-Dependent and -Independent Mechanisms over a Wide Range in Serum Potassium.

BACKGROUND: Aldosterone activates the intercalated cell mineralocorticoid receptor, which is enhanced with hypokalemia. Whether this receptor directly regulates the intercalated cell chloride/bicarbonate exchanger pendrin is unclear, as are potassium's role in this response and the receptor's effect on intercalated and principal cell function in the cortical collecting duct (CCD). METHODS: We measured CCD chloride absorption, transepithelial voltage, epithelial sodium channel activity, and pendrin abundance and subcellular distribution in wild-type and intercalated cell-specific mineralocorticoid receptor knockout mice. To determine if the receptor directly regulates pendrin, as well as the effect of serum aldosterone and potassium on this response, we measured pendrin label intensity and subcellular distribution in wild-type mice, knockout mice, and receptor-positive and receptor-negative intercalated cells from the same knockout mice. RESULTS: Ablation of the intercalated cell mineralocorticoid receptor in CCDs from aldosterone-treated mice reduced chloride absorption and epithelial sodium channel activity, despite principal cell mineralocorticoid receptor expression in the knockout mice. With high circulating aldosterone, intercalated cell mineralocorticoid receptor gene ablation directly reduced pendrin's relative abundance in the apical membrane region and pendrin abundance per cell whether serum potassium was high or low. Intercalated cell mineralocorticoid receptor ablation blunted, but did not eliminate, aldosterone's effect on pendrin total and apical abundance and subcellular distribution. CONCLUSIONS: With high circulating aldosterone, intercalated cell mineralocorticoid receptor ablation reduces chloride absorption in the CCD and indirectly reduces principal cell epithelial sodium channel abundance and function. This receptor directly regulates pendrin's total abundance and its relative abundance in the apical membrane region over a wide range in serum potassium concentration. Aldosterone regulates pendrin through mechanisms both dependent and independent of the IC MR receptor.

Bacillus subtilis strain L1 promotes nitrate reductase activity in Arabidopsis and elicits enhanced growth performance in Arabidopsis, lettuce, and wheat.

Plant growth promoting rhizobacteria (PGPR) are a group of bacteria that promote plants growth in the rhizosphere. PGPRs are involved in various mechanisms that reinforce plant development. In this study, we screened for PGPRs that were effective in early growth of Arabidopsis thaliana when added to the media and one Bacillus subtilis strain L1 (Bs L1) was selected for further study. When Bs L1 was placed near the roots, seedlings showed notably stronger growth than that in the control, particularly in biomass and root hair. Quantitative reverse transcription polymerase chain reaction analysis revealed a high level of expression of the high affinity nitrate transporter gene, NRT2.1 in A. thaliana treated with Bs L1. After considering how Bs L1 could promote plant growth, we focused on nitrate, which is essential to plant growth. The nitrate content was lower in A. thaliana treated with Bs L1. However, examination of the activity of nitrate reductase revealed higher activity in plants treated with PGPR than in the control. Bs L1 had pronounced effects in representative crops (wheat and lettuce). These results suggest that Bs L1 promotes the assimilation and use of nitrate and plant growth.

MtMOT1.2 is responsible for molybdate supply to Medicago truncatula nodules.

Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss-of-function mot1.2-1 mutant showed reduced growth compared with wild-type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with a wild-type MtMOT1.2 gene or molybdate-fortification of the nutrient solution, both restored wild-type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.

The role of pendrin in renal physiology.

Pendrin is a Na(+)-independent Cl(-)/HCO3(-) exchanger that localizes to type B and non-A, non-B intercalated cells, which are expressed within the aldosterone-sensitive region of the nephron, i.e., the distal convoluted tubule, the connecting tubule, and the cortical collecting duct. Type B cells mediate Cl(-) absorption and HCO3(-) secretion primarily through pendrin-mediated Cl(-)/HCO3(-) exchange. At least in some treatment models, pendrin acts in tandem with the Na(+)-dependent Cl(-)/HCO3(-) exchanger (NDCBE) encoded by Slc4a8 to mediate NaCl absorption. The pendrin-mediated Cl(-)/HCO3(-) exchange process is greatly upregulated in models of metabolic alkalosis, such as following aldosterone administration or dietary NaHCO3 loading. It is also upregulated by angiotensin II. In the absence of pendrin [Slc26a4 (-/-) or pendrin null mice], aldosterone-stimulated NaCl absorption is reduced, which lowers the blood pressure response to aldosterone and enhances the alkalosis that follows the administration of this steroid hormone. Pendrin modulates aldosterone-induced Na(+) absorption by changing ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function at least in part by altering luminal HCO3(-) and ATP concentrations. Thus, aldosterone and angiotensin II also stimulate pendrin expression and function, which likely contributes to the pressor response of these hormones. This review summarizes the contribution of the Cl(-)/HCO3(-) exchanger pendrin in distal nephron function.

The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron.

Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.

Acute genetic ablation of pendrin lowers blood pressure in mice.

BACKGROUND: Pendrin, the chloride/bicarbonate exchanger of ß-intercalated cells of the renal connecting tubule and the collecting duct, plays a key role in NaCl reabsorption by the distal nephron. Therefore, pendrin may be important for the control of extracellular fluid volume and blood pressure. METHODS: Here, we have used a genetic mouse model in which the expression of pendrin can be switched-on in vivo by the administration of doxycycline. Pendrin can also be rapidly removed when doxycycline administration is discontinued. Therefore, our genetic strategy allows us to test selectively the acute effects of loss of pendrin function. RESULTS: We show that acute loss of pendrin leads to a significant decrease of blood pressure. In addition, acute ablation of pendrin did not alter significantly the acid-base status or blood K + concentration. CONCLUSION: By using a transgenic mouse model, avoiding off-target effects related to pharmacological compounds, this study suggests that pendrin could be a novel target to treat hypertension.

Prestin and the good vibrations.

In a recent paper published in the Biochemical Journal, Lolli et al. presented evidence that the C-terminal STAS (sulfate transporter and anti-sigma factor antagonist) domain of the motor protein prestin possesses an anion-binding site. This discovery might shed light on an aspect of the function of this mysterious and fascinating protein that is crucial for the human hearing system.

Nuclear Translocation of SGPP-1 and Decrease of SGPL-1 Activity Contribute to Sphingolipid Rheostat Regulation of Inflammatory Dendritic Cells.

A balanced sphingolipid rheostat is indispensable for dendritic cell function and survival and thus initiation of an immune response. Sphingolipid levels are dynamically maintained by the action of sphingolipid enzymes of which sphingosine kinases, S1P phosphatases (SGPP-1/2) and S1P lyase (SGPL-1), are pivotal in the balance of S1P and sphingosine levels. In this study, we present that SGPP-1 and SGPL-1 are regulated in inflammatory dendritic cells and contribute to S1P fate. TLR-dependent activation caused SGPL-1 protein downregulation with subsequent decrease of enzymatic activity by two-thirds. In parallel, confocal fluorescence microscopy revealed that endogenous SGPP-1 was expressed in nuclei of naive dendritic cells and was translocated into the cytoplasmatic compartment upon inflammatory stimulation resulting in dephosphorylation of S1P. Mass spectrometric determination showed that a part of the resulting sphingosine was released from the cell, increasing extracellular levels. Another route of diminishing intracellular S1P was possibly taken by its export via ATP-binding cassette transporter C1 which was upregulated in array analysis, while the S1P transporter, spinster homolog 2, was not relevant in dendritic cells. These investigations newly describe the sequential expression and localization of the endogenous S1P regulators SGPP-1 and SGPL-1 and highlight their contribution to the sphingolipid rheostat in inflammation.

The Arabidopsis root stele transporter NPF2.3 contributes to nitrate translocation to shoots under salt stress.

In most plants, NO(3)(-) constitutes the major source of nitrogen, and its assimilation into amino acids is mainly achieved in shoots. Furthermore, recent reports have revealed that reduction of NO(3)(-) translocation from roots to shoots is involved in plant acclimation to abiotic stress. NPF2.3, a member of the NAXT (nitrate excretion transporter) sub-group of the NRT1/PTR family (NPF) from Arabidopsis, is expressed in root pericycle cells, where it is targeted to the plasma membrane. Transport assays using NPF2.3-enriched Lactococcus lactis membranes showed that this protein is endowed with NO(3)(-) transport activity, displaying a strong selectivity for NO(3)(-) against Cl(-). In response to salt stress, NO(3)(-) translocation to shoots is reduced, at least partly because expression of the root stele NO(3)(-) transporter gene NPF7.3 is decreased. In contrast, NPF2.3 expression was maintained under these conditions. A loss-of-function mutation in NPF2.3 resulted in decreased root-to-shoot NO(3)(-) translocation and reduced shoot NO(3)(-) content in plants grown under salt stress. Also, the mutant displayed impaired shoot biomass production when plants were grown under mild salt stress. These mutant phenotypes were dependent on the presence of Na(+) in the external medium. Our data indicate that NPF2.3 is a constitutively expressed transporter whose contribution to NO(3)(-) translocation to the shoots is quantitatively and physiologically significant under salinity.

N availability modulates the role of NPF3.1, a gibberellin transporter, in GA-mediated phenotypes in Arabidopsis.

MAIN CONCLUSION: AtNPF3.1 gene expression is promoted by limiting nitrogen nutrition. Atnpf3.1 mutants are affected in hypocotyl elongation and seed germination under conditions of low-nitrate availability. The NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) family encodes nitrate or peptides transporters, some of which are also able to transport hormones. AtNPF3.1 has been described as a nitrate/nitrite/gibberellin transporter. Until now only its gibberellins (GAs) transport capacity have been proven in planta. We further analyzed its substrate specificity towards different GA species using a yeast heterologous system which revealed that (1) NPF3.1 transported not only bioactive GAs but also their precursors and metabolites and (2) the GAs' import activity of NPF3.1 was not affected by the presence of exogenous nitrate. Gene expression analysis along with germination assays and hypocotyl length measurements of loss of function mutants was used to understand the in planta role of NPF3.1. GUS staining revealed that this gene is expressed mainly in the endodermis of roots and hypocotyls, in shoots, stamens, and dry seeds. Germination assays in the presence of paclobutrazol, a GA biosynthesis inhibitor, revealed that the germination rate of npf3.1 mutants was lower compared to wild type when GA was added at the same time. Likewise, hypocotyl length measurements showed that the npf3.1 mutants were less sensitive to exogenous GA addition in the presence of paclobutrazol, compared to wild type. Moreover, this phenotype was observed only when plants were grown on low-nitrate supply. In addition, NPF3.1 gene expression was upregulated by low exogenous nitrate concentrations and the npf3.1 mutants exhibited a not yet described GA-related phenotype under these conditions. All together, these results indicated that NPF3.1 is indeed involved in GAs transport in planta under low-nitrate conditions.

Spinster 2, a sphingosine-1-phosphate transporter, plays a critical role in inflammatory and autoimmune diseases.

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite that regulates numerous processes important for immune responses. S1P is made within cells and must be transported out of cells to exert its effects through activation of 5 specific cell surface GPCRs in an autocrine or paracrine fashion. Spinster 2 (Spns2) transports S1P out of cells, and its deletion in mice reduces circulating levels of S1P, alters immune cell trafficking, and induces lymphopenia. Here we examined the effects of Spns2 deletion on adaptive immune responses and in autoimmune disease models. Airway inflammation and hypersensitivity as well as delayed-type contact hypersensitivity were attenuated in Spns2(-/-) mice. Similarly, Spns2 deletion reduced dextran sodium sulfate- and oxazolone-induced colitis. Intriguingly, Spns2(-/-) mice were protected from the development of experimental autoimmune encephalopathy, a model of the autoimmune disease multiple sclerosis. Deletion of Spns2 also strongly alleviated disease development in collagen-induced arthritis. These results point to a broad role for Spns2-mediated S1P transport in the initiation and development of adaptive immune related disorders.

A putative molybdate transporter LjMOT1 is required for molybdenum transport in Lotus japonicus.

Molybdenum (Mo) is an essential micronutrient that is required for plant growth and development, and it affects the formation of root nodules and nitrogen fixation in legumes. In this study, Lotus japonicus was grown on MS solid media containing 0 nmol l-1 (-Mo), 103 nmol l-1 (+Mo) and 1030 nmol l-1 (10 × Mo) of Mo. The phenotypes of plants growing on the three different media showed no obvious differences after 15 days, but the plants growing on -Mo for 45 days presented typical symptoms of Mo depletion, such as a short taproot, few lateral roots and yellowing leaves. A Mo transporter gene, LjMOT1, was isolated from L. japonicus. It encoded 468 amino acids, including two conserved motifs, and was predicted to locate to chromosome 3 of the L. japonicus genome. A homology comparison indicated that LjMOT1 had high similarities to other MOT1 proteins and was closely related to GmMOT1. Subcellular localization indicated that LjMOT1 is localized to the plasma membrane. qRT-PCR analyses showed that increasing Mo concentrations regulated the relative expression level of LjMOT1. Moreover, the Mo concentration in shoots was positively correlated to the expression of LjMOT1, but there was no such evident correlation in the roots. In addition, changes in the nitrate reductase activity were coincident with changes in the Mo concentration. These results suggest that LjMOT1 may be involved in the transport of Mo and provide a theoretical basis for further understanding of the mechanism of Mo transport in higher plants.

Chloride and salicylate influence prestin-dependent specific membrane capacitance: support for the area motor model.

The outer hair cell is electromotile, its membrane motor identified as the protein SLC26a5 (prestin). An area motor model, based on two-state Boltzmann statistics, was developed about two decades ago and derives from the observation that outer hair cell surface area is voltage-dependent. Indeed, aside from the nonlinear capacitance imparted by the voltage sensor charge movement of prestin, linear capacitance (Clin) also displays voltage dependence as motors move between expanded and compact states. Naturally, motor surface area changes alter membrane capacitance. Unit linear motor capacitance fluctuation (δCsa) is on the order of 140 zeptofarads. A recent three-state model of prestin provides an alternative view, suggesting that voltage-dependent linear capacitance changes are not real but only apparent because the two component Boltzmann functions shift their midpoint voltages (Vh) in opposite directions during treatment with salicylate, a known competitor of required chloride binding. We show here using manipulations of nonlinear capacitance with both salicylate and chloride that an enhanced area motor model, including augmented δCsa by salicylate, can accurately account for our novel findings. We also show that although the three-state model implicitly avoids measuring voltage-dependent motor capacitance, it registers δCsa effects as a byproduct of its assessment of Clin, which increases during salicylate treatment as motors are locked in the expanded state. The area motor model, in contrast, captures the characteristics of the voltage dependence of δCsa, leading to a better understanding of prestin.

The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants.

Nitrogen is a key mineral nutrient playing a crucial role in plant growth and development. Understanding the mechanisms of nitrate uptake from the soil and distribution through the plant in response to nitrogen starvation is an important step on the way to improve nitrogen uptake and utilization efficiency for better growth and productivity of plants, and to prevent negative effects of nitrogen fertilizers on the environment and human health. In this study, we show that Arabidopsis NITRATE TRANSPORTER 2.5 (NRT2.5) is a plasma membrane-localized high-affinity nitrate transporter playing an essential role in adult plants under severe nitrogen starvation. NRT2.5 expression is induced under nitrogen starvation and NRT2.5 becomes the most abundant transcript amongst the seven NRT2 family members in shoots and roots of adult plants after long-term starvation. GUS reporter analyses showed that NRT2.5 is expressed in the epidermis and the cortex of roots at the root hair zone and in minor veins of mature leaves. Reduction of NRT2.5 expression resulted in a decrease in high-affinity nitrate uptake without impacting low-affinity uptake. In the background of the high-affinity nitrate transporter mutant nrt2.4, an nrt2.5 mutation reduced nitrate levels in the phloem of N-starved plants further than in the single nrt2.4 mutants. Growth analyses of multiple mutants between NRT2.1, NRT2.2, NRT2.4, and NRT2.5 revealed that NRT2.5 is required to support growth of nitrogen-starved adult plants by ensuring the efficient uptake of nitrate collectively with NRT2.1, NRT2.2 and NRT2.4 and by taking part in nitrate loading into the phloem during nitrate remobilization.

Targeting novel chemical and constitutive primed metabolites against Plectosphaerella cucumerina.

Priming is a physiological state for protection of plants against a broad range of pathogens, and is achieved through stimulation of the plant immune system. Various stimuli, such as beneficial microbes and chemical induction, activate defense priming. In the present study, we demonstrate that impairment of the high-affinity nitrate transporter 2.1 (encoded by NRT2.1) enables Arabidopsis to respond more quickly and strongly to Plectosphaerella cucumerina attack, leading to enhanced resistance. The Arabidopsis thaliana mutant lin1 (affected in NRT2.1) is a priming mutant that displays constitutive resistance to this necrotroph, with no associated developmental or growth costs. Chemically induced priming by ß-aminobutyric acid treatment, the constitutive priming mutant ocp3 and the constitutive priming present in the lin1 mutant result in a common metabolic profile within the same plant-pathogen interactions. The defense priming significantly affects sugar metabolism, cell-wall remodeling and shikimic acid derivatives levels, and results in specific changes in the amino acid profile and three specific branches of Trp metabolism, particularly accumulation of indole acetic acid, indole-3-carboxaldehyde and camalexin, but not the indolic glucosinolates. Metabolomic analysis facilitated identification of three metabolites in the priming fingerprint: galacturonic acid, indole-3-carboxylic acid and hypoxanthine. Treatment of plants with the latter two metabolites by soil drenching induced resistance against P. cucumerina, demonstrating that these compounds are key components of defense priming against this necrotrophic fungus. Here we demonstrate that indole-3-carboxylic acid induces resistance by promoting papillae deposition and H2 O2 production, and that this is independent of PR1, VSP2 and PDF1.2 priming.

A key role of the mitochondrial citrate carrier (SLC25A1) in TNFα- and IFNγ-triggered inflammation.

The chronic induction of inflammation underlies multiple pathological conditions, including metabolic, autoimmune disorders and cancer. The mitochondrial citrate carrier (CIC), encoded by the SLC25A1 gene, promotes the export of citrate from the mitochondria to the cytoplasm, a process that profoundly influences energy balance in the cells. We have previously shown that SLC25A1 is a target gene for lipopolysaccharide signaling and promotes the production of inflammatory mediators. We now demonstrate that SLC25A1 is induced at the transcriptional level by two key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ), and such induction involves the activity of the nuclear factor kappa B and STAT1 transcription factors. By studying the down-stream events following SLC25A1 activation during signals that mimic inflammation, we demonstrate that CIC is required for regulating the levels of nitric oxide and of prostaglandins by TNFα or IFNγ. Importantly, we show that the citrate exported from mitochondria via CIC and its downstream metabolic intermediate, acetyl-coenzyme A, are necessary for TNFα or IFNγ to induce nitric oxide and prostaglandin production. These findings provide the first line of evidence that the citrate export pathway, via CIC, is central for cytokine-induced inflammatory signals and shed new light on the relationship between energy metabolism and inflammation.

Pendrin gene ablation alters ENaC subcellular distribution and open probability.

The present study explored whether the intercalated cell Cl(-)/HCO3(-) exchanger pendrin modulates epithelial Na(+) channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na(+) absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na(+) absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddle's syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddle's syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddle's syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability.

Inhibition of nitrate transporter 1.1-controlled nitrate uptake reduces cadmium uptake in Arabidopsis.

Identification of mechanisms that decrease cadmium accumulation in plants is a prerequisite for minimizing dietary uptake of cadmium from contaminated crops. Here, we show that cadmium inhibits nitrate transporter 1.1 (NRT1.1)-mediated nitrate (NO3 (-)) uptake in Arabidopsis (Arabidopsis thaliana) and impairs NO3 (-) homeostasis in roots. In NO3 (-)-containing medium, loss of NRT1.1 function in nrt1.1 mutants leads to decreased levels of cadmium and several other metals in both roots and shoots and results in better biomass production in the presence of cadmium, whereas in NO3 (-)-free medium, no difference is seen between nrt1.1 mutants and wild-type plants. These results suggest that inhibition of NRT1.1 activity reduces cadmium uptake, thus enhancing cadmium tolerance in an NO3 (-) uptake-dependent manner. Furthermore, using a treatment rotation system allowing synchronous uptake of NO3 (-) and nutrient cations and asynchronous uptake of cadmium, the nrt1.1 mutants had similar cadmium levels to wild-type plants but lower levels of nutrient metals, whereas the opposite effect was seen using treatment rotation allowing synchronous uptake of NO3 (-) and cadmium and asynchronous uptake of nutrient cations. We conclude that, although inhibition of NRT1.1-mediated NO3 (-) uptake by cadmium might have negative effects on nitrogen nutrition in plants, it has a positive effect on cadmium detoxification by reducing cadmium entry into roots. NRT1.1 may regulate the uptake of cadmium and other cations by a common mechanism.