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A number of artificial cation ionophores (or transporters) have been developed for basic research and biomedical applications. However, their mechanisms of action and the putative correlations between changes in intracellular cation concentrations and induced cell death remain poorly understood. Here we show that three hemispherand-strapped calix[4]pyrrole based ion pair receptors act as efficient Na+/K+ exchangers in the presence of Cl- in liposomal models, and promote Na+ influx and K+ efflux (Na+/K+ exchange) in cancer cells to induce apoptosis. Mechanistic studies reveal that these cation exchangers induce endoplasmic reticulum (ER) stress in cancer cells by perturbing intracellular cation homeostasis, promote generation of reactive oxygen species, and eventually enhance mitochondria-mediated apoptosis. However, they neither induce osmotic stress nor affect autophagy. The present study provides support for the notion that synthetic receptors which perturb cellular cation homeostasis may provide a new approach to generating agents with potentially useful apoptotic activity.

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain (TMD), in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal, or multiple metals, from the cytoplasm, and it is not known if the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the role of the CTD in metal recognition and selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of their binding sites, thermodynamics and binding-dependent conformations, both in crystal form and in solution, which suggests a varying level of functional discrimination between CDF domains. Furthermore, these results provide the first direct evidence that CDF CTDs play a role in metal selectivity. We demonstrate that MamM's CTD can discriminate against Mn2+, supporting its postulated role in preventing magnetite formation poisoning in magnetotactic bacteria via Mn2+ incorporation.

Mammalian Na+/H+ exchanger type I isoform (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH (pHi) by removing one intracellular proton in exchange for one extracellular sodium ion. Abnormal activity of the protein occurs in cardiovascular disease and breast cancer. The purpose of this study is to examine the role of negatively charged amino acids of extracellular loop 3 (EL3) in the activity of the NHE protein. We mutated glutamic acid 217 and aspartic acid 226 to alanine, and to glutamine and asparagine, respectively. We examined effects on expression levels, cell surface targeting and activity of NHE1, and also characterized affinity for extracellular sodium and lithium ions. Individual mutation of these amino acids had little effect on protein function. However, mutation of both these amino acids together impaired transport, decreasing the Vmax for both Na+ and Li+ ions. We suggested that amino acids E217 and D226 form part of a negatively charged coordination sphere, which facilitates cation transport in the NHE1 protein.

Manganese (Mn) is an essential micronutrient for plants. This study elucidates the physiological consequences and characterization of TaNRAMP1 transporter in Mn-starved wheat. The cellular integrity, redox status, chlorophyll score, and Fv/Fm were severely affected, accompanied by decreased Mn concentration in root and shoot in Mn-deficient wheat. However, Fe concentration and root phytosiderophore release were not affected, contradicting the interactions of Fe status with Mn under Mn shortage. The genome-wide identification of TaNRAMP1 (natural resistance-associated macrophage protein 1), known as high-affinity Mn transporter, showed several polymorphisms within genome A, B, and D. The expression of TaNRAMP1 significantly decreased in roots of genome A and B but was constitutively expressed in genome D due to Mn-deficiency. The TaNRAMP1 was located in the plasma membrane and showed six motifs matched to Nramp (divalent metal transport). Further, TaNRAMP1 showed a close partnership with cation transporter associated with P-type ATPase/cation transport network. In the RNASeq platform, TaNRAMP1, located in all three genomes, showed the highest expression potential in microspore. Besides, only TaNRAMP1 in genome D was upregulated due to heat and drought stress but showed downregulation in response to excess sulfur and Puccinia triticina infection in all three genomes. The cis-regulatory analysis implies the transcriptional regulation of TaNRAMP1 linked to methyl jasmonate and abscisic acid synthesis. Furthermore, TaNRAMP1 proteins showed similar physicochemical properties, but the C-terminus position of genome D was different than genome A and B. This is the first study on the responses and genome-wide characterization of TaNRAMP1 in Mn-starved wheat.

Background Cotton (Gossypium hirsutum) is considered a fairly salt tolerant crop however, salinity can still cause significant economic losses by affecting the yield and deteriorating the fiber quality. We studied a salt-tolerant upland cotton cultivar under temporal salt stress to unfold the salt tolerance molecular mechanisms. Biochemical response to salt stress (400 mM) was measured at 0 h, 3 h, 12 h, 24 h and 48 h post stress intervals and single-molecule long-read sequencing technology from Pacific Biosciences (PacBio) combined with the unique molecular identifiers approach was used to identify differentially expressed genes (DEG). Results Antioxidant enzymes including, catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) were found significantly induced under temporal salt stress, suggesting that reactive oxygen species scavenging antioxidant machinery is an essential component of salt tolerance mechanism in cotton. We identified a wealth of novel transcripts based on the PacBio long reads sequencing approach. Prolonged salt stress duration induces high number of DEGs. Significant numbers of DEGs were found under key terms related to stress pathways such as “response to oxidative stress”, “response to salt stress”, “response to water deprivation”, “cation transport”, “metal ion transport”, “superoxide dismutase”, and “reductase”. Key DEGs related to hormone (abscisic acid, ethylene and jasmonic acid) biosynthesis, ion homeostasis (CBL-interacting serine/threonine-protein kinase genes, calcium-binding proteins, potassium transporter genes, potassium channel genes, sodium/hydrogen exchanger or antiporter genes), antioxidant activity (POD, SOD, CAT, glutathione reductase), transcription factors (myeloblastosis, WRKY, Apetala 2) and cell wall modification were found highly active in response to salt stress in cotton. Expression fold change of these DEGs showed both positive and negative responses, highlighting the complex nature of salt stress tolerance mechanisms in cotton. Conclusion Collectively, this study provides a good insight into the regulatory mechanism under salt stress in cotton and lays the foundation for further improvement of salt stress tolerance.

Cases of congenital disorders of glycosylation (CDG) have been associated with specific mutations within the gene encoding the human Golgi TMEM165 (transmembrane protein 165), belonging to UPF0016 (uncharacterized protein family 0016), a family of secondary ion transporters. To date, members of this family have been reported to be involved in calcium, manganese, and pH homeostases. Although it has been suggested that TMEM165 has cation transport activity, direct evidence for its Ca(2+)- and Mn(2+)-transporting activities is still lacking. Here, we functionally characterized human TMEM165 by heterologously expressing it in budding yeast (Saccharomyces cerevisiae) and in the bacterium Lactococcus lactis. Protein production in these two microbial hosts was enhanced by codon optimization and truncation of the putatively autoregulatory N terminus of TMEM165. We show that TMEM165 expression in a yeast strain devoid of Golgi Ca(2+) and Mn(2+) transporters abrogates Ca(2+)- and Mn(2+)-induced growth defects, excessive Mn(2+) accumulation in the cell, and glycosylation defects. Using bacterial cells loaded with the fluorescent Fura-2 probe, we further obtained direct biochemical evidence that TMEM165 mediates Ca(2+) and Mn(2+) influxes. We also used the yeast and bacterial systems to evaluate the impact of four disease-causing missense mutations identified in individuals with TMEM165-associated CDG. We found that a mutation leading to a E108G substitution within the conserved UPF0016 family motif significantly reduces TMEM165 activity. These results indicate that TMEM165 can transport Ca(2+) and Mn(2+), which are both required for proper protein glycosylation in cells. Our work also provides tools to better understand the pathogenicity of CDG-associated TMEM165 mutations.

Aims (i) To analyse the increase in calcium ion uptake caused by several cationic dyes on Candida albicans, (ii) to postulate a mechanism, (iii) to define the effects of Zn ions on the phenomenon, and (iv) to propose the use of the dyes or their derivatives against C. albicans. Methods and results Cells were grown in yeast peptone dextrose medium and starved. We measured the hydrophobic solvent/water partition coefficients and the dyes uptake by the cells and found no correlation with their hydrophobicity. Most of the dyes caused an increase in K+ efflux (in correlation with a decrease in 86 Rb+ uptake), and a raise in Ca2+ uptake except for those used as Zn salts, but not of their HCl salts. Respiration and acidification of the medium were modified only with few dyes and interestingly, when exposing cultures to nile blue, neutral red and toluidine blue ZnCl2 a decrease in C. albicans growth was observed. Conclusions We propose a general mechanism for the stimulation of Ca2+ uptake by the dyes used. Some of the dyes tested might be used as agents against C. albicans, probably combined with other agents. Moreover, the effects of Zn ions on Ca2+ uptake and on cell growth open possibilities of further studies, not only of their effects, but also of the mechanism of Ca2+ transport in C. albicans and other yeasts. Significance and impact of the study This study, in conjunction with previously published results, contribute to the basic research regarding ion transport in C. albicans and the role of zinc in this process. Besides, suggests the additional use of dyes, along with other antifungals agents, as combined therapy against candidiasis. Derived dyes from those used also might be possible therapeutic agents against this disease.

Due to their preferential cation transport, dense cation exchange membranes like Nafion membranes are unsuitable for Zn/Br redox flow batteries which require bi-ionic transport of Zn2+ and Br- ions. This work shows that scaling the water cluster size of Nafion membranes by a pre-hydration treatment can achieve not only a high ionic conductivity but also a bi-ionic transport property. Small angle X-Ray scattering, diffusion cell, and electrochemical analysis verify that by increasing the pre-hydration temperature, the water clusters are expanded, resulting in an increase of water uptake, ionic conductivity and the anion transference number. The bi-ionic transport and low area specific resistance induced by the pretreatment enable the successful operation of a Zn/Br redox flow battery with a NRE-212 membrane. Compared with a conventional porous membrane, the properly treated Nafion membrane has an 11.3% higher energy efficiency indicating that the dense structured ion exchange membrane can be used for Zn/Br flow batteries by scaling the water cluster size.

International audience; This paper addresses hybrid ion exchange membranes fabricated by the synthesis of amorphous zirconiumphosphate (dopant contents from 0.5 to 24 wt%) directly in the pore and channel system of heterogeneouscation-exchange membrane RALEX® CM (by in situ technique). The incorporation of zirconium phosphate nanoparticlesinto the membrane system of pores and channels leads to the displacement of the pore water. As aresult, the cation transport numbers increase. The hybrid materials thus obtained are characterized by increasedionic resistance and enhanced monovalent ion selectivity. The former effect was eliminated by fabrication of asurface-modified membrane. The relative simplicity of modification, together with the benefits of the hybridmaterials make them promising for some membrane processes. Using 31P MAS NMR and elemental analysis,considerable difference between the zirconium phosphate composition inside and outside the membrane wasfound.

Ion exchange membranes (IEMs) have been established as a key component in industrial water desalination and electrolysis processes. Thus, nowadays, they are being studied and developed for application in new energy conversion and storage systems as well as efficient desalination and wastewater treatment processes. These processes include redox flow batteries, reverse electrodialysis, membrane capacitive deionization, microbial fuel cells, and ion exchange membrane bioreactors. Ion permselectivity between counter- and co-ions, the most essential property in IEMs, makes these processes possible and/or efficient. Additionally, ion selectivity between different counter-ions is required for these novel processes to be efficient. This review aims to provide a comprehensive overview of ion exchange membrane permselectivity by summarizing the developments in this field over the past decade. Membrane microstructure and possible mechanisms for ion transport in the membrane phase are discussed with respect to permselectivity, along with the influence of current density and related membrane-solution boundary conditions. A selectivity order for the transport of common anions through conventional anion exchange membranes (AEMs) is generalized, the same is done for cation transport through conventional cation exchange membranes (CEMs). Two types of experimental methods for the determination of ion permselectivity: electrodialysis and the membrane potential method, are summarized. Finally, relevant membrane preparation methods and surface modification of IEMs reported over the past decade are classified and discussed. In summary, we conclude that synthetic methods have evolved rapidly; however, development of fundamental knowledge on the many complex phenomena occurring in the adaptive membrane bulk and solution-interface environments is not sufficiently established. Thus, much effort is required to combine experimental, theoretical, and simulation data for a more comprehensive and coherent understanding of these phenomena.

The present work describes a novel and cleaner approach for large scale (litre) separation of cesium from simulated nuclear waste solutions by electrodriven transport through a hollow fiber supported liquid membrane (HFSLM) containing chlorinated cobalt dicarbollide as the carrier. To the best of our knowledge, this is the first report on electrodriven cation transport through HFSLM. The transport selectivity of Cs + over Na + and other fission products was studied from synthetic nuclear waste solutions. An attempt has also been made to pre-concentrate radio-cesium from an environmental sample containing very low level of 137 Cs (≤ 10 –12 M) with excellent transport efficiency. A very high decontamination factor of cesium over Na + other metal ions have been obtained for all types of feed solution. For neutral feed solution, the current efficiency for the Cs + transport has been found to be 100%. The present work certainly adds a new dimension to the ongoing membrane based separations.

The transport selectivity of different cations through cation exchange membranes (CEMs) could be estimated with the partitioning selectivity factor ( K j i ) and the cation mobility ratio in the membrane ( u i m / u j m ), which in turn can be related to corresponding membrane conductivity and dimensional swelling ratio data [Journal of Membrane Science, 2020, 597, 117645]. This method has been validated in two hydrocarbon polymer-based CEMs, and the obtained K+/Na+ selectivity equals to the one obtained with conventional electrodialysis (ED) method. However, the K+/Na+ selectivity of perfluorosulfonic acid (PFSA) membranes, and the bi-/monovalent cation (Mg2+/Na+) selectivity of all three types of CEMs estimated with this ionic conductivity experimental approach deviate noticeably from corresponding values obtained with ED. In this work, it is proved that this deviation is partly due to the simplification of cation activity coefficients in the membrane. Here, the cation activity coefficients in three types of CEMs are calculated according to Manning's counter-ion condensation model. In this model, the Manning parameter ( ξ ) characterizing the dimensionless linear charge density is determined by the average distance between two adjacent fixed sulfonate groups ( b ) and the permittivity of hydrated membranes ( e ). In hydrocarbon polymer-based CEMs, the average distance between fixed sulfonate groups can be estimated by assuming homogeneous distribution of the fixed groups, while in PFSA membranes three representative structure models are employed to estimate this average distance. After accounting for the cation activity coefficients in the membrane, the Mg2+/Na+ cation transport selectivity obtained with the ionic conductivity experimental approach gets closer to, but is still smaller than the selectivity obtained with the ED method. This work shows the importance of cation activity coefficients in the membrane phase in interpreting the membrane transport properties, and complements the proposed conductivity approach to characterize the counter-ion selectivity of ion exchange membranes.

Reverse electrodialysis of saline solutions (RED) is a renewable energy technology with large potential. Key components in the system are the ion exchange membranes. This work evaluates the efficiency of commercially available anion and cation selective membranes for RED. Their efficiency is often described with the apparent transport number for the ion in question, and this number depends on the salt concentration. It is known since long that the water transference coefficient contributes to the apparent transport number, but much of the recent literature on RED takes the permselectivity as defined by the apparent transport number as a direct measure of the membrane selectivity. The purpose of this paper is to clarify that situation with new data on Fumasep membranes. Concentration cell potentials were measured for anion- and cation exchange membranes from Fumasep (FAD and FKS, respectively, Fumatech, Germany) in the temperature range 12−45°C12−45°C in salt solutions relevant for reverse electrodialysis. The results show that the anion exchange membrane is a perfectly selective ion exchange membrane, and has a water transference coefficient of −6±1−6±1. The cation selective membrane has a cation transport number of 0.93 and a water transference coefficient of 8±8± 7. We suggest that the developers of membranes should pay more attention to the water transference coefficient. To enhance the performance of ion selective membranes and RED, it is beneficial to have water transference coefficients as close to zero as possible. Attribution 4.0 International (CC BY 4.0)

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state. Fil: Faraj, Santiago Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Centeno, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Wild-type cells of Candida albicans, the most common human fungal pathogen, are able to grow at very low micromolar concentrations of potassium in the external milieu. One of the reasons behind that behaviour is the existence of three different types of K+ transporters in their plasma membrane: Trk1, Acu1 and Hak1. This work shows that the transporters are very differently regulated at the transcriptional level upon exposure to saline stress, pH alterations or K+ starvation. We propose that different transporters take the lead in the diverse environmental conditions, Trk1 being the “house-keeping” one, and Acu1/Hak1 dominating upon K+ limiting conditions. Heterologous expression of the genes coding for the three transporters in a Saccharomyces cerevisiae strain lacking its endogenous potassium transporters showed that all of them mediated cation transport but with very different efficiencies. Moreover, expression of the transporters in S. cerevisiae also affected other physiological characteristics such as sodium and lithium tolerance, membrane potential or intracellular pH, being, in general, CaTrk1 the most effective in keeping these parameters close to the usual wild-type physiological levels.

The present paper is devoted to the properties of sulfonated cation-exchange membranes obtained by radiation chemical grafting polymerization of styrene with divinylbenzene on a polyethylene film. The mechanical and transport properties (conductivity, diffusion permeability, and transport numbers) of the membranes obtained with various amounts of a cross-linking agent (0–3.5%) and degrees of polystyrene grafting (23–32%) were investigated in comparison with commercially available membranes, i.e. Nafion® 117 and Neosepta® CMX. Tensile strength values around 15–20 MPa were obtained, and they increased with increasing cross-linker amounts. The conductivity of membranes containing no cross-linking agent reached 8.4·10 −2 Ω −1 cm −1 at 25 °C and high relative humidity. As the amount of the cross-linking agent increased, the water uptake in membranes and their conductivity at high relative humidity decreased, whereas the activation energy of conductivity increased. At low humidity (RH=30%), the water uptake and conductivity increased with increasing amounts of the cross-linking agent. The membranes based on cross-linked polystyrene had low diffusion permeability, while the associated cation transport numbers were higher than those with commercial Nafion® 117 membrane. The calculated theoretical power of reverse electrodialysis batteries based on the best membranes was 10% higher than that of the battery based on the Neosepta® CMX membrane.

Two-pore channels (TPCs) constitute a family of intracellular cation channels with diverse permeation properties and functions in animals and plants. In the model plant Arabidopsis, the vacuolar cation channel TPC1 is involved in propagation of calcium waves and in cation homeostasis. Here, we discovered that the dimerization of a predicted helix within the carboxyl-terminus (CTH) is essential for the activity of TPC1. Bimolecular fluorescence complementation and co-immunoprecipitation demonstrated the interaction of the two C-termini and pointed towards the involvement of the CTH in this process. Synthetic CTH peptides dimerized with a dissociation constant of 3.9 µM. Disruption of this domain in TPC1 either by deletion or point mutations impeded the dimerization and cation transport. The homo-dimerization of the CTH was analyzed in silico using coarse-grained molecular dynamics (MD) simulations for the study of aggregation, followed by atomistic MD simulations. The simulations revealed that the helical region of the wild type, but not a mutated CTH forms a highly stable, antiparallel dimer with characteristics of a coiled-coil. We propose that the voltage- and Ca2+-sensitive conformation of TPC1 depends on C-terminal dimerization, adding an additional layer to the complex regulation of two-pore cation channels. Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2131-3) contains supplementary material, which is available to authorized users.

Outer-membrane c-type cytochrome (OM c-Cyt) complexes in several genera of iron-reducing bacteria, such as Shewanella and Geobacter, are capable of transporting electrons from the cell interior to extracellular solids as a terminal step of anaerobic respiration. The kinetics of this electron transport has implications for controlling the rate of microbial electron transport during bioenergy or biochemical production, iron corrosion, and natural mineral cycling. Herein, we review the findings from in-vivo and in-vitro studies examining electron transport kinetics through single OM c-Cyt complexes in Shewanella oneidensis MR-1. In-vitro electron flux via a purified OM c-Cyt complex, comprised of MtrA, B, and C proteins from S. oneidensis MR-1, embedded in a proteoliposome system is reported to be 10- to 100-fold faster compared with in-vivo estimates based on measurements of electron flux per cell and OM c-Cyts density. As the proteoliposome system is estimated to have 10-fold higher cation flux via potassium channels than electrons, we speculate that the slower rate of electron-coupled cation transport across the OM is responsible for the significantly lower electron transport rate that is observed in-vivo. As most studies to date have primarily focused on the energetics or kinetics of interheme electron hopping in OM c-Cyts in this microbial electron transport mechanism, the proposed model involving cation transport provides new insight into the rate detemining step of EET, as well as the role of self-secreted flavin molecules bound to OM c-Cyt and proton management for energy conservation and production in S. oneidensis MR-1.

Electrodialysis (ED) technique is a typical and promising membrane process, for applications like water treatment and ion separation. Especially, the separation between monovalent and multivalent cations is a current central issue in many industries and academic researches. Herein, a series of novel cation exchange membranes were prepared from quaternized polyaniline modified sulfonated polyphenyl sulfone (SPPSU) by in-situ polymerization-deposited polyaniline followed by quaternizing with methyl iodide. Restricted divalent cation penetration and regulated monovalent cation transport were achieved by elevating the kinetic effect of electrostatic repulsion and narrowing water channel. Importantly, positive charge density of modification layer could be adjusted by controlling the degree of quaternization, which was accompanied by the change of water channel. When evaluated in a simulated mixed salt system (Mg2+/Na+ and Mg2+/Li+), the optimal quaternized polyaniline membrane exhibited a higher perm-selectivity (PNaMg=4.1, PLiMg=1.75) than initial polyaniline modified cation exchange membrane (PNaMg=0.8, PLiMg=0.75), and commercial monovalent-selective cations exchange membrane CIMS (PNaMg=3.56, PLiMg=1.11). This presented strategy is straightforward and effective, demonstrating the effect of surface positive charge layer on perm-selectivity.

Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.

7 páginas, 3 figuras, Here, we review and update the recent advances in the metabolic control during the adaptive response of budding yeast to hyperosmotic and salt stress, which is one of the best understood signaling events at the molecular level. This environmental stress can be easily applied and hence has been exploited in the past to generate an impressively detailed and comprehensive model of cellular adaptation. It is clear now that this stress modulates a great number of different physiological functions of the cell, which altogether contribute to cellular survival and adaptation. Primary defense mechanisms are the massive induction of stress tolerance genes in the nucleus, the activation of cation transport at the plasma membrane, or the production and intracellular accumulation of osmolytes. At the same time and in a coordinated manner, the cell shuts down the expression of housekeeping genes, delays the progression of the cell cycle, inhibits genomic replication, and modulates translation efficiency to optimize the response and to avoid cellular damage. To this fascinating interplay of cellular functions directly regulated by the stress, we have to add yet another layer of control, which is physiologically relevant for stress tolerance. Salt stress induces an immediate metabolic readjustment, which includes the up-regulation of peroxisomal biomass and activity in a coordinated manner with the reinforcement of mitochondrial respiratory metabolism. Our recent findings are consistent with a model, where salt stress triggers a metabolic shift from fermentation to respiration fueled by the enhanced peroxisomal oxidation of fatty acids. We discuss here the regulatory details of this stress-induced metabolic shift and its possible roles in the context of the previously known adaptive functions., The work of the authors was supported by grants from Ministerio de Economía y Competitividad (BFU2011-23326 and BFU2016-75792-R).

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes. ispartof: MOLECULAR OMICS vol:14 issue:3 pages:181-196 ispartof: location:England status: published

The ability of prokaryotic microbes to produce and respond to neurochemicals that are more often associated with eukaryotic systems is increasingly recognized through the concept of microbial endocrinology. Most studies have described the phenomena of neurochemical production by bacteria, but there remains an incomplete understanding of the mechanisms by which microbe- or host-derived neuroactive substances can be recognized by bacteria. Based on the evolutionary origins of eukaryotic solute carrier transporters, we hypothesized that bacteria may possess an analogous uptake function for neuroactive biogenic amines. Using specific fluorescence-based assays, Lactobacillus salivarius biofilms appear to express both plasma membrane monoamine transporter (PMAT)- and organic cation transporter (OCT)-like uptake of transporter-specific fluorophores. This phenomenon is not distributed throughout the genus Lactobacillus as L. rhamnosus biofilms did not take up these fluorophores. PMAT probe uptake into L. salivarius biofilms was attenuated by the protonophore CCCP, the cation transport inhibitor decynium-22, and the natural substrates norepinephrine, serotonin and fluoxetine. These results provide the first evidence, to our knowledge, for the existence of PMAT- and OCT-like uptake systems in a bacterium. They also suggest the existence of a hitherto unrecognized mechanism by which a probiotic bacterium may interact with host signals and may provide a means to examine microbial endocrinology-based interactions in health and disease that are part of the larger microbiota-gut-brain axis.

Chinese mitten crabs (Eriocheir japonica sinensis) are catadromous, spending most of their lives in fresh water, but moving to a mixed salt-fresh water environment for reproduction. The characteristics of this life history might imply a rapidly evolutionary transition model for adaptation to marine from freshwater habitats. In this study, transcriptome-wide identification and differential expression on Chinese mitten crab groups were analysed. Results showed: clean reads that were obtained totalled 93,833,096 (47,440,998 in Group EF, the reference, and 46,392,098 in Group ES, the experimental) and 14.08G (7.12G in Group EF 6.96G in Group ES); there were 11,667 unigenes (15.29%) annotated, and they were located to 230 known KEGG pathways in five major categories; in differential expression analysis, most of the top 20 up-regulated pathways were connected to the immune system, disease, and signal transduction, while most of the top 20 down-regulated pathways were related to the metabolism system; meanwhile, 8 representative osmoregulation-related genes (14-3-3 epsilon, Cu2+ transport ATPase, Na+/K+ ATPase, Ca2+ transporting ATPase, V-ATPase subunit A, Putative arsenite-translocating ATPase, and Cation transport ATPase, Na+/K+ symporter) showed up-regulation, and 1 osmoregulation-related gene (V-ATPase subunit H) showed down-regulation. V-ATPase subunit H was very sensitive to the transition of habitats. These results were consistent with the tests of qRT-PCR. The present study has provided a foundation to further understand the molecular mechanism in response to salinity changing in water.

Rutile TiO 2 was synthesized using the sol gel method and used for the preparation of polymer composite electrolyte membrane of TiO 2 +SPEEK. The composite membranes prepared by solvent casting method showed improved oxygen mass transfer coefficient values and the transport of cations other than protons. The antifouling property of the composite membranes was observed in SEM images which were additionally supported by hemocytometer results. The improved ion exchange and water absorption capacity of composite membranes showed enhanced proton conductivity and electrochemical properties for the composite membranes. The prepared composite membranes were evaluated in single chamber microbial fuel cell and the results were compared with that of the Nafion 117 ® under similar conditions. The results revealed that all the prepared composite membranes showed higher maximum power density and current density than that of the commercially available Nafion117 ® . Among the prepared membranes, SPEEK+7.5% TiO 2 composite membrane showed the highest power density (98.1 mW/m 2 ) and current density (300 mA/m 2 ) along with high proton conductivity, antifouling property.

Bothrops jararacussu venom drastically decreases sarcoplasmic Ca(2+)-ATPase (SERCA) protein expression in vivo and inhibits its activity in vitro, in contrast to a slight increase of Na(+)/K(+)-ATPase expression in murine EDL. We investigated the effect of myotoxins bothropstoxin-I and/or -II (BthTX-I, BthTX-II and BthTX-I+II) on this model. No changes were seen in SERCA1, SERCA2 and Na(+)/K(+)-ATPase α1 protein expression as well as (2+)Ca-ATPase activity, but BthTX-II (1 μg/g) reduced Na(+)/K(+)-ATPase α2 expression by 50% one day after perimuscular injection. Interestingly, BthTX-II inhibited Ca(2+)-ATPase activity (IC50 around 6 nM). Our findings suggest that only BthTX-II affects ion transport ATPases, being a potent SERCA inhibitor and a putative target for antivenom drug development.

The P1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. An important feature of P1B-ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity. Only one type of MBD has been characterized extensively, but bioinformatics analyses indicate that a diversity of MBDs may exist in nature. Here we report the biochemical, structural and functional characterization of a new MBD from the Cupriavidus metallidurans P1B-4-ATPase CzcP (CzcP MBD). The CzcP MBD binds two Cd(2+), Co(2+) or Zn(2+) ions in distinct and unique sites and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Both in vitro and in vivo activity assays using full-length CzcP, truncated CzcP and several variants indicate a regulatory role for the MBD and distinct functions for the two metal binding sites. Taken together, these findings elucidate a previously unknown MBD and suggest new regulatory mechanisms for metal transport by P1B-ATPases.

Robust oligopeptides that mimic natural ion channels are attractive for use as molecular switches or model systems to study ion transport. Herein, we report octapeptides derived from aminobenzoic acid and l/d amino acids. Two of the alanine containing peptides were found to be most active and the peptide containing p-aminobenzoic acid was found to be most active (2.4 times its m-analog). Experimental studies indicate the peptides do not transport halides and transport alkali metal ions. � 2015 Elsevier Ltd. All rights reserved.

Mdm31p is an inner mitochondrial membrane (IMM) protein with unknown function in Saccharomyces cerevisiae. Mutants lacking Mdm31p contain only a few giant spherical mitochondria with disorganized internal structure, altered phospholipid composition and disturbed ion homeostasis, accompanied by increased resistance to the electroneutral K+/H+ ionophore nigericin. These phenotypes are interpreted as resulting from diverse roles of Mdm31p, presumably in linking mitochondrial DNA (mtDNA) to the machinery involved in segregation of mitochondria, in mediating cation transport across IMM and in phospholipid shuttling between mitochondrial membranes. To investigate which of the roles of Mdm31p are conserved in ascomycetous yeasts, we analysed the Mdm31p orthologue in Schizosaccharomyces pombe. Our results demonstrate that, similarly to its S. cerevisiae counterpart, SpMdm31 is a mitochondrial protein and its absence results in increased resistance to nigericin. However, in contrast to S. cerevisiae, Sz. pombe cells lacking SpMdm31 are also less sensitive to the electrogenic K+ ionophore valinomycin. Moreover, mitochondria of the fission yeast mdm31Δ mutant display no changes in morphology or phospholipid composition. Therefore, in terms of function, the two orthologous proteins appear to have considerably diverged between these two evolutionarily distant yeast species, possibly sharing only their participation in ion homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.

The fungal cell membrane is one of the targets for some groups of antifungal agents: the polyenes, lipodepsinonapeptides, amino acids and other peptides, which interferes with the structural integrity of the lipid bilayer. Polyene antifungals show effectiveness against sterol-containing organisms by interacting directly with the fungal ergosterol. Amphotericin B exhibits activity against most species of Candida, Cryptococcus neoformans, most Aspergillus sp., hyaline molds, Penicillium, Paecilomyces, and Scopulariopsis, dimorphic fungi including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Paracoccidioides brasiliensis. Nystatin binds to sterols in the cell membrane of yeasts resulting in electrolyte leakages. Statins are competitive inhibitors of HMG-CoA reductase, which is involved in the ergosterol synthesis. The azoles specifically inhibits the cytochrome P450 enzyme CYP51, resulting in interruption of membrane synthesis, depletion of ergosterol leading to an increase in toxic methylated sterol precursors in the cell membrane. The morpholines are synthetic antifungals and inhibits the reductase and isomerase enzymes in ergosterol biosynthesis triggering accumulation of dimethyllanosterol and depletion of ergosterol. Aureobasidins are highly lipophilic and act by inhibiting inositol phosphoceramide (IPC) synthase, critical in fungal sphingolipid biosynthesis thereby depleting essential sphingolipids in the fungal cells. The lipopeptide iturin acts by increasing membrane permeabilization in fungal cell. Lipodepsinonapeptides produced by Pseudomonas syringae interacts with the fungal cell membrane by pore formation resulting in fatal electrolytic leakage. Syringomycins form ion channels in the fungal plasma membrane employing a novel sphingolipid-modulated channel formation mechanism while Pseudomycin A alters several membrane functions like membrane potential, protein phosphorylation, H1-ATPase activity, and cation transport fluxes.

Biodegradable magnesium (Mg) materials are considered ideal as osteosynthesis implants. However, clinical application has proven complex. This is primarily associated with the issue of reducing the extent of implant degradation to a range acceptable for the human body, while simultaneously enhancing osteogenesis or osteoinduction. In the present study, a combination of Mg ions and low‑intensity pulsed ultrasound (LIPUS) treatment was applied in hFOB 1.19 human osteoblast cells as a potential strategy to resolve this issue. A total of 7,314 differentially expressed genes (DEGs) and 826 shared DEGs in hFOB1.19 osteoblast cells were identified by microarray analysis following treatment with Mg and/or LIPUS. Gene Ontology analysis demonstrated that among cells treated with a combination of Mg and LIPUS, DEGs were significantly enriched in various functional annotations, including 'wound healing', 'transforming growth factor beta receptor signaling pathway', 'transcription, DNA‑templated', 'receptor complex', 'nucleus', 'SMAD protein complex', 'DNA binding', 'metal ion binding' and 'GTPase activator activity'. Notably, the transforming growth factor (TGF)‑β, mitogen‑activated protein kinase (MAPK) and tumor necrosis factor (TNF) signaling pathways were preferentially overrepresented in the Mg and LIPUS combination group, which was subsequently confirmed by reverse transcription‑quantitative polymerase chain reaction. Furthermore, genes involved in osteoblast mineralization promotion, including bone morphogenetic protein 6, noggin, bone morphogenetic protein receptor (BMPR)1A, BMPR2 and SMAD 5/8, were significantly upregulated following combination treatment compared with the control group. Genes involved in the promotion of migration, including c‑Jun N‑terminal kinase, doublecortin, paxillin and Jun proto‑oncogene AP‑1 transcription factor subunit, were also upregulated in the combination treatment group compared with the control group. The DEG data were supported by morphological observations of the osteoblasts using alizarin red S staining and wound healing assays, which indicated that Mg and LIPUS combinative treatment had a synergistic effect on osteoblast mineralization and migration. Additionally, the combined treatment significantly upregulated metal transporter genes associated with Mg entry, including ATPase Na+/K+‑transporting subunit α1, cyclin and CBS domain divalent metal cation transport mediator 2, K+ voltage‑gated channel subfamily J member 14, transient receptor potential cation channel (TRP) subfamily M member 7 and TRP subfamily V member 2. In summary, the findings of the present study revealed that combined stimulation with Mg and LIPUS may exhibit a synergistic effect on human osteoblast bone formation through the TGF‑β, MAPK and TNF signaling pathways, while also facilitating Mg influx. The present study demonstrated the potential of combinative LIPUS and Mg treatment as a novel therapeutic strategy for enhancing the osteoinduction, biocompatibility and biosafety of biodegradable Mg implants.

The ability of cyanobacteria to survive many environmental stress factors is a testament to their resilience in nature. Of these environmental stress factors, overexposure to zinc is important to study since excessive zinc intake can be a severe hazard. Zinc toxicity in freshwater has been demonstrated to affects organisms such as invertebrates, algae and cyanobacteria. Cyanobacteria which possess increased resistance to zinc have been isolated. It is therefore important to elucidate the mechanism of survival and response to determine what factors allow their survival; as well as any remediation implications they may have. To characterize the effects of zinc in freshwater cyanobacteria, we investigated the response of Synechococcus sp. IU 625 (S. IU 625) over 29 days to various concentrations (10, 25, and 50 mg/L) of ZnCl2. S. IU 625 was shown to be tolerant up to 25 mg/L ZnCl2 exposure, with 10 mg/L ZnCl2 having no outward physiological change and 50 mg/L ZnCl2 proving lethal to the cells. To determine a potential mechanism Inductive Coupled Plasma–Mass Spectrometry (ICP–MS) and RNA-seq analysis were performed on zinc exposed cells. Analysis performed on days 4 and 7 indicated that response is dose-dependent, with 10 mg/L ZnCl2 exhibiting nearly all zinc extracellular, corresponding with upregulation of cation transport response. Whereas the 25 mg/L ZnCl2 exhibited half of total zinc sequestered by the cells, which corresponds with the upregulation of sequestering proteins such as metallothionein and the downregulation of genes involved with ATP synthesis and phycobilisome assembly. These analyses were combined with growth monitoring, microscopy, quantitative polymerase chain reaction (qPCR) and flow cytometry to present a full spectrum of mechanisms behind zinc response in S. IU 625.

Maintenance of the appropriate intracellular concentrations of monovalent cations, mostly potassium and sodium, is a requirement for living cells, and this is particularly relevant in cells that, like unicellular fungi, are directly exposed to a changing environment. These organisms are endowed with a variety of cation influx and efflux systems. Import of potassium, which is accumulated intracellularly, is achieved by means of at least three different kind of transporters, whereas removal of the surplus of sodium (a relatively toxic cation) is accomplished by a combination of Na + -ATPases and antiporters. In addition, toxic cations can be confined in intracellular organelles, such as the vacuole, with the aid of intracellular transporters. Fine regulation of these elements allows a relatively constant ionic intracellular composition in spite of sudden changes in the external medium.

The purpose of this study is to determine the behavioral and proteomic consequences of shock-induced stress in zebrafish (Danio rerio) as a vertebrate model. Here we describe the behavioral effects of exposure to predictable and unpredictable electric shock, together with quantitative tandem mass tag isobaric labeling workflow to detect altered protein candidates in response to shock exposure. Behavioral results demonstrate a hyperactivity response to electric shock and a suppression of activity to a stimulus predicting shock. On the basis of the quantitative changes in protein abundance following shock exposure, eight proteins were significantly up-regulated (HADHB, hspa8, hspa5, actb1, mych4, atp2a1, zgc:86709, and zgc:86725). These proteins contribute crucially in catalytic activities, stress response, cation transport, and motor activities. This behavioral proteomic driven study clearly showed that besides the rapid induction of heat shock proteins, other catalytic enzymes and cation transporters were rapidly elevated as a mechanism to counteract oxidative stress conditions resulting from elevated fear/anxiety levels.

The colonic epithelium is a highly dynamic system important for the regulation of ion and water homeostasis via absorption and secretion and for the maintenance of a protective barrier between the outer milieu and the inside of the body. These processes are known to gradually change along the length of the colon, although a complete characterization at the protein level is lacking. We therefore analyzed the membrane proteome of isolated human (n = 4) colonic epithelial cells from biopsies obtained via routine colonoscopy for four segments along the large intestine: ascending, transverse, descending, and sigmoid colon. Label-free quantitative proteomic analyses using high-resolution mass spectrometry were performed on enriched membrane proteins. The results showed a stable level for the majority of membrane proteins but a distinct decrease in proteins associated with bacterial sensing, cation transport, and O-glycosylation in the proximal to distal regions. In contrast, proteins involved in microbial defense and anion transport showed an opposing gradient and increased toward the distal end. The gradient of ion-transporter proteins could be directly related to previously observed ion transport activities. All individual glycosyltransferases required for the O-glycosylation of the major colonic mucin MUC2 were observed and correlated with the known glycosylation variation along the colon axis. This is the first comprehensive quantitative dataset of membrane protein abundance along the human colon and will add to the knowledge of the physiological function of the different regions of the colonic mucosa. Mass spectrometry data have been deposited to the ProteomeXchange with the identifier PXD000987.

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.

Metabolic intermediates, such as succinate and citrate, regulate important processes ranging from energy metabolism to fatty acid synthesis. Cytosolic concentrations of these metabolites are controlled, in part, by members of the SLC13 gene family. The molecular mechanism underlying Na+-coupled di- and tricarboxylate transport by this family is understood poorly. The human Na+/dicarboxylate cotransporter NaDC3 (SLC13A3) is found in various tissues, including the kidney, liver, and brain. In addition to citric acid cycle intermediates such as α-ketoglutarate and succinate, NaDC3 transports other compounds into cells, including N-acetyl aspartate, mercaptosuccinate, and glutathione, in keeping with its dual roles in cell nutrition and detoxification. In this study, we construct a homology structural model of NaDC3 on the basis of the structure of the Vibrio cholerae homolog vcINDY. Our computations are followed by experimental testing of the predicted NaDC3 structure and mode of interaction with various substrates. The results of this study show that the substrate and cation binding domains of NaDC3 are composed of residues in the opposing hairpin loops and unwound portions of adjacent helices. Furthermore, these results provide a possible explanation for the differential substrate specificity among dicarboxylate transporters that underpin their diverse biological roles in metabolism and detoxification. The structural model of NaDC3 provides a framework for understanding substrate selectivity and the Na+-coupled anion transport mechanism by the human SLC13 family and other key solute carrier transporters. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Acidity of water molecules coordinated to Co ions in CoFe Prussian blue analogues (PBA) has been used to reversibly activate the Co(III)Fe(II) ↔ Co(II)Fe(III) electron transfer. The study of the structure and the electronic structure shows that the process implies an original PCET reaction between a solid-state porous coordination polymer and hydroxide ions in solution. The PCET reaction spreads throughout the solid network thanks to a long-range H(+) and Rb(+) transport within the pore channels of PBA taking advantage of the hydrogen-bonding network of zeolitic water molecules acting as proton wires.

Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress.

Membrane-spanning M5 and M6 segments, which play a role in the formation of cation transport sites in H(+)-, Ca2(+)-, K(+)-, Na(+)-, and other P2-ATPases, are connected by a short extracytoplasmic loop. In the yeast plasma membrane H(+)-ATPase, which belongs to a family of P2-ATPases, the loop is connected to M5 and M6 through the Asp-714 and Asp-720 residues. In this work, the effect of point amino, acidreplacements of Asp-714 and Asp-720 by Ala, Val, Asn, and Glu residues on the function of the enzyme was studied. The Asp714Asn point mutant possessed activities similar to those of the wild-type enzyme, whereas the replacement of Asp-714 by other amino acid residues disrupted biogenesis and led to a loss of activity. All mutants with substitution of Asp-720 were expressed and possessed relatively high activity. The D720V mutant displayed significantly reduced expression levels, activity, H+ transport, and ATP hydrolyzing activity. Thus, substitutions of Asp-714, except for the D714N mutant, led to significant defects in biogenesis and/or function of the enzyme. The results indicate the important role for the Asp-714 residue in biogenesis, structure stability, and enzyme function.

Aims: Carbon monoxide (CO) delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas. The metal carbonyl Ru(CO)3Cl(glycinate) (CORM-3) is a novel, potent antimicrobial agent. Here, we established its mode of action. Results: CORM-3 inhibits respiration in several bacterial and yeast pathogens. In anoxic Escherichia coli suspensions, CORM-3 first stimulates, then inhibits respiration, but much higher concentrations of CORM-3 than of a classic protonophore are required for stimulation. Proton translocation measurements (H+/O quotients, i.e., H+ extrusion on pulsing anaerobic cells with O2) show that respiratory stimulation cannot be attributed to true “uncoupling,” that is, dissipation of the protonmotive force, or to direct stimulation of oxidase activity. Our data are consistent with CORM-3 facilitating the electrogenic transmembrane movement of K+ (or Na+), causing a stimulation of respiration and H+ pumping to compensate for the transient drop in membrane potential (ΔΨ). The effects on respiration are not mimicked by CO gas or control Ru compounds that do not release CO. Inhibition of respiration and loss of bacterial viability elicited by CORM-3 are reversible by white light, unambiguously identifying heme-containing oxidase(s) as target(s). Innovation: This is the most complete study to date of the antimicrobial action of a CO-RM. Noteworthy are the demonstration of respiratory stimulation, electrogenic ion transport, and photosensitive activity, establishing terminal oxidases and ion transport as primary targets. Conclusion: CORM-3 has multifaceted effects: increased membrane permeability, inhibition of terminal oxidases, and perhaps other unidentified mechanisms underlie its effectiveness in tackling microbial pathogenesis. Antioxid. Redox Signal. 19, 497–509.

Intracellular cations are essential for the physiology of all living organisms including bacteria. Cations such as potassium ion (K(+)), sodium ion (Na(+)) and proton (H(+)) are involved in nearly all aspects of bacterial growth and survival. K(+) is the most abundant cation and its homeostasis in Escherichia coli and Salmonella is regulated by three major K(+) transporters: high affinity transporter Kdp and low affinity transporters Kup and Trk. Previous studies have demonstrated the roles of cations and cation transport in the physiology of Escherichia coli; their roles in the virulence and physiology of pathogenic bacteria are not well characterized. We have previously reported that the Salmonella K(+) transporter Trk is important for the secretion of effector proteins of the type III secretion system (TTSS) of Salmonella pathogenicity island 1 (SPI-1). Here we further explore the role of Salmonella cation transport in virulence in vitro and pathogenesis in animal models. Impairment of K(+) transport through deletion of K(+) transporters or exposure to the chemical modulators of cation transport, gramicidin and valinomycin, results in a severe defect in the TTSS of SPI-1, and this defect in the TTSS was not due to a failure to regulate intrabacterial pH or ATP. Our results also show that K(+) transporters are critical to the pathogenesis of Salmonella in mice and chicks and are involved in multiple growth and virulence characteristics in vitro, including protein secretion, motility and invasion of epithelial cells. These results suggest that cation transport of the pathogenic bacterium Salmonella, especially K(+) transport, contributes to its virulence in addition to previously characterized roles in maintaining homeostasis of bacteria.

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.

Copper ferrocyanide (CFCN) crystals, well known for its selectivity for Cs+ ions, have been synthesized within the pores of a polycarbonate track etched membrane (PTEM) by two compartment permeation method using CuSO4 and K4Fe(CN)6. The synthesized material has been characterized by X-ray Diffraction (XRD), Small Angle X-ray Scattering (SAXS), Energy Dispersive X-ray Fluorescence (EDXRF) and Transmission Electron Microscopy (TEM) techniques. X-ray techniques confirmed the presence of nanosized ferrocyanide particles (radius ∼40 nm) within the pores of the PTEM. It has been observed from TEM analysis that the CFCN crystals are almost uniformly filling the conical shaped pores of the PTEM with the dimension of each conical shaped structure being ∼300 nm diameter at surface and ∼140 nm diameter inside. This CFCN loaded PTEM (CFCNm) has been used to study the selective transport of Cs+ over Na+ in presence of nitric acid. The rate of cation transport has been enhanced (∼300%) by application of electric field across the membrane. A cation exchange membrane (Nafion-1135) has been coupled with the CFCNm to protect the ferrocyanide crystals from early degradation. The transport behavior of the Nafion-CFCNm composite system under different experimental conditions has been studied and explanation has been given for the differential transport of Cs+ over Na+ through this membrane. The Cs+ transport rate of the membrane has been found to be limited by the ion exchange capacity of the CFCNm.

Anion exchange membranes (PE/VBC) are successfully synthesized by pore-filling a porous polyethylene (PE) substrate with poly(4-vinylbenzyl chloride) followed by amination with pyridyl functional groups for vanadium redox flow battery (VRFB) applications. The membrane crosslinked with 10% of DVB content (PE/VBC(10)), 33 μm in thickness, exhibits a low swelling ratio of 3.4% and a water uptake of 23%. In addition, the vanadium crossover rate of the PE/VBC(10) membrane is lower than Nafion 117 by 48% at a similar electrical area resistance. Electrochemical properties of the PE/VBC membranes are examined in terms of the transport number and migration of ions as well as the electrical area resistance. Transport numbers for anions of the PE/VBC(10) membranes are 0.96 and 0.97 in NaCl and NaOH solutions, respectively. In acidic solutions of HCl and H2SO4, the membrane shows remarkably higher cation transport numbers of 0.20 and 0.72, which imply that the membrane allows high proton permeation and it is advantageous in VRFB operations. Furthermore, the migration of ions reveals reasonably low fluxes of other cations through the anion exchange membrane. Finally, the energy efficiency of the PE/VBC (10) membrane is 90.2%, higher than Nafion 117 by 5.4%.

Members of the ZIP (SLC39A) and ZnT (SLC30A) families of transmembrane domain proteins are predicted to transport the essential transition metal zinc across membranes, regulating cellular zinc content and distribution via uptake and efflux at the outer plasma and organellar membranes. Twenty-four ZIP and ZnT proteins are encoded in mammalian genomes, raising questions of whether all actually transport zinc, whether several function together in the same tissues/cell types, and how the activity of these transporters is coordinated. To address these questions, we have taken advantage of the ability to manipulate several genes simultaneously in targeted cell types in Drosophila. Previously we reported zinc toxicity phenotypes caused by combining overexpression of a zinc uptake gene, dZip42C.1, with suppression of a zinc efflux gene, dZnT63C. Here we show that these phenotypes can be used as a sensitized in vivo system to detect subtle alterations in zinc transport activity that would be buffered in healthy cells. Using two adult tissues, the fly eye and midline (thorax/abdomen), we find that when overexpressed, most of the 17 Drosophila Zip and ZnT genes modify the zinc toxicity phenotypes in a manner consistent with their predicted zinc transport activity. In most cases, we can reconcile that activity with the cellular localization of an enhanced green fluorescent protein tagged version of the protein. Additionally, targeted suppression of each gene by RNA interference reveals several of the fly Zip and ZnT genes are required in the eye, indicating that numerous independent zinc transport genes are acting together in a single tissue.

In limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates that S. oneidensis MR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of the mtrA and mtrC homologs mtrF and mtrD either remains unaffected or decreases under these conditions. The ompW gene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream of mtrC and omcA . The transcriptome and mutant analyses of S. oneidensis MR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires. IMPORTANCE Shewanella oneidensis MR-1 has the capacity to transfer electrons to its external surface using extensions of the outer membrane called bacterial nanowires. These bacterial nanowires link the cell's respiratory chain to external surfaces, including oxidized metals important in bioremediation, and explain why S. oneidensis can be utilized as a component of microbial fuel cells, a form of renewable energy. In this work, we use differential gene expression analysis to focus on which genes function to produce the nanowires and promote extracellular electron transfer during oxygen limitation. Among the genes that are expressed at high levels are those encoding cytochrome proteins necessary for electron transfer. Shewanella coordinates the increased expression of regulators, metabolic pathways, and transport pathways to ensure that cytochromes efficiently transfer electrons along the nanowires.

Cation transport regulator homolog 1 (Chac1) is an endoplasmic reticulum (ER) stress inducible gene that has a function as a γ-glutamyl cyclotransferase involved in the degradation of glutathione. To characterize the translation and stability of Chac1, we found that the Kozak-like sequence present in the 5′ untranslated region (5′UTR) of the Chac1 mRNA was responsible for Chac1 translation. In addition, the short form (ΔChac1), which translated from the second ATG codon, was generated in the absence of the 5′UTR. The proteasome pathway predominantly participated in the stability of the Chac1 protein; however, its expression was remarkably up-regulated by co-transfection with ubiquitin genes. Using an immunoprecipitation assay, we revealed that ubiquitin molecule was directly conjugated to Chac1 and that mutated Chac1 with all lysine residues replaced by arginine was also ubiquitinated. Finally, we showed that WT Chac1 but not ΔChac1 reduced the intracellular level of glutathione. Taken together, our results suggest that the Chac1 protein expression is regulated in translational and post-translational fashion due to the Kozak-like sequence in the 5′UTR and the ubiquitin-mediated pathways. The bidirectional roles of ubiquitination in regulating Chac1 stabilization might give us a new insight into understanding the homeostasis of glutathione under pathophysiological conditions.