Active Ion Transport Research Articles

Cactus-like NC/CoxP electrode enables efficient and stable hydrogen evolution for saline water splitting

Designing efficient and robust catalysts for hydrogen evolution reaction (HER) is imperative for saline water electrolysis technology. A catalyst composed of CoxP nanowires array with N-doped carbon nanosheets (NC) was fabricated on Ni foam (NF) by an in-situ growth strategy. The material is designated as NC/CoxP@NF. In the preparation process, Co(OH)2 nanowires were transformed into a metal organic framework of cobalt (ZIF-67) on NF by the dissolution-coordination of endogenous Co2+ and 2-methylimidazole. The resulting cactus-like microstructure gives NC/CoxP@NF abundant exposed active sites and ion transport channels, which improve the HER catalytic reaction kinetics. Furthermore, the interconnected alternating nanowires and free-standing nanosheets in NC/CoxP@NF improve its structural stability, and the formation of surface polyanions (phosphate) and a NC nanosheet protective layer improve the anti-corrosive properties of catalysts. Thus, the NC/CoxP@NF has an excellent performance, requiring overpotentials of 107 and 133 mV for HER to achieve 10 mA cm−2 in 1.0 mol L−1 KOH and 1.0 mol L−1 KOH + 0.5 mol L−1 NaCl, respectively. This in-situ transformation strategy is a new way of constructing highly-efficient HER catalysts for saline water electrolysis.

Drosophila ClC-c Is a Homolog of Human CLC-5 and a New Model for Dent Disease Type 1.

Drosophila serve as exceptional alternative models for in vivo and ex vivo research and may provide an avenue for in-depth investigation for human ClC-5 and Dent disease type 1 (DD1). The Drosophila ClC-c (CG5284) has sequence homology with human ClC-5 and is hypothesized to encompass similar functional and phenotypical roles with ClC-5 and variants that cause DD1. Ion transport function and activity of Drosophila ClC-c and homologous DD1 variants were assessed by voltage clamp electrophysiology. Membrane localization was demonstrated in Drosophila expressing a GFP-labeled construct of ClC-c. Genetic expression of an RNAi against ClC-c mRNA was used to generate a knock-down fly that serves as a DD1 disease model. Tubule secretion of cations and protein were assessed as well as the crystal formation in the Malpighian tubules. Voltage clamp experiments demonstrate that ClC-c is voltage-gated with Cl--dependent and pH-sensitive currents. Inclusion of homologous DD1 mutations pathogenic variants (S393L, R494W, and Q777X) impairs ClC-c ion transport activity. In vivo expression of ClC-c-eGFP in Malpighian tubules reveals that the membrane transporter localizes to the apical membrane and nearby cytosolic regions. RNAi knock-down of ClC-c (48% decreased mRNA expression) causes increased secretion of both urinary protein and Ca2+ as well as increased occurrence of spontaneous tubule crystals. Drosophila ClC-c shows orthologous function and localization to human ClC-5. Thus, Drosophila and ClC-c regulation may be useful for future investigations of Cl- transport, Ca2+ homeostasis and urinary protein loss in DD1.

Bio-inspired High-Performance Artificial Ion Pump Mediated by Subnanoscale Dehydration Hydration Effects.

The energy conversion in plant chloroplast is carried out by pumping protons into the thylakoid for driving ATP synthesis. Inspired by ion active transport in living organisms, we attempted to design an artificial ion pump induced by subnanoconfinement effects. This ionic device uses two polarity functional nanoporous films as ion-selective valves at both ends and UiO-66 metal-organic framework-filled microchannels as ion storage cavities. In the charging process, ions could be pumped into the central cavities by nanovalves, which produced an ion gradient 10 to 100 times higher than the bulk, and were trapped within the subnanocages by dehydration. In the discharging process, the enriched ions were rehydrated and slowly released by the surface charge of the nanovalves, producing a sustainable ion current. The ion storage efficiency of this nanofluidic device could be improved to 60.3%, and the release time of ion current was also prolonged by 1 order of magnitude. This work combines the active and passive transport of ions to realize fast storage and slow release of ionic current, which provides an ion gradient-mediated novel energy conversion strategy.

Phosphoregulation of the yeast Pma1 H+-ATPase autoinhibitory domain involves the Ptk1/2 kinases and the Glc7 PP1 phosphatase and is under TORC1 control.

Plasma membrane (PM) H+-ATPases of the P-type family are highly conserved in yeast, other fungi, and plants. Their main role is to establish an H+ gradient driving active transport of small ions and metabolites across the PM and providing the main component of the PM potential. Furthermore, in both yeast and plant cells, conditions have been described under which active H+-ATPases promote activation of TORC1, the rapamycin-sensitive kinase complex controlling cell growth. Fungal and plant PM H+-ATPases are self-inhibited by their respective cytosolic carboxyterminal tails unless this domain is phosphorylated at specific residues. In the yeast H+-ATPase Pma1, neutralization of this autoinhibitory domain depends mostly on phosphorylation of the adjacent Ser911 and Thr912 residues, but the kinase(s) and phosphatase(s) controlling this tandem phosphorylation remain unknown. In this study, we show that S911-T912 phosphorylation in Pma1 is mediated by the largely redundant Ptk1 and Ptk2 kinase paralogs. Dephosphorylation of S911-T912, as occurs under glucose starvation, is dependent on the Glc7 PP1 phosphatase. Furthermore, proper S911-T912 phosphorylation in Pma1 is required for optimal TORC1 activation upon H+ influx coupled amino-acid uptake. We finally show that TORC1 controls S911-T912 phosphorylation in a manner suggesting that activated TORC1 promotes feedback inhibition of Pma1. Our results shed important new light on phosphoregulation of the yeast Pma1 H+-ATPase and on its interconnections with TORC1.

Protein localization of aquaporins in the adult female disease vector mosquito, Aedes aegypti.

The female Aedes aegypti mosquito is a vector for several arboviral diseases, due to their blood feeding behavior and their association with urban communities. While ion transport in Ae. aegypti has been studied, much less is known about mechanisms of water transport. Rapid water and ion excretion occurs in the adult female mosquito post blood meal and involves a set of organs including the midgut, Malpighian tubules (MTs), and hindgut. The MTs are responsible for the formation of primary urine and are considered the most important site for active transport of ions. Within the cells of the MTs, along with various ion transporters, there are aquaporin water channels that aid in the transport of water across the tubule cell membrane. Six aquaporin genes have been molecularly identified in Ae. aegypti (AQP1-6) and found to be responsible for the transport of water and in some cases, small solutes such as glycerol. In this study, we used immunohistochemistry to localize AaAQP1, 2, 4, 5, and 6 in the adult female Ae. aegypti, in non-blood fed and post blood feeding (0.5 and 24hr) conditions. We further examined the main water transporting aquaporin, AaAQP1, using western blotting to determine protein abundance changes in isolated MTs pre- and post-blood feeding. Using fluorescence in situ hybridization, aqp1 mRNA was found exclusively in the principal cells of female MTs. Finally, we used immunogold staining with transmission electron microscopy to determine subcellular localization of AaAQP1 in the Malpighian tubules under non-blood fed conditions. Interestingly, AaAQP1 was found to be predominantly in the principal cells of the MTs, dispersed throughout the brush border; however, there was also evidence of some AaAQP1 localization in the stellate cells of the MTs.

Design of Chiral β-Double Helices from γ-Peptide Foldamers.

Chirality is ubiquitous in nature, and homochirality is manifested in many biomolecules. Although β-double helices are rare in peptides and proteins, they consist of alternating L- and D-amino acids. No peptide double helices with homochiral amino acids have been observed. Here, we report chiral β-double helices constructed from γ-peptides consisting of alternating achiral (E)-α,β-unsaturated 4,4-dimethyl γ-amino acids and chiral (E)-α,β-unsaturated γ-amino acids in both single crystals and in solution. The two independent strands of the same peptide intertwine to form a β-double helix structure, and it is stabilized by inter-strand hydrogen bonds. The peptides with chiral (E)-α,β-unsaturated γ-amino acids derived from α-L-amino acids adopt a (P)-β-double helix, whereas peptides consisting of (E)-α,β-unsaturated γ-amino acids derived from α-D-amino acids adopt an (M)-β-double helix conformation. The circular dichroism (CD) signature of the (P) and (M)-β-double helices and the stability of these peptides at higher temperatures were examined. Furthermore, ion transport studies suggested that these peptides transport ions across membranes. Even though the structural analogy suggests that these new β-double helices are structurally different from those of the α-peptide β-double helices, they retain ion transport activity. The results reported here may open new avenues in the design of functional foldamers.

Design of Chiral β‐Double Helices from γ‐Peptide Foldamers

AbstractChirality is ubiquitous in nature, and homochirality is manifested in many biomolecules. Although β‐double helices are rare in peptides and proteins, they consist of alternating L‐ and D‐amino acids. No peptide double helices with homochiral amino acids have been observed. Here, we report chiral β‐double helices constructed from γ‐peptides consisting of alternating achiral (E)‐α,β‐unsaturated 4,4‐dimethyl γ‐amino acids and chiral (E)‐α,β‐unsaturated γ‐amino acids in both single crystals and in solution. The two independent strands of the same peptide intertwine to form a β‐double helix structure, and it is stabilized by inter‐strand hydrogen bonds. The peptides with chiral (E)‐α,β‐unsaturated γ‐amino acids derived from α‐L‐amino acids adopt a (P)‐β‐double helix, whereas peptides consisting of (E)‐α,β‐unsaturated γ‐amino acids derived from α‐D‐amino acids adopt an (M)‐β‐double helix conformation. The circular dichroism (CD) signature of the (P) and (M)‐β‐double helices and the stability of these peptides at higher temperatures were examined. Furthermore, ion transport studies suggested that these peptides transport ions across membranes. Even though the structural analogy suggests that these new β‐double helices are structurally different from those of the α‐peptide β‐double helices, they retain ion transport activity. The results reported here may open new avenues in the design of functional foldamers.

AAPH-induced oxidative damage reduced anion exchanger 1 (SLC4A1/AE1) activity in human red blood cells: protective effect of an anthocyanin-rich extract.

Introduction: During their lifespan in the bloodstream, red blood cells (RBCs) are exposed to multiple stressors, including increased oxidative stress, which can affect their morphology and function, thereby contributing to disease. Aim: This investigation aimed to explore the cellular and molecular mechanisms related to oxidative stress underlying anion exchanger 1 activity (band 3, SLC4A1/AE1) in human RBCs. To achieve this aim, the relationship between RBC morphology and functional and metabolic activity has been explored. Moreover, the potential protective effect of an anthocyanin-enriched fraction extracted from Callistemon citrinus flowers was studied. Methods: Cellular morphology, parameters of oxidative stress, as well as the anion exchange capability of band 3 have been analyzed in RBCs treated for 1h with 50mM of the pro-oxidant 2,2'-azobis (2-methylpropionamide)-dihydrochloride (AAPH). Before or after the oxidative insult, subsets of cells were exposed to 0.01μg/mL of an anthocyanin-enriched fraction for 1h. Results: Exposure to AAPH caused oxidative stress, exhaustion of reduced glutathione, and over-activation of the endogenous antioxidant machinery, resulting in morphological alterations of RBCs, specifically the formation of acanthocytes, increased lipid peroxidation and oxidation of proteins, as well as abnormal distribution and hyper-phosphorylation of band 3. Expected, oxidative stress was also associated with a decreased band 3 ion transport activity and an increase of oxidized haemoglobin, which led to abnormal clustering of band 3. Exposure of cells to the anthocyanin-enriched fraction prior to, but not after, oxidative stress efficiently counteracted oxidative stress-related alterations. Importantly, protection of band3 function from oxidative stress could only be achieved in intact cells and not in RBC ghosts. Conclusion: These findings contribute a) to clarify oxidative stress-related physiological and biochemical alterations in human RBCs, b) propose anthocyanins as natural antioxidants to neutralize oxidative stress-related modifications, and 3) suggest that cell integrity, and therefore a cytosolic component, is required to reverse oxidative stress-related pathophysiological derangements in human mature RBCs.

Porous powder synthesis of (FeCoCrMnNi)3O4 high entropy ceramic and its supercapacitor applications

High entropy materials are becoming more and more popular in energy storage field of supercapacitors because of their unique structure and modulated functional characteristics, but their low conductivity and instability of electrode structure limit their further electrochemical performance. In this paper, we use polyethylene glycol (PEG) with different molecular weights to modify the surface morphology of the high entropy oxides (FeCoCrMnNi)3O4 in an attempt to improve their supercapacitors performance. The PEG was used as a pore-making agent to prepare porous high entropy oxides powder with a large specific surface area of 92.7559 m2 g−1, providing more active sites and ion transport channels. A high specific capacitance of 204 F g−1 at 0.5 A g−1 and good cycle performance of 60.4 % capacitance retention after 10,000 cycles were obtained. Besides, an asymmetric device with improved energy density of 53 W h kg−1 and the power density of 400.8 W kg−1 were reached via the designed microstructure of the high entropy oxide electrode.

An outward-rectifying plant K+ channel SPORK2 exhibits temperature-sensitive ion-transport activity

Temperature sensing is critical for the survival of living organisms.1,2 Thermosensitive transient receptor-potential (TRP) cation channels function as thermosensors in mammals.2,3,4,5,6 In contrast to animals, landplants lack TRP genes.7,8,9 Previous patch-clamp studies in plant cells suggested the presence of ion channels whose activities are related to temperature, implying the presence of TRP-like channels.10,11,12,13,14 However, the molecular entities of such temperature-sensitive ion channels were still unknown in land plants. In this study, we observed that the unique rainfall-induced leaf-folding movement of the legumetree Samanea saman15 was temperature-sensitive by using a rainfall-mimicking assay. Chilling-induced leaf folding in S.saman was shown to be related to the swelling of the motor cells16,17 at the base of the leaflet. This swelling suggested involvement of temperature-sensitive inactivation of K+ currents, independent of fluctuations in ion channel gene expression in motor cells. These findings led us to examine the temperature sensitivity of an outward-rectifying K+ channel, SPORK2, which was reported as an ion channel responsible for the nyctinastic (circadian-rhythmic) leaf movement of S.saman.18 We alsodiscovered that SPORK2 exhibits temperature-sensitive K+ transport activity in the Xenopus oocyte expression system. Using chimeric channels, we showed that two domains of SPORK2 regulated the temperaturesensitivity. Furthermore, heterologously expressed SPORK2 in Arabidopsis guard cells induced temperature-dependent stomatal closure. Therefore, SPORK2 is an ion channel in land plantswith temperature-sensitive ion-transport activity that functions similarly to mammalian TRP channels. Our current findings advance the molecular understanding of temperature-sensing mechanisms in plants.

MPA-Pred: A machine learning approach for predicting the binding affinity of membrane protein-protein complexes.

Membrane protein-protein interactions are essential for several functions including cell signaling, ion transport, and enzymatic activity. These interactions are mainly dictated by their binding affinities. Although several methods are available for predicting the binding affinity of protein-protein complexes, there exists no specific method for membrane protein-protein complexes. In this work, we collected the experimental binding affinity data for a set of 114 membrane protein-protein complexes and derived several structure and sequence-based features. Our analysis on the relationship between binding affinity and the features revealed that the important factors mainly depend on the type of membrane protein and the functional class of the protein. Specifically, aromatic and charged residues at the interface, and aromatic-aromatic and electrostatic interactions are found to be important to understand the binding affinity. Further, we developed a method, MPA-Pred, for predicting the binding affinity of membrane protein-protein complexes using a machine learning approach. It showed an average correlation and mean absolute error of 0.83 and 0.91 kcal/mol, respectively, using the jack-knife test on a set of 114 complexes. We have also developed a web server and it is available at https://web.iitm.ac.in/bioinfo2/MPA-Pred/. This method can be used for predicting the affinity of membrane protein-protein complexes at a large scale and aid to improve drug design strategies.

The impact of salt stress on the physiology and the transcriptome of the model streptophyte green alga Chara braunii.

Chara braunii is a model for early land plant evolution and terrestrialization. Salt stress has a profound effect on water and ion transport activities, thereby interacting with many other processes, including inorganic carbon acquisition for photosynthesis. In this study, we analyzed the impact of salt stress (5 practical salt units, PSU) on the physiology and gene expression in C. braunii. Photosynthesis was only slightly affected 6 h after salt addition and returned to control levels after 48 h. Several organic compounds such as proline, glutamate, sucrose, and 2-aminobutyrate accumulated in salt-treated thalli and might contribute to osmotic potential acclimation, whereas the amount of K+ decreased. We quantified transcript levels for 17,387 genes, of which 95 were up-regulated and 44 down-regulated after salt addition. Genes encoding proteins of the functional groups ion/solute transport and cell wall synthesis/modulation were enriched among the up-regulated genes 24-48 h after salt stress, indicating their role in osmotic acclimation. However, a homolog to land plant ERD4 osmosensors was transiently upregulated after 6 h, and phylogenetic analyses suggested that these sensors evolved in Charophyceae. Down-regulated genes were mainly related to photosynthesis and carbon metabolism/fixation, consistent with the observed lowered growth after extended cultivation. The changed expression of genes encoding proteins for inorganic carbon acquisition might be related to the impact of salt on ionic relations and inorganic carbon uptake. The results indicate that C. braunii can tolerate enhanced salt concentrations in a defined acclimation process, including distinct gene expression changes to achieve new metabolic homeostasis.

56 Early Weaning Stress Relocates Epithelial Enteroendocrine Cells and Alters Intestinal Nutrient Sensing and Transport Functions in Pigs

Abstract Intestinal nutrient sensing and enteroendocrine functions are critical in controlling feed intake and nutrient transport, yet have been overlooked in agricultural animals. Enteroendocrine cells (EECs) residing on intestinal epithelium monitor luminal chemical environment and orchestrate body neuronal and hormonal responses. After nutrient stimulation, the EECs release hormones, such as glucagon-like peptide 2 (GLP2), which dynamically regulates intestinal nutrient transport and epithelial barrier homeostasis. In pigs, early weaning stress has negatively impacted intestinal development and functions. The objective of the present study is to evaluate effects of early weaning on intestinal nutrient sensing functions, EEC-derived GLP2 production, and epithelial nutrient transport in pigs. Commercial pig littermates (females, n = 9) were split-weaned at the age of d15 (early weaning, EW) and d 26 (late weaning, LW). Piglets were all raised under the same conditions and euthanized at 35 days of age. Intestinal tissues were collected and preserved for later immunohistochemistry staining and gene expression analyses. Immediately post euthanasia, the mucosal layers from jejunum and ileum were mounted on the Ussing chambers to investigate physiological functions. Nutrients transport activities were measured by short circuit current changes (ΔIsc) in response to luminal nutrients (glucose, alanine, lysine, and di-peptide) applications. Nutrient sensing function was measured by GLP2 release rate following glucose stimulation in Ussing chambers. Serum and intestinal tissue concentrations of GLP2 were also measured by ELISA. Data were analyzed by the student’s t-test. Our results showed altered electrogenic nutrients and ion transport activities between EW and LW pigs. The ileal basal Isc was significantly less in EW pigs, indicating a reduction in ions transport. The EW pigs significantly downregulated electrogenic lysine transport, while upregulated active transport of dipeptide. The EECs number in ileum was reduced in EW pigs, indicated by less ChgA (EEC cell marker) mRNA and significantly less ChgA positive-stained cells. In addition, EW pigs exhibited a pattern of EECs relocation, in which the ChgA-positive cells enriched towards the lower half of villi. Nutrient sensing activities of the EECs were suppressed in both jejunum and ileum in EW pigs, indicated by a significant reduction in the glucose-stimulated GLP2 release on Ussing chambers. Moreover, in contrast with the significantly greater GLP2 concentration in ileum, the serum GLP2 concentration was significantly less in the EW pigs, which reflected a decreased GLP2 release rate. In summary, our results suggested that early weaning stress altered EECs number and locations on epithelium, reduced EECs nutrient sensing activities and endogenous GLP2 release, and changed nutrient transport activities. Understanding the mechanism by which early weaning stress regulates intestinal nutrient sensing and transport functions will lead to novel feeding strategies that improve intestinal health and animal production.

Regulation of water flow in the ocular lens: new roles for aquaporins.

The ocular lens is an important determinant of overall vision quality whose refractive and transparent properties change throughout life. The lens operates an internal microcirculation system that generates circulating fluxes of ions, water and nutrients that maintain the transparency and refractive properties of the lens. This flow of water generates a substantial hydrostatic pressure gradient which is regulated by a dual feedback system that uses the mechanosensitive channels TRPV1 and TRPV4 to sense decreases and increases, respectively, in the pressure gradient. This regulation of water flow (pressure) and hence overall lens water content, sets the two key parameters, lens geometry and the gradient of refractive index, which determine the refractive properties of the lens. Here we focus on the roles played by the aquaporin family of water channels in mediating lens water fluxes, with a specific focus on AQP5 as a regulated water channel in the lens. We show that in addition to regulating the activity of ion transporters, which generate local osmotic gradients that drive lens water flow, the TRPV1/4-mediated dual feedback system also modulates the membrane trafficking of AQP5 in the anterior influx pathway and equatorial efflux zone of the lens. Since both lens pressure and AQP5-mediated water permeability ( ) can be altered by changes in the tension applied to the lens surface via modulating ciliary muscle contraction we propose extrinsic modulation of lens water flow as a potential mechanism to alter the refractive properties of the lens to ensure light remains focused on the retina throughout life.

Molecular basis of ClC-6 function and its impairment in human disease.

ClC-6 is a late endosomal voltage-gated chloride-proton exchanger that is predominantly expressed in the nervous system. Mutated forms of ClC-6 are associated with severe neurological disease. However, the mechanistic role of ClC-6 in normal and pathological states remains largely unknown. Here, we present cryo-EM structures of ClC-6 that guided subsequent functional studies. Previously unrecognized ATP binding to cytosolic ClC-6 domains enhanced ion transport activity. Guided by a disease-causing mutation (p.Y553C), we identified an interaction network formed by Y553/F317/T520 as potential hotspot for disease-causing mutations. This was validated by the identification of a patient with a de novo pathogenic variant p.T520A. Extending these findings, we found contacts between intramembrane helices and connecting loops that modulate the voltage dependence of ClC-6 gating and constitute additional candidate regions for disease-associated gain-of-function mutations. Besides providing insights into the structure, function, and regulation of ClC-6, our work correctly predicts hotspots for CLCN6 mutations in neurodegenerative disorders.

Effects of Salinity Stress on Histological Changes, Glucose Metabolism Index and Transcriptomic Profile in Freshwater Shrimp, Macrobrachium nipponense.

Salinity is an important factor in the aquatic environment and affects the ion homeostasis and physiological activities of crustaceans. Macrobrachium nipponense is a shrimp that mainly lives in fresh and low-salt waters and plays a huge economic role in China's shrimp market. Currently, there are only a few studies on the effects of salinity on M. nipponense. Therefore, it is of particular importance to study the molecular responses of M. nipponense to salinity fluctuations. In this study, M. nipponense was set at salinities of 0, 8, 14 and 22‱ for 6 weeks. The gills from the control (0‱) and isotonic groups (14‱) were used for RNA extraction and transcriptome analysis. In total, 593 differentially expressed genes (DEGs) were identified, of which 282 were up-regulated and 311 were down-regulated. The most abundant gill transcripts responding to different salinity levels based on GO classification were organelle membrane (cellular component), creatine transmembrane transporter activity (molecular function) and creatine transmembrane transport (biological function). KEGG analysis showed that the most enriched and significantly affected pathways included AMPK signaling, lysosome and cytochrome P450. In addition, 15 DEGs were selected for qRT-PCR verification, which were mainly related to ion homeostasis, glucose metabolism and lipid metabolism. The results showed that the expression patterns of these genes were similar to the high-throughput data. Compared with the control group, high salinity caused obvious injury to gill tissue, mainly manifested as contraction and relaxation of gill filament, cavity vacuolation and severe epithelial disintegration. Glucose-metabolism-related enzyme activities (e.g., pyruvate kinase, hexokinase, 6-phosphate fructose kinase) and related-gene expression (e.g., hexokinase, pyruvate kinase, 6-phosphate fructose kinase) in the gills were significantly higher at a salinity of 14‱. This study showed that salinity stress activated ion transport channels and promoted an up-regulated level of glucose metabolism. High salinity levels caused damage to the gill tissue of M. nipponense. Overall, these results improved our understanding of the salt tolerance mechanism of M. nipponense.

Synthesis, Microstructure, and Electrical Conductivity of Eutectic Composites in MF2–RF3 (M = Ca, Sr, Ba; R = La–Nd) Systems

Multiphase fluoride polycrystalline eutectics pRF3 × qMF2 forming in the MF2–RF3 (M = Ca, Sr, Ba; R = La–Nd) binary systems were synthesized by the directional crystallization technique from a melt. The phase composition, morphology, and temperature dependences of fluorine ionic conductivity in fabricated composites were studied in detail. The pRF3 × qMF2 (p and q are the mole percentages of components) eutectic composites consist of both extremely saturated fluorite-type structure M1−xRxF2+x solid solutions and the tysonite-type R1−yMyF3−y ones. Microsized growth blocks with a fine lamellar structure are typical for synthesized composites. The thinnest (from 3 μm) and longest lamellae are observed in the 68LaF3 × 32BaF2 composition. The ionic conductivity values of pRF3 × qMF2 composites are determined by the phase composition, practically do not depend on their morphological features, and reach 10−3–10−2 S/cm at 500 K (with an ion transport activation enthalpy of about 0.5–0.6 eV). Crystallized eutectics are superior to any single-phase M1−xRxF2+x solid solutions and ball-milling R1−yMyF3−y nanoceramics in terms of ion-conducting properties. These fluoride materials represent an alternative to widely applied tysonite-type ceramic composites in various electrochemical devices and require further in-depth studies.

ATP modulates the activity of the voltage-gated proton channel through direct binding interaction.

ATP is an important molecule implicated in diverse biochemical processes, including the modulation of ion channel and transporter activity. The voltage-gated proton channel (Hv1) controls proton flow through the transmembrane pathway in response to membrane potential, and various molecules regulate its activity. Although it is believed that ATP is not essential for Hv1 activity, a report has indicated that cytosolic ATP may modulate Hv1. However, the detailed molecular mechanism underlying the effect of ATP on Hv1 is unknown, and whether ATP is involved in the physiological regulation of Hv1 activity remains unclear. Here, we report that cytosolic ATP is required to maintain Hv1 activity. To gain insight into the underlying mechanism, we analysed the effects of ATP on the mouse Hv1 channel (mHv1) using electrophysiological and microscale thermophoresis (MST) methods. Intracellular ATP accelerated the activation kinetics of mHv1, thereby increasing the amplitude of the proton current within the physiological concentration range. The increase in proton current was reproduced with a non-hydrolysable ATP analogue, indicating that ATP directly influences Hv1 activity without an enzymatic reaction. The direct molecular interaction between the purified mHv1 protein and ATP was analysed and demonstrated through MST. In addition, ATP facilitation was observed for the endogenous proton current flowing through Hv1 in the physiological concentration range of ATP. These results suggest that ATP influences Hv1 activity via direct molecular interactions and is required for the physiological function of Hv1. KEY POINTS: We found that ATP is required to maintain the activity of voltage-gated proton channels (Hv1) and investigated the underlying molecular mechanism. Application of intracellular ATP increased the amplitude of the proton current flowing through Hv1, accompanied by an acceleration of activation kinetics. The direct interaction between purified Hv1 protein and ATP was quantitatively analysed using microscale thermophoresis. ATP enhanced endogenous proton currents in breast cancer cell lines. These results suggest that ATP influences Hv1 activity via direct molecular interactions and that its functional characteristics are required for the physiological activity of Hv1.

NAD(P)H:Quinone Acceptor Oxidoreductase 1 (NQO1) Activatable Salicylamide H+/Cl- Transporters.

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a detoxifying enzyme overexpressed in tumors, plays a key role in protecting cancer cells against oxidative stress and thus has been considered an attractive candidate for activating prodrug(s). Herein, we report the first use of NQO1 for the selective activation of 'protransporter' systems in cancer cells leading to the induction of apoptosis. Salicylamides, easily synthesizable small molecules, have been effectively used for efficient H+/Cl- symport across lipid membranes. The ion transport activity of salicylamides was efficiently abated by caging the OH group with NQO1 activatable quinones via either ether or ester linkage. The release of active transporters, following the reduction of quinone caged 'protransporters' by NQO1, was verified. Both the transporters and protransporters exhibited significant toxicity towards the MCF-7 breast cancer line, mediated via the induction of oxidative stress, mitochondrial membrane depolarization, and lysosomal deacidification. Induction of cell death via intrinsic apoptotic pathway was verified by monitoring PARP1 cleavage.

In Vitro Study of the Multimodal Effect of Na+/K+ ATPase Blocker Ouabain on the Tumor Microenvironment and Malignant Cells.

Na+/K+ ATPase is a protein involved in the active transport of ions across the cellular membrane. Ouabain is a cardiotonic glycoside that, by inhibiting the Na+/K+ pump, interferes with cell processes mediated directly by the pump, but also indirectly influences other cellular processes such as cell cycle and proliferation, growth, cell differentiation, angiogenesis, migration, adhesion, and invasion. We used the SK-BR-3 breast cancer cell line, mesenchymal stem cells (MSCs), and tumor-associated fibroblasts (TAFs) in vitro to determine the effects of ouabain exposure on these cellular types. The results showed a multi-level effect of ouabain mainly on tumor cells, in a dose-dependent manner, while the TAFs and their normal counterparts were not significantly influenced. Following exposure to ouabain, the SK-BR-3 cells changed their morphologic appearance, decreased the expression of immunophenotypic markers (CD29, Her2, VEGF), the proliferation rate was significantly decreased (Ki67 index), the cells were blocked in the G0 phase of the cell cycle and suffered necrosis. These data were correlated with the variable expression of α and β Na+/K+ pump subunits in tumor cells, resulting in decreased ability to adhere to the VCAM-1 substrate in functional flow chamber studies. Being indicative of the pro-apoptotic and inhibitory effect of ouabain on tumor invasion and metastasis, the results support the addition of ouabain to the oncological therapeutic arsenal, trailing the "repurposing drugs" approach.