G-Quadruplexes as Sensors of Intracellular Na+/K+ Ratio: Potential Role in Regulation of Transcription and Translation.

Data on the structure of G-quadruplexes, noncanonical nucleic acid forms, supporting an idea of their potential participation in regulation of gene expression in response to the change in intracellular Na+i/K+i ratio are considered in the review. Structural variety of G-quadruplexes, role of monovalent cations in formation of this structure, and thermodynamic stability of G-quadruplexes are described. Data on the methods of their identification in the cells and biological functions of these structures are presented. Analysis of information about specific interactions of G-quadruplexes with some proteins was conducted, and their potential participation in the development of some pathological conditions, in particular, cancer and neurodegenerative diseases, is considered. Special attention is given to the plausible role of G-quadruplexes as sensors of intracellular Na+i/K+i ratio, because alteration of this parameter affects folding of G-quadruplexes changing their stability and, thereby, organization of the regulatory elements of nucleic acids. The data presented in the conclusion section demonstrate significant change in the expression of some early response genes under certain physiological conditions of cells and tissues depending on the intracellular Na+i/K+i ratio.

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport.

The Na+/K+-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na+ for extracellular K+ to the hydrolysis of ATP. The asymmetric distribution of Na+ and K+ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers-Post model. It involves the presence of gates alternatively exposing Na+/K+-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K+, information is lacking about Na+-occluded intermediates, as occluded Na+ was only detected in states incapable of performing a catalytic cycle, including two Na+-containing crystallographic structures. The current knowledge is that intracellular Na+ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na+-occluded intermediates, we isolated species with tightly bound Na+ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form of the Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na+ occlusion as it would have been expected; on the contrary, occluded Na+ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.

Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations.

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.

Effect of Dynamic and Static Load on the Concentration of Myokines in the Blood Plasma and Content of Sodium and Potassium in Mouse Skeletal Muscles.

Modulation of cytokine production by physical activity is of considerable interest, since it might be a promising strategy for correcting metabolic processes at both cellular and systemic levels. The content of IL-6, IL-8, and IL-15 in the plasma and the concentration of monovalent cations in the skeletal muscles of trained and untrained mice were studied at different periods after static and dynamic exercises. Dynamic loads caused an increase in the IL-6 content and decrease in the IL-15 content in the plasma of untrained mice, but produced no effect on the concentration of IL-8. In trained mice, the effect of a single load on the concentration of IL-6 and IL-15 in the plasma was enhanced, while the concentration of IL-8 decreased. Static loads produced a similar, but more pronounced effect on the plasma concentration of IL-6 and IL-15 compared the dynamic exercises; however, the concentration of IL-8 in response to the static exercise increased significantly. Prior training reinforced the described response for all the myokines studied. Dynamic load (swimming) increased the intracellular content of sodium but decreased the content of potassium in the mouse musculus soleus. Similar response was observed after the static load (grid hanging) in the musculus biceps; but no correlation of this response with the prior training was found. Possible mechanisms involved in the regulation of cytokine secretion after exercise are discussed, including triggering of gene transcription in response to changes in the [Na+]i/[K+]I ratio.

Selective regulation of human TRAAK channels by biologically active phospholipids.

TRAAK is an ion channel from the two-pore domain potassium (K2P) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K2P4.1 toward lipids remains poorly understood. Here we show the two isoforms of K2P4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K2P4.1. Liposome potassium flux assays with K2P4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K2P4.1.

Bioglass could increase cell membrane fluidity with ion products to develop its bioactivity.

OBJECTIVES: Silicate bioactive glass (BG) has been widely demonstrated to stimulate both of the hard and soft tissue regeneration, in which ion products released from BG play important roles. However, the mechanism by which ion products act on cells on cells is unclear. MATERIALS AND METHODS: Human umbilical vein endothelial cells and human bone marrow stromal cells were used in this study. Fluorescence recovery after photobleaching and generalized polarization was used to characterize changes in cell membrane fluidity. Migration, differentiation and apoptosis experiments were carried out. RNA and protein chip were detected. The signal cascade is simulated to evaluate the effect of increased cell membrane fluidity on signal transduction. RESULTS: We have demonstrated that ion products released from BG could effectively enhance cell membrane fluidity in a direct and physical way, and Si ions may play a major role. Bioactivities of BG ion products on cells, such as migration and differentiation, were regulated by membrane fluidity. Furthermore, we have proved that BG ion products could promote apoptosis of injured cells based on our conclusion that BG ion products increased membrane fluidity. CONCLUSIONS: This study proved that BG ion products could develop its bioactivity on cells by directly enhancing cell membrane fluidity and subsequently affected cell behaviours, which may provide an explanation for the general bioactivities of silicate material.

Cytotoxicity of Exogenous Acetoacetate in Lithium Salt Form Is Mediated by Lithium and Not Acetoacetate.

BACKGROUND/AIM: The ketogenic diet has recently gained interest as potential adjuvant therapy for cancer. Many researchers have endeavored to support this claim in vitro. One common model utilizes treatment with exogenous acetoacetate in lithium salt form (LiAcAc). We aimed to determine whether the effects of treatment with LiAcAc on cell viability, as reported in the literature, accurately reflect the influence of acetoacetate. MATERIALS AND METHODS: Breast cancer and normal cell lines were treated with acetoacetate, in lithium and sodium salt forms, and cell viability was assessed. RESULTS: The effect of LiAcAc on cells was mediated by Li ions. Our results showed that the cytotoxic effects of LiAcAc treatment were significantly similar to those caused by LiCl, and also treatment with NaAcAc did not cause any significant cytotoxic effect. CONCLUSION: Treatment of cells with LiAcAc is not a convincing in vitro model for studying ketogenic diet. These findings are highly important for interpreting previously published results, and for designing new experiments to study the ketogenic diet in vitro.

NEK7 mediated assembly and activation of NLRP3 inflammasome downstream of potassium efflux in ventilator-induced lung injury.

Disordered immune regulation and persistent inflammatory damage are the key mechanisms of ventilator-induced lung injury (VILI). NLR family pyrin domain containing 3 (NLRP3) inflammasome activation causes VILI by mediating the formation of inflammatory mediators and infiltration of inflammatory cells, increasing pulmonary capillary membrane permeability, which leads to pulmonary edema and lung tissue damage. What mediates activation of NLRP3 inflammasome in VILI? In this study, we constructed an in vitro cyclic stretch (CS)-stimulated mouse lung epithelial (MLE-12) cell model that was transfected with NIMA-related kinase 7 (NEK7) small interfering RNA (siRNA) or scramble siRNA (sc siRNA) and pretreated with or without glibenclamide (glb). We also established a VILI mouse model, which was pretreated with glibenclamide or oridonin (Ori). Our goal was to investigate the regulatory effects of NEK7 on NLRP3 inflammasome activation and the anti-inflammatory effects of glibenclamide and oridonin on VILI. Mechanical stretch exaggerated the interaction between NEK7 and NLRP3, leading to assembly and activation of NLRP3 inflammasome downstream of potassium efflux. NEK7 depletion and treatment with glibenclamide or oridonin exerted anti-inflammatory effects that alleviated VILI by blocking the interaction between NEK7 and NLRP3, inhibiting NLRP3 inflammasome activation. NEK7 is a vital mediator of NLRP3 inflammasome activation, and glibenclamide or oridonin may be candidates for the development of new therapeutics against VILI driven by the interaction between NEK7 and NLRP3.

d-Alanine-d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations.

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine-d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl-d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+ Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

Airway submucosal glands from cystic fibrosis swine suffer from abnormal ion transport across the serous acini, collecting duct, and ciliated duct.

The human airway is protected by an efficient innate defense mechanism that requires healthy secretion of airway surface liquid (ASL) to clear pathogens from the lungs. Most of the ASL in the upper airway is secreted by submucosal glands. In cystic fibrosis (CF), the function of airway submucosal glands is abnormal, and these abnormalities are attributed to anomalies in ion transport across the epithelia lining the different sections of the glands that function coordinately to produce the ASL. However, the ion transport properties of most of the anatomical regions of the gland have never been measured, and there is controversy regarding which segments express CFTR. This makes it difficult to determine the glandular abnormalities that may contribute to CF lung disease. Using a noninvasive, extracellular self-referencing ion-selective electrode technique, we characterized ion transport properties in all four segments of submucosal glands from wild-type and CFTR-/- swine. In wild-type airways, the serous acini, mucus tubules, and collecting ducts secrete Cl- and Na+ into the lumen in response to carbachol and forskolin stimulation. The ciliated duct also transports Cl- and Na+ but in the opposite direction, i.e., reabsorption from the ASL, which may contribute to lowering Na+ and Cl- activities in the secreted fluid. In CFTR-/- airways, the serous acini, collecting ducts, and ciliated ducts fail to transport ions after forskolin stimulation, resulting in the production of smaller volumes of ASL with normal Cl-, Na+, and K+ concentration.

Synthesis, Structural, and Cytotoxic Properties of New Water-Soluble Copper(II) Complexes based on 2,9-Dimethyl-1,10-Phenanthroline and Their One Derivative Containing 1,3,5-Triaza-7-Phosphaadamantane-7-Oxide.

A series of water-soluble copper(II) complexes based on 2,9-dimethyl-1,10-phenanthroline (dmphen) and mixed-ligands, containing PTA=O (1,3,5-triaza-7-phosphaadamantane-7-oxide) have been synthesized and fully characterized. Two types of complexes have been obtained, monocationic [Cu(NO3)(O-PTA=O)(dmphen)][PF6] (1), [Cu(Cl)(dmphen)2][PF6] (2), and neutral [Cu(NO3)2(dmphen)] (3). The solid-state structures of all complexes have been determined by single-crystal X-ray diffraction. Magnetic studies for the complex 1-3 indicated a very weak antiferromagnetic interaction between copper(II) ions in crystal lattice. Complexes were successfully evaluated for their cytotoxic activities on the normal human dermal fibroblast (NHDF) cell line and the antitumor activity using the human lung carcinoma (A549), epithelioid cervix carcinoma (HeLa), colon (LoVo), and breast adenocarcinoma (MCF-7) cell lines. Complexes 1 and 3 revealed lower toxicity to NHDF than A549 and HeLa cells, meanwhile compound 2 appeared to be more toxic to NHDF cell line in comparison to all cancer lines. Additionally, interactions between the complexes and human apo-transferrin (apo-Tf) using fluorescence and circular dichroism (CD) spectroscopy were also investigated. All compounds interacted with apo-transferrin, causing same changes of the protein conformation. Electrostatic interactions dominate in the 1/2 - apo- Tf systems and hydrophobic and ionic interactions in the case of 3.

Structure and Mechanism of P-Type ATPase Ion Pumps.

P-type ATPases are found in all kingdoms of life and constitute a wide range of cation transporters, primarily for H+, Na+, K+, Ca2+, and transition metal ions such as Cu(I), Zn(II), and Cd(II). They have been studied through a wide range of techniques, and research has gained very significant insight on their transport mechanism and regulation. Here, we review the structure, function, and dynamics of P2-ATPases including Ca2+-ATPases and Na,K-ATPase. We highlight mechanisms of functional transitions that are associated with ion exchange on either side of the membrane and how the functional cycle is regulated by interaction partners, autoregulatory domains, and off-cycle states. Finally, we discuss future perspectives based on emerging techniques and insights.

The effect of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations on rapeseed oleosome (oil body) extraction and stability at pH 7.

Oleosomes are storage vehicles of TAGs in plant seeds. They are protected with a phospholipid-protein monolayer and extracted with alkaline aqueous media; however, pH adjustment intensifies the extraction process. Therefore, the aim of this work was to investigate the extraction mechanism of rapeseed oleosomes at pH 7 and at the presence of monovalent and divalent cations (Na+, K+, Mg2+, and Ca+2). The oleosome yield at pH 9.5 was 64 wt%, while the yield at pH 7 with H2O was just 43 wt.%. The presence of cations at pH 7, significantly enhanced the yield, with K+ giving the highest yield (64 wt.%). The cations affected the oleosome interface and their interactions. The presence of monovalent cations resulted in aggregation and minor coalescence, while divalent cations resulted in extensive coalescence. These results help to understand the interactions of oleosomes in their native matrix and design simple extraction processes at neutral conditions.

Search for Intracellular Sensors Involved in the Functioning of Monovalent Cations as Secondary Messengers.

Maintenance of non-equilibrium Na+ and K+ distribution between cytoplasm and extracellular medium suggests existence of sensors responding with conformational transitions to the changes of these monovalent cations' intracellular concentration. Molecular nature of monovalent cation sensors has been established in Na,K-ATPase, G-protein-coupled receptors, and heat shock proteins structural studies. Recently, it was found that changes in Na+ and K+ intracellular concentration are the key factors in the transcription and translation control, respectively. In this review, we summarize results of these studies and discuss physiological and pathophysiological significance of Na+i,K+i-dependent gene expression regulation mechanism.

Endotracheal tube mucus as a source of airway mucus for rheological study.

Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.

C-terminal Cysteines of CueR Act as Auxiliary Metal Site Ligands upon HgII Binding-A Mechanism To Prevent Transcriptional Activation by Divalent Metal Ions?

Intracellular CuI is controlled by the transcriptional regulator CueR, which effectively discriminates between monovalent and divalent metal ions. It is intriguing that HgII does not activate transcription, as bis-thiolate metal sites exhibit high affinity for HgII . Here the binding of HgII to CueR and a truncated variant, ΔC7-CueR, without the last 7 amino acids at the C-terminus including a conserved CCHH motif is explored. ESI-MS demonstrates that up to two HgII bind to CueR, while ΔC7-CueR accommodates only one HgII . 199m Hg PAC and UV absorption spectroscopy indicate HgS2 structure at both the functional and the CCHH metal site. However, at sub-equimolar concentrations of HgII at pH 8.0, the metal binding site displays an equilibrium between HgS2 and HgS3 , involving cysteines from both sites. We hypothesize that the C-terminal CCHH motif provides auxiliary ligands that coordinate to HgII and thereby prevents activation of transcription.

Impact of Sodium Cationization on Gas-Phase Conformations of DNA and RNA Cytidine Mononucleotides.

Gas-phase conformations of the sodium-cationized forms of the 2'-deoxycytidine and cytidine mononucleotides, [pdCyd+Na]+ and [pCyd+Na]+, are examined by infrared multiple photon dissociation action spectroscopy. Complimentary electronic structure calculations at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory provide candidate conformations and their respective predicted IR spectra for comparison across the IR fingerprint and hydrogen-stretching regions. Comparisons of the predicted IR spectra and the measured infrared multiple photon dissociation action spectra provide insight into the impact of sodium cationization on intrinsic mononucleotide structure. Further, comparison of present results with those reported for the sodium-cationized cytidine nucleoside analogues elucidates the impact of the phosphate moiety on gas-phase structure. Across the neutral, protonated, and sodium-cationized cytidine mononucleotides, a preference for stabilization of the phosphate moiety and nucleobase orientation is observed, although the details of this stabilization differ with the state of cationization. Several low-energy conformations of [pdCyd+Na]+ and [pCyd+Na]+ involving several different orientations of the phosphate moiety and sugar puckering modes are observed experimentally.

Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis.

RCK (regulating conductance of K+) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K+ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.

3D Droplet-Based Microfluidic Device Easily Assembled from Commercially Available Modules Online Coupled with ICPMS for Determination of Silver in Single Cell.

More recently, single-cell analysis based on ICPMS has made considerable headway while a challenge remains to differentiate single cell from doublets during the analysis. One burgeoning solution is to encapsulate single cell into droplets on the platform of the microfluidic chip. However, the manufacture of the droplet-based microfluidic chip requires sophisticated fabrication and limits its potential application. In this paper, we presented an off-the-shelf three-dimensional (3D) microfluidic device by assembling commercially available parts without any proficient manufacturing process. Uniform monodisperse microdroplet was generated from the 3D microfluidic device with a size variation of 1.5%, and the innner diameter of the 3D microfluidic device was the same as the nebulizer (150 µm). The proposed 3D microfluidic device-time-resolved ICPMS system was applied to detect silver in single AgNPs (51 nm), and the result is in good agreement with conventional acid digestion method, demonstrating the accuracy of the method. Silver uptake behaviors in HepG2 cells were then studied by incubating with Ag+ or AgNPs under biocompatible conditions. The results revealed that the cell-to-cell variability in terms of the diversity of cells incubated with AgNPs was wider than those cells incubated with Ag+ from the aspect of the content distribution of silver at the single-cell level.

Monovalent cation transporters at the plasma membrane in yeasts.

Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H+ -ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na+ , K+ , and H+ , both under normal growth conditions and in response to stress.