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We studied the impact of Ba2+ ions on the function and structure of large conductance potassium (BK) channels. Ion composition has played a crucial role in the physiological studies of BK channels due to their ability to couple ion composition and membrane voltage signaling. Unlike Ca2+, which activates BK channels through all Regulator of K+Conductance (RCK) domains, Ba2+ has been described as specifically interacting with the RCK2 domain. It has been shown that Ba2+ also blocks potassium permeation by binding to the channel’s selectivity filter. The Cryo-EM structure of the Aplysia BK channel in the presence of high concentration Ba2+ here presented (PDBID: 7RJT) revealed that Ba2+ occupies the K+ S3 site in the selectivity filter. Densities attributed to K+ ions were observed at sites S2 and S4. Ba2+ ions were also found bound to the high-affinity Ca2+ binding sites RCK1 and RCK2, which agrees with functional work suggesting that the Ba2+ increases open probability through the Ca2+ bowl site (RCK2). A comparative analysis with a second structure here presented (PDBID: 7RK6), obtained without additional Ba2+, shows localized changes between the RCK1 and RCK2 domains, suggestive of coordinated dynamics between the RCK ion binding sites with possible relevance for the activation/blockade of the channel. The observed densities attributed to Ba2+ at RCK1 and RCK2 sites and the selectivity filter contribute to a deeper understanding of the structural basis for Ba2+'s dual role in BK channel modulation, adding to the existing knowledge in this field.

Actinomyces oris plays an important role in oral biofilm development. Like many gram-positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA-a genetic suppressor of srtA deletion lethality-not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild-type cells, under elevated concentrations of mono- and divalent cations the formation of mono- and multi-species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, compared to the wild-type strain. Altogether, the results support the notion that the cell wall-anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris.

Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge-based design of cancer-targeting molecular tools, such as pH-low insertion peptide (pHLIP). Here, we explore the effects of PS, Ca2+ , and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond-timescale all-atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide-lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH-induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+ -induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to linking cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+ -dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid-coupled regulation of protein-membrane insertion by divalent cations.

Exosomes are considered potential biomarkers for early screening and accurate non-invasive diagnosis of cancer, so development of innovatively facile approaches for the detection of cancer cell-derived exosomes has become increasingly important. Herein, we propose a facile electrochemical biosensor based on divalent aptamer-functionalized nanochannels for highly efficient detection of cancer cell-derived exosomes. The aptamer against transmembrane receptor protein CD63 and the aptamer targeting membrane protein EpCAM are simultaneously immobilized on the nanochannels to construct the divalent aptamer-functionalized nanochannels. Thus, the target exosomes can be recognized and selectively captured by the functionalized nanochannels in a divalent collaborative manner. The combined exosomes overlay the ion channel effectively and hinder the ionic flow through the nanochannels, resulting in an evidently varied ionic transport behavior corresponding to the abundance of exosomes. The divalent aptamer-functionalized nanochannels can substantially promote the binding stability and enhance the detection specificity, while the sensitivity of detection is improved greatly by virtue of the amplified response of array channels synergized with the electrochemical technique. Therefore, the developed biosensor provides a highly specific, sensitive, and accurate approach for the detection of cancer cell-derived exosomes, which may hold great potential for application in early clinical cancer diagnosis. [ABSTRACT FROM AUTHOR]

Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.

Exosomes are considered potential biomarkers for early screening and accurate non-invasive diagnosis of cancer, so development of innovatively facile approaches for the detection of cancer cell-derived exosomes has become increasingly important. Herein, we propose a facile electrochemical biosensor based on divalent aptamer-functionalized nanochannels for highly efficient detection of cancer cell-derived exosomes. The aptamer against transmembrane receptor protein CD63 and the aptamer targeting membrane protein EpCAM are simultaneously immobilized on the nanochannels to construct the divalent aptamer-functionalized nanochannels. Thus, the target exosomes can be recognized and selectively captured by the functionalized nanochannels in a divalent collaborative manner. The combined exosomes overlay the ion channel effectively and hinder the ionic flow through the nanochannels, resulting in an evidently varied ionic transport behavior corresponding to the abundance of exosomes. The divalent aptamer-functionalized nanochannels can substantially promote the binding stability and enhance the detection specificity, while the sensitivity of detection is improved greatly by virtue of the amplified response of array channels synergized with the electrochemical technique. Therefore, the developed biosensor provides a highly specific, sensitive, and accurate approach for the detection of cancer cell-derived exosomes, which may hold great potential for application in early clinical cancer diagnosis.

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca2+ and inhibition by Zn2+, which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca2+ and Zn2+, which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.

Ion channels control the ability of human sperm to fertilize the egg by triggering hyperactivated motility, which is regulated by membrane potential, intracellular pH, and cytosolic calcium. Previous studies unraveled three essential ion channels that regulate these parameters: (1) the Ca2+ channel CatSper, (2) the K+ channel KSper, and (3) the H+ channel Hv1. However, the molecular identity of the sperm Na+ conductance that mediates initial membrane depolarization and, thus, triggers downstream signaling events is yet to be defined. Here, we functionally characterize DSper, the Depolarizing Channel of Sperm, as the temperature-activated channel TRPV4. It is functionally expressed at both mRNA and protein levels, while other temperature-sensitive TRPV channels are not functional in human sperm. DSper currents are activated by warm temperatures and mediate cation conductance, that shares a pharmacological profile reminiscent of TRPV4. Together, these results suggest that TRPV4 activation triggers initial membrane depolarization, facilitating both CatSper and Hv1 gating and, consequently, sperm hyperactivation.

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A2A GPCR. While Na+ reinforces an inactive ensemble and a partial-agonist stabilized state, Ca2+ and Mg2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.

Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms.

Defective Ca2+ handling is a key mechanism underlying hepatic endoplasmic reticulum (ER) dysfunction in obesity. ER Ca2+ level is in part monitored by the store-operated Ca2+ entry (SOCE) system, an adaptive mechanism that senses ER luminal Ca2+ concentrations through the STIM proteins and facilitates import of the ion from the extracellular space. Here, we show that hepatocytes from obese mice displayed significantly diminished SOCE as a result of impaired STIM1 translocation, which was associated with aberrant STIM1 O-GlycNAcylation. Primary hepatocytes deficient in STIM1 exhibited elevated cellular stress as well as impaired insulin action, increased glucose production and lipid droplet accumulation. Additionally, mice with acute liver deletion of STIM1 displayed systemic glucose intolerance. Conversely, over-expression of STIM1 in obese mice led to increased SOCE, which was sufficient to improve systemic glucose tolerance. These findings demonstrate that SOCE is an important mechanism for healthy hepatic Ca2+ balance and systemic metabolic control.

Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.

Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression.

BackgroundDevelopment of hypocalcemia after treatment of hyperparathyroidism results in increased costs and risk of poorer outcomes. Previous studies have shown conflicting data about predictors of hypocalcemia after these procedures.Hypothesis/objectivesThe objective of this study was to investigate whether ionized calcium (iCa) concentrations before treatment are predictive of hypocalcemia or its clinical signs after surgical removal or heat ablation in dogs with primary hyperparathyroidism.AnimalsFifty-four dogs with primary hyperparathyroidism (29 female, 25 male; 49 retrospective, 5 prospective).MethodsDogs were enrolled if they met the inclusion criteria: persistent hypercalcemia (iCa >1.41 mmol/L) due to primary hyperparathyroidism and absence of preemptive calcitriol treatment. All dogs were treated with parathyroidectomy (n = 37) or percutaneous ultrasound-guided heat ablation (n = 17). After treatment, iCa was monitored twice daily until plateau or intervention.ResultsThere was a moderate correlation between before-treatment hypercalcemia and after-treatment hypocalcemia. The prospective study was terminated due to ethical concerns given findings in the retrospective section. All dogs were placed into groups according to their pretreatment iCa: 1.46-1.61 mmol/L, 1.62-1.71 mmol/L, iCa 1.72-1.81 mmol/L, or >1.81 mmol/L. After treatment, the mean lowest iCa for each group, respectively, was 1.19, 1.18, 1.13, and 1.01 mmol/L. There was a significant association between higher group and proportion of dogs with iCa

Cost-effective and controllable synthesis of M0.25Ni0.15Cu0.25Co0.35Fe2O4 (M2+ = Mg2+, Mn2+, and Cd2+) ferrites via the sol–gel auto-combustion technique. The impact of divalent cations on the structural, dielectric, and optoelectrical properties of ferrites was examined by XRD, FTIR, Raman, LCR, UV–Vis, and two probe I-V measurement techniques. The crystallite size was 52.66 nm, and the minimum specific surface area was observed 5.1507 m2/g for Mg2+ doped NCCF ferrite. The FTIR and Raman analysis also confirmed the substitution of divalent cations (M2+ = Mg2+, Mn2+, and Cd2+) at their respective lattice sites. The maximum energy bandgap was 1.67 eV Mg2+-doped NCCF ferrite as compared to other divalent ion-doped ferrites. The dielectric loss decreased while the ac conductivity increased with increasing frequency, and the minimum values were observed for Mg2+-doped NCCF ferrite. The activation energy was observed maximum for Mg2+-doped NCCF ferrite (0.2234 eV). Due to incredible properties including small specific surface area, large energy band gap, high resistivity, and loss dielectric loss of Mg2+-doped NCCF ferrite have potential applications in different fields. [ABSTRACT FROM AUTHOR]

Histone deacetylases (HDACs) are responsible for the removal of acetyl groups from histones, resulting in gene silencing. Overexpression of HDACs is associated with cancer, and their inhibitors are of particular interest as chemotherapeutics. However, HDACs remain a target of mechanistic debate. HDAC class 8 is the most studied HDAC, and of particular importance due to its human oncological relevance. HDAC8 has traditionally been considered to be a Zn-dependent enzyme. However, recent experimental assays have challenged this assumption and shown that HDAC8 is catalytically active with a variety of different metals, and that it may be a Fe-dependent enzyme in vivo. We studied two opposing mechanisms utilizing a series of divalent metal ions in physiological abundance (Zn(2+), Fe(2+), Co(2+), Mn(2+), Ni(2+), and Mg(2+)). Extensive sampling of the entire protein with different bound metals was done with the mixed quantum-classical QM/DMD method. Density functional theory (DFT) on an unusually large cluster model was used to describe the active site and reaction mechanism. We have found that the reaction profile of HDAC8 is similar among all metals tested, and follows one of the previously published mechanisms, but the rate-determining step is different from the one previously claimed. We further provide a scheme for estimating the metal binding affinities to the protein. We use the quantum theory of atoms in molecules (QTAIM) to understand the different binding affinities for each metal in HDAC8 as well as the ability of each metal to bind and properly orient the substrate for deacetylation. The combination of this data with the catalytic rate constants is required to reproduce the experimentally observed trend in metal-depending performance. We predict Co(2+) and Zn(2+) to be the most active metals in HDAC8, followed by Fe(2+), and Mn(2+) and Mg(2+) to be the least active.

Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.

The neural activity patterns of suprachiasmatic nucleus (SCN) neurons are dynamically regulated throughout the circadian cycle with highest levels of spontaneous action potentials during the day. These rhythms in electrical activity are critical for the function of the circadian timing system and yet the mechanisms by which the molecular clockwork drives changes in the membrane are not well understood. In this study, we sought to examine how the clock gene Period1 (Per1) regulates the electrical activity in the mouse SCN by transiently and selectively decreasing levels of PER1 through use of an antisense oligodeoxynucleotide. We found that this treatment effectively reduced SCN neural activity. Direct current injection to restore the normal membrane potential partially, but not completely, returned firing rate to normal levels. The antisense treatment also reduced baseline [Ca(2+)]i levels as measured by Fura2 imaging technique. Whole cell patch clamp recording techniques were used to examine which specific potassium currents were altered by the treatment. These recordings revealed that the large conductance [Ca(2+)]i-activated potassium currents were reduced in antisense-treated neurons and that blocking this current mimicked the effects of the anti-sense on SCN firing rate. These results indicate that the circadian clock gene Per1 alters firing rate in SCN neurons and raise the possibility that the large conductance [Ca(2+)]i-activated channel is one of the targets.

Current treatments for cutaneous and visceral leishmaniasis are toxic, expensive, difficult to administer, and limited in efficacy and availability. Disulfiram has primarily been used to treat alcoholism. More recently, it has shown some efficacy as therapy against protozoan pathogens and certain cancers, suggesting a wide range of biological activities. We used an ex vivo system to screen several thiuram disulfide compounds for antileishmanial activity. We found five compounds (compound identifier [CID] 7188, 5455, 95876, 12892, and 3117 [disulfiram]) with anti-Leishmania activity at nanomolar concentrations. We further evaluated these compounds with the addition of divalent metal salts based on studies that indicated these salts could potentiate the action of disulfiram. In addition, clinical studies suggested that zinc has some efficacy in treating cutaneous leishmaniasis. Several divalent metal salts were evaluated at 1 μM, which is lower than the normal levels of copper and zinc in plasma of healthy individuals. The leishmanicidal activity of disulfiram and CID 7188 were enhanced by several divalent metal salts at 1 μM. The in vitro therapeutic index (IVTI) of disulfiram and CID 7188 increased 12- and 2.3-fold, respectively, against L. major when combined with ZnCl2. The combination of disulfiram with ZnSO4 resulted in a 1.8-fold increase in IVTI against L. donovani. This novel combination of thiuram disulfides and divalent metal ions salts could have application as topical and/or oral therapies for treatment of cutaneous and visceral leishmaniasis.

Cellular edema (cell swelling) is a principal component of numerous brain disorders including ischemia, cortical spreading depression, hyponatremia, and epilepsy. Cellular edema increases seizure-like activity in vitro and in vivo, largely through nonsynaptic mechanisms attributable to reduction of the extracellular space. However, the types of excitability changes occurring in individual neurons during the acute phase of cell volume increase remain unclear. Using whole-cell patch clamp techniques, we report that one of the first effects of osmotic edema on excitability of CA1 pyramidal cells is the generation of slow inward currents (SICs), which initiate after approximately 1 min. Frequency of SICs increased as osmolarity decreased in a dose-dependent manner. Imaging of real-time volume changes in astrocytes revealed that neuronal SICs occurred while astrocytes were still in the process of swelling. SICs evoked by cell swelling were mainly nonsynaptic in origin and NMDA receptor-dependent. To better understand the relationship between SICs and changes in neuronal excitability, recordings were performed in increasingly physiological conditions. In the absence of any added pharmacological reagents or imposed voltage clamp, osmotic edema induced excitatory postsynaptic potentials and burst firing over the same timecourse as SICs. Like SICs, action potentials were blocked by NMDAR antagonists. Effects were more pronounced in adult (8-20 weeks old) compared with juvenile (P15-P21) mice. Together, our results indicate that cell swelling triggered by reduced osmolarity rapidly increases neuronal excitability through activation of NMDA receptors. Our findings have important implications for understanding nonsynaptic mechanisms of epilepsy in relation to cell swelling and reduction of the extracellular space.

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.

Guanine-rich sequences can, under appropriate conditions, adopt a distinctive, four-stranded, helical fold known as a G-quadruplex. Interest in quadruplex folds has grown in recent years as evidence of their biological relevance has accumulated from both sequence analysis and function-specific assays. The folds are unusually stable and their formation appears to require close management to maintain cell health; regulatory failure correlates with genomic instability and a number of cancer phenotypes. Biologically relevant quadruplex folds are anticipated to form transiently in mRNA and in single-stranded, unwound DNA. To elucidate factors, including bound solvent, that contribute to the stability of RNA quadruplexes, we examine, by X-ray crystallography and small-angle X-ray scattering, the structure of a previously reported tetramolecular quadruplex, UGGGGU stabilized by Sr(2+) ions. Crystal forms of the octameric assembly formed by this sequence exhibit unusually strong diffraction and anomalous signal enabling the construction of reliable models to a resolution of 0.88Å. The solvent structure confirms hydration patterns reported for other nucleic acid helical conformations and provides support for the greater stability of RNA quadruplexes relative to DNA. Novel features detected in the octameric RNA assembly include a new crystal form, evidence of multiple conformations and structural variations in the 3' U tetrad, including one that leads to the formation of a hydrated internal cavity.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

We have scrutinized eighteen new derivatives of carbenes with one, two, and three carbenic centers and compared their structural and thermodynamic parameters, at B3LYP/6-311++G** level of theory. All novel carbene scrutinized show singlet ground stat, and the highest stability belongs to 1,2,3,4-tetraazabicyclo[1.1.1]-5-pentanylene (9) (ΔEs-t = 47.95 kcal/mol). This stability can be related to their σ−bond and intermolecular interactions. This bond arose from a tendency of nonbonding electrons of nitrogen (N(E or G)) to empty p orbital of the carbenic center. In addition, carbene 16s and 17s because of three coordinate covalent bonds with their carbenic centers have low nucleophilicity (N). The increase in band gaps (ΔΕHOMO–LUMO) is correlated with an increase in ΔΕs–t values. The purpose of the present work was to assess the influence of 1 to 4 nitrogen substituents on the stability (ΔEs-t), ΔΕHOMO–LUMO, N, electrophilicity (ω), and isodesmic reactions. Finally, our investigation introduces novel carbenes that can be applied as cumulated multi-dentate ligands. [ABSTRACT FROM AUTHOR]

Substitution effects on stability (ΔΕs-t) of novel singlet and triplet forms of bicyclo[2.2.1]hepta-7-silylenes are compared and contrasted, at B3LYP/6–311++G** level of theory. All species appear as ground state minima on their energy surface, for showing no negative force constant. Singlets (1s-24s) are ground state and more stable than their corresponding triplets (1t-24t). The most stable scrutinized silylenes appear to be 2,3-disilabicyclo[2.2.1]hepta-7-silylene (9) for showing the highest value of ΔEs-t. This stability can be related to our imposed topology and β-silicon effect. The band gaps (ΔΕHOMO–LUMO) show the same trend as ΔEs-t and the lowest unoccupied molecular orbital energies. Also, the electrophilicity appears inverse correlation with our results of ΔΕs–t. The purpose of the present work was to assess the influence of 1 to 6 silicon substitutions on the stability, band gaps, nucleophilicity, electrophilicity, and proton affinity. Finally, our investigation introduces novel silylenes with possible applications in chemistry such as semiconductors, cumulated multidentate ligands, etc. [ABSTRACT FROM AUTHOR]

Metal ion assimilation is essential for all forms of life. However, organisms must properly control the availability of these nutrients within the cell to avoid inactivating proteins by mismetallation. To safeguard against an imbalance between supply and demand in eukaryotes, intracellular compartments contain metal transporters that load and unload metals. Although the vacuoles of Saccharomyces cerevisiae and Arabidopsis thaliana are well established locales for the storage of copper, zinc, iron, and manganese, related compartments are emerging as important mediators of metal homeostasis. Here we describe these compartments and review their metal transporter complement.

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β3 integrins αIIbβ3 and αVβ3, but a potential role of this interaction on stability of the metal ion at LIMBS in β3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbβ3 versus αVβ3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVβ3. Replacing the αIIb-LIMBS interface residue Phe(191) in αIIb (equivalent to Trp(179) in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbβ3; a Trp(179) to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbβ3 and a W179/F substitution in αVβ3 reduced and increased, respectively, the apparent affinity of Mn(2+) to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVβ3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.

HDV-like self-cleaving ribozymes have been found in a wide variety of organisms, implicated in diverse biological processes, and their activity typically shows a strong divalent metal dependence, but little metal specificity. Recent studies suggested that very short variants of these ribozymes exist in nature, but their distribution and biochemical properties have not been established. To map out the distribution of small HDV-like ribozymes, the drz-Spur-3 sequence was minimized to yield a core construct for structure-based bioinformatic searches. These searches revealed several microbial ribozymes, particularly in the human microbiome. Kinetic profile of the smallest ribozyme revealed two distinct metal binding sites, only one of which promotes fast catalysis. Furthermore, this ribozyme showed markedly reduced activity in Ca(2+), even in the presence of physiological Mg(2+) concentrations. Our study substantially expands the number of microbial HDV-like ribozymes and provides an example of cleavage regulation by divalent metals.

Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca(2+)-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca(2+), Sr(2+), and Ba(2+), and discovered that Mg(2+) competes with Ca(2+) in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore-as revealed by the permeability ratios of these anions-appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.

Transient receptor potential vanilloid type 1 (TRPV1) channel responds to a wide spectrum of physical and chemical stimuli. In doing so, it serves as a polymodal cellular sensor for temperature change and pain. Many chemicals are known to strongly potentiate TRPV1 activation, though how this is achieved remains unclear. In this study we investigated the molecular mechanism underlying the gating effects of divalent cations Mg(2+) and Ba(2+). Using a combination of fluorescence imaging and patch-clamp analysis, we found that these cations potentiate TRPV1 gating by most likely promoting the heat activation process. Mg(2+) substantially lowers the activation threshold temperature; as a result, a significant fraction of channels are heat-activated at room temperature. Although Mg(2+) also potentiates capsaicin- and voltage-dependent activation, these processes were found either to be not required (in the case of capsaicin) or insufficient (in the case of voltage) to mediate the activating effect. In support of a selective effect on heat activation, Mg(2+) and Ba(2+) cause a Ca(2+)-independent desensitization that specifically prevents heat-induced channel activation but does not prevent capsaicin-induced activation. These results can be satisfactorily explained within an allosteric gating framework in which divalent cations strongly promote the heat-dependent conformational change or its coupling to channel activation, which is further coupled to the voltage- and capsaicin-dependent processes.

Divalent cations Mg(2+) and Ba(2+) selectively and directly potentiate transient receptor potential vanilloid type 1 heat activation by lowering the activation threshold into the room temperature range. We found that Mg(2+) potentiates channel activation only from the extracellular side; on the intracellular side, Mg(2+) inhibits channel current. By dividing the extracellularly accessible region of the channel protein into small segments and perturbing the structure of each segment with sequence replacement mutations, we observed that the S1-S2 linker, the S3-S4 linker, and the pore turret are all required for Mg(2+) potentiation. Sequence replacements at these regions substantially reduced or eliminated Mg(2+)-induced activation at room temperature while sparing capsaicin activation. Heat activation was affected by many, but not all, of these structural alternations. These observations indicate that extracellular linkers and the turret may interact with each other. Site-directed fluorescence resonance energy transfer measurements further revealed that, like heat, Mg(2+) also induces structural changes in the pore turret. Interestingly, turret movement induced by Mg(2+) precedes channel activation, suggesting that Mg(2+)-induced conformational change in the extracellular region most likely serves as the cause of channel activation instead of a coincidental or accommodating structural adjustment.

Single-molecule experiments indicate that integrin affinity is cation-type-dependent, but in spread cells integrins are engaged in complex focal adhesions (FAs), which can also regulate affinity. To better understand cation-type-dependent adhesion in fully spread cells, we investigated attachment strength by application of external shear. While cell attachment strength is indeed modulated by cations, the regulation of integrin-mediated adhesion is also exceedingly complex, cell specific, and niche dependent. In the presence of magnesium only, fibroblasts and fibrosarcoma cells remodel their cytoskeleton to align in the direction of applied shear in an α5-integrin/fibronectin-dependent manner, which allows them to withstand higher shear. In the presence of calcium or on collagen in modest shear, fibroblasts undergo piecewise detachment but fibrosarcoma cells exhibit increased attachment strength. These data augment the current understanding of force-mediated detachment by suggesting a dynamic interplay in situ between cell adhesion and integrins depending on local niche cation conditions.

We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na(+) ion binding in the ribozyme's active site. At least two such Na(+) ions are observed. The first Na(+) ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na(+) ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na(+), but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na(+) directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank.

Two virtually identical acireductone dioxygenases, ARD and ARD', catalyze completely different oxidation reactions of the same substrate, 1,2-dihydroxy-3-keto-5-(methylthio)pentene, depending exclusively on the nature of the bound metal. Fe(2+)-dependent ARD' produces the α-keto acid precursor of methionine and formate and allows for the recycling of methionine in cells. Ni(2+)-dependent ARD instead produces methylthiopropionate, CO, and formate, and exits the methionine salvage cycle. This mechanistic difference has not been understood to date but has been speculated to be due to the difference in coordination of the substrate to Fe(2+)versus Ni(2+): forming a five-membered ring versus a six-membered ring, respectively, thus exposing different carbon atoms for the attack by O2. Here, using mixed quantum-classical molecular dynamics simulations followed by the density functional theory mechanistic investigation, we show that, contrary to the old hypothesis, both metals preferentially bind the substrate as a six-membered ring, exposing the exact same sites to the attack by O2. It is the electronic properties of the metals that are then responsible for the system following different reaction paths, to yield the respective products. We fully explain the puzzling metal-induced difference in functionality between ARD and ARD' and, in particular, propose a new mechanism for ARD'. All results are in agreement with available isotopic substitution and other experimental data.

The NCS protein Visinin-like Protein 1 (VILIP-1) transduces calcium signals in the brain and serves as an effector of the non-retinal receptor guanylyl cyclases (GCs) GC-A and GC-B, and nicotinic acetyl choline receptors (nAchR). Analysis of the quaternary structure of VILIP-1 in solution reveals the existence of monomeric and dimeric species, the relative contents of which are affected but not exclusively regulated by divalent metal ions and Redox conditions. Using small-angle X-ray scattering, we have investigated the low resolution structure of the calcium-bound VILIP-1 dimer under reducing conditions. Scattering profiles for samples with high monomeric and dimeric contents have been obtained. The dimerization interface involves residues from EF-hand regions EF3 and EF4.Using monolayer adsorption experiments, we show that myristoylated and unmyristoylated VILIP-1 can bind lipid membranes. The presence of calcium only marginally improves binding of the protein to the monolayer, suggesting that charged residues at the protein surface may play a role in the binding process.In the presence of calcium, VILIP-1 undergoes a conformational re-arrangement, exposing previously hidden surfaces for interaction with protein partners. We hypothesise a working model where dimeric VILIP-1 interacts with the membrane where it binds membrane-bound receptors in a calcium-dependent manner.

Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A 2 (GIA PLA 2) was carried out in the presence of metal ions Ca (2+) and Ba (2+) and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large alpha-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca (2+) or Ba (2+) ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca (2+). The crystal structure of the N. naja naja GIA PLA 2 contains a single Ca (2+) bound in the catalytic site, but the crystal structures of related PLA 2s contain a second Ca (2+) binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA 2 does in fact bind two Ca (2+) ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 microM Ca (2+) present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.

Sedum alfredii (Crasulaceae) is the only known Cd-hyperaccumulating species that are not in the Brassica family; the mechanism of Cd hyperaccumulation in this plant is, however, little understood. Here, a combination of radioactive techniques, metabolic inhibitors, and fluorescence imaging was used to contrast Cd uptake and translocation between a hyperaccumulating ecotype (HE) and a non-hyperaccumulating ecotype (NHE) of S. alfredii. The K(m) of (109)Cd influx into roots was similar in both ecotypes, while the V(max) was 2-fold higher in the HE. Significant inhibition of Cd uptake by low temperature or metabolic inhibitors was observed in the HE, whereas the effect was less pronounced in the NHE. (109)Cd influx into roots was also significantly decreased by high Ca in both ecotypes. The rate of root-to-shoot translocation of (109)Cd in the HE was >10 times higher when compared with the NHE, and shoots of the HE accumulated dramatically higher (109)Cd concentrations those of the NHE. The addition of the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) resulted in a significant reduction in Cd contents in the shoots of the HE, and in the roots of the NHE. Cd was distributed preferentially to the root cylinder of the HE but not the NHE, and there was a 3-5 times higher Cd concentration in xylem sap of the HE in contrast to the NHE. These results illustrate that a greatly enhanced rate of root-to-shoot translocation, possibly as a result of enhanced xylem loading, rather than differences in the rate of root uptake, was the pivotal process expressed in the Cd hyperaccumulator HE S. alfredii.

• Ruthenium is electrodeposited from a non-aqueous electrolyte based on ethylene glycol. • Ruthenium is deposited from a solution containing the metal in its divalent state. • Ru(III) is partially reduced to Ru(II) using ascorbic acid. • Compact and crack-free layers are obtained under galvanostatic deposition. • The layers are highly pure and characterized by good electrical properties. The electrodeposition of ruthenium from an ethylene glycol based electrolyte containing the metal in its divalent state is investigated for the first time. Ascorbic acid is used to reduce a fraction of the Ru(III) ions added to the non-aqueous solvent to Ru(II). Its effect is studied by means of UV–visible spectroscopy and cyclic voltammetry, demonstrating the presence of divalent ions and the stability of the electrolyte over time. Ruthenium coatings are deposited on gold-sputtered silicon wafers under galvanostatic conditions at various current densities and deposition times. The characterizations carried out evidence the significant purity of the ruthenium film, while the morphological analysis reveals its compact and crack-free morphology. Indeed, thicknesses up to 2.35 µm are successfully plated with no sign of delamination or cracking. The good electrical properties observed for the deposited layers validate the possible applicability of the Ru(II) containing non-aqueous electrolyte here described to the manufacturing of integrated circuits interconnects or contacts for electrical applications. [Display omitted] [ABSTRACT FROM AUTHOR]

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.

Manganese toxicity can evoke neuropsychiatric and neuromotor symptoms, which have frequently been attributed to profound oxidative stress in the dopaminergic system. However, the characterization of manganese as a pro-oxidant remains controversial because antioxidant properties also have been associated with this metal. The current study was designed to address these disparate findings concerning the oxidative properties of manganese. The apparent ability of manganese in its divalent form to promote formation of reactive oxygen species (ROS) within a cortical mitochondrial-synaptosomal (P2) fraction was completely abolished by the addition of one five hundredth of its molarity of desferroxamine (DFO), a trivalent metal chelator. This large ratio and the high specificity of DFO for trivalent metal ions discounted the possibility of inhibition of ROS generation by direct sequestration of divalent manganese, and implied the trace presence of a trivalent metal. Further analysis suggested that this trace metal was manganic rather than ferric ion. Ferric ion was able to dampen the reactive oxygen species-generating capacity of manganous chloride, whereas manganic ion markedly promoted this property attributed to manganous ion. Such findings of the potent effects of trace amounts of trivalent cations upon Mn2+-related free radical generation offer resolution of earlier disparate findings concerning the oxidative character of manganese.

We have constructed a model structure that we believe represents the strongest possible physically and chemically reasonable representation of a hypothesized catalytically active hammerhead ribozyme structure in which a single divalent metal ion bridges the A9 and scissile phosphate groups. It has been proposed that such a structure arises from a conformational change in which the so-called ground-state structure (as observed by X-ray crystallography) rearranges in such a way that the pro-R oxygen atoms of both the A9 and scissile phosphate groups are directly coordinated by a single divalent metal ion in the transition-state of the hammerhead ribozyme cleavage reaction. We show that even the small subset of possible model structures that are consistent with these requirements, and that are stereochemically and sterically reasonable, are contradicted by experimental evidence. We also demonstrate that even a minimal subset of assumptions, i.e. that stems I and II are helical and that the two phosphate groups are coordinated by a divalent metal ion in the standard octahedral geometry, are sufficient to lead to this contradiction. We therefore conclude that such a mechanism of hammerhead ribozyme catalysis is untenable, at least in its present formulation.

Extracellular Zn2+ was found to reversibly inhibit the ClC-0 Cl- channel. The apparent on and off rates of the inhibition were highly temperature sensitive, suggesting an effect of Zn2+ on the slow gating (or inactivation) of ClC-0. In the absence of Zn2+, the rate of the slow-gating relaxation increased with temperature, with a Q10 of approximately 37. Extracellular Zn2+ facilitated the slow-gating process at all temperatures, but the Q10 did not change. Further analysis of the rate constants of the slow-gating process indicates that the effect of Zn2+ is mostly on the forward rate (the rate of inactivation) rather than the backward rate (the rate of recovery from inactivation) of the slow gating. When ClC-0 is bound with Zn2+, the equilibrium constant of the slow-gating process is increased by approximately 30-fold, reflecting a 30-fold higher Zn2+ affinity in the inactivated channel than in the open-state channel. As examined through a wide range of membrane potentials, Zn2+ inhibits the opening of the slow gate with equal potency at all voltages, suggesting that a two-state model is inadequate to describe the slow-gating transition. Following a model originally proposed by Pusch and co-workers (Pusch, M., U. Ludewig, and T.J. Jentsch. 1997. J. Gen. Physiol. 109:105-116), the effect of Zn2+ on the activation curve of the slow gate can be well described by adding two constraints: (a) the dissociation constant for Zn2+ binding to the open channel is 30 microM, and (b) the difference in entropy between the open state and the transition state of the slow-gating process is increased by 27 J/ mol/ degreesK for the Zn2+-bound channel. These results together indicate that extracellular Zn2+ inhibits ClC-0 by facilitating the slow-gating process.

Abstract Novel electrolytes with wide electrochemical potential window and high thermal stability have great potential for aqueous rechargeable lithium-ion batteries (ARLBs). Herein, we report the synthesis of two ionic salts of lithium sulfonylbis(fluorosulfonyl)imide (LiSFSI) and lithium carbonylbis(fluorosulfonyl)imide (LiCFSI) with divalent Li+ for ARLBs. These ionic compounds are the derivatives of monovalent lithium bis(fluorosulfonyl)imide (LiFSI). The LiSFSI and LiCFSI exhibit the kinetic electrochemical stability window of ca. 3.78 and 3.52 V, respectively, which can be further expanded due to the formation of a stable solid electrolyte interface (SEI) layer. While LiFSI exhibits the kinetic electrochemical stability window of ca. 2.22 V without the formation of an SEI layer. Full ARLBs based on LiSFSI and LiCFSI electrolytes with a LiCoO 2 cathode and graphite anode can deliver the specific discharge capacity of ca. 113.50 and 95.0 mAh/g, respectively, at 0.1C rate. Whereas, it is ca. 52.53 mAh/g for LiFSI at 0.1C rate. The capacity retention for LiSFSI, LiCFSI, and LiFSI based ARLBs are ca. 97.3, 89.6, and 67.8%, respectively, after 500 cycles. Furthermore, both LiSFSI and LiCFSI reveal much higher thermal stability compared to LiFSI. Thus, the derivatization of conventional ionic compounds is an effective strategy to enhance the battery performance and its lifetime. Graphical abstract Image 1 Highlights • Novel divalent organo-lithium electrolytes (LiSFSI and LiCFSI) were prepared and characterized. • The electrolytes exhibited enhanced thermal and electrochemical stability. • Both LiSFSI and LiCFSI have formed a stable and dense SEI layer. • A maximum specific discharge capacity of 113.50 mAh/g was attained with LiSFSI in ARLBs. [ABSTRACT FROM AUTHOR]