Genome-Wide Analysis of Cation/Proton Antiporter Family in Soybean (Glycine max) and Functional Analysis of GmCHX20a on Salt Response.

Monovalent cation proton antiporters (CPAs) play crucial roles in ion and pH homeostasis, which is essential for plant development and environmental adaptation, including salt tolerance. Here, 68 CPA genes were identified in soybean, phylogenetically dividing into 11 Na+/H+ exchangers (NHXs), 12 K+ efflux antiporters (KEAs), and 45 cation/H+ exchangers (CHXs). The GmCPA genes are unevenly distributed across the 20 chromosomes and might expand largely due to segmental duplication in soybean. The GmCPA family underwent purifying selection rather than neutral or positive selections. The cis-element analysis and the publicly available transcriptome data indicated that GmCPAs are involved in development and various environmental adaptations, especially for salt tolerance. Based on the RNA-seq data, twelve of the chosen GmCPA genes were confirmed for their differentially expression under salt or osmotic stresses using qRT-PCR. Among them, GmCHX20a was selected due to its high induction under salt stress for the exploration of its biological function on salt responses by ectopic expressing in Arabidopsis. The results suggest that the overexpression of GmCHX20a increases the sensitivity to salt stress by altering the redox system. Overall, this study provides comprehensive insights into the CPA family in soybean and has the potential to supply new candidate genes to develop salt-tolerant soybean varieties.

Monovalent metal ion binding promotes the first transesterification reaction in the spliceosome.

Cleavage and formation of phosphodiester bonds in nucleic acids is accomplished by large cellular machineries composed of both protein and RNA. Long thought to rely on a two-metal-ion mechanism for catalysis, structure comparisons revealed many contain highly spatially conserved second-shell monovalent cations, whose precise function remains elusive. A recent high-resolution structure of the spliceosome, essential for pre-mRNA splicing in eukaryotes, revealed a potassium ion in the active site. Here, we employ biased quantum mechanics/ molecular mechanics molecular dynamics to elucidate the function of this monovalent ion in splicing. We discover that the K+ ion regulates the kinetics and thermodynamics of the first splicing step by rigidifying the active site and stabilizing the substrate in the pre- and post-catalytic state via formation of key hydrogen bonds. Our work supports a direct role for the K+ ion during catalysis and provides a mechanistic hypothesis likely shared by other nucleic acid processing enzymes.

Regulation of Mitochondrial Permeability Transition Pore Opening by Monovalent Cations in Liver Mitochondria.

The opening of the permeability transition pore (PTP) in mitochondria is a key event in the initiation of cell death in various pathologic states, including ischemia/reperfusion. The activation of K+ transport into mitochondria protects cells from ischemia/reperfusion. However, the role of K+ transport in PTP regulation is unclear. Here, we studied the role of K+ and other monovalent cations in the regulation of the PTP opening in an in vitro model. The registration of the PTP opening, membrane potential, Ca2+-retention capacity, matrix pH, and K+ transport was performed using standard spectral and electrode techniques. We found that the presence of all cations tested in the medium (K+, Na+, choline+, and Li+) strongly stimulated the PTP opening compared with sucrose. Several possible reasons for this were examined: the effect of ionic strength, the influx of cations through selective and non-selective channels and exchangers, the suppression of Ca2+/H+ exchange, and the influx of anions. The data obtained indicate that the mechanism of PTP stimulation by cations includes the suppression of K+/H+ exchange and acidification of the matrix, which facilitates the influx of phosphate. Thus, the K+/H+ exchanger and the phosphate carrier together with selective K+ channels compose a PTP regulatory triad, which might operate in vivo.

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.

Calcium decreases cell wall swelling in sweet cherry fruit.

Swelling of epidermal cell walls decreases cell-to-cell adhesion and increases cracking susceptibility in sweet cherry. Ca is suggested to decrease cracking susceptibility by crosslinking of cell wall components and, possibly, by decreasing swelling. The objective is to test this hypothesis. The effect of Ca on swelling of anticlinal epidermal cell walls was quantified microscopically in vivo using excised skin sections and in vitro using extracted cell walls. After removal of turgor, cell wall thickness increased. Incubation in CaCl2 decreased cell wall thickness up to 3 mM CaCl2. At higher concentrations thickness remained constant. Decreased cell wall swelling in vivo also occurred with other salts of divalent and trivalent cations, but not with those of monovalent cations. Decreased swelling was due to the Ca cation, the anions had no effect. Ca also decreased swelling of cell walls that were already swollen. CaCl2 also decreased swelling of extracted cell walls in vitro. There was no effect on swelling pressure. The effect on swelling increased as the CaCl2 concentration increased. Chlorides of divalent and trivalent cations, but not those of monovalent cations decreased swelling in vitro. The decrease in swelling among the divalent cations was linearly related to the radius of the cation. The results indicate that Ca decreases cracking susceptibility by decreasing swelling.

Carboxylate binding prefers two cations to one.

Almost all studies of specific ion binding by carboxylates (-COO-) have considered only a single cation, but clustering of ions and ligands is a common phenomenon. We apply density functional theory to investigate how variations in the number of acetate ligands in binding to two monovalent cations affects ion binding preferences. We study a series of monovalent (Li+, Na+, K+, Cs+) ions relevant to experimental work on many topics, including ion channels, battery storage, water purification and solar cells. We find that the preferred optimal structure has 3 acetates except for Cs+, which has 2 acetates. The optimal coordination of the cation by the carboxylate O atoms is 4 for both Na+ and K+, and 3 for Li+ and Cs+. There is a 4-fold coordination minimum just a few kcal mol-1 higher than the optimal 3-fold structure for Li+. For two cations, multiple minima occur in the vicinity of the lowest free energy state. We find that, for Li, Na and K, the preferred optimal structure with two cations is favored over a mixture of single cation complexes, providing a basis for understanding ionic cluster formation that is relevant for engineering proteins and other materials for rapid, selective ion transport.

A DFT/PCM Study on the Affinity of Salinomycin to Bind Monovalent Metal Cations.

The affinity of the polyether ionophore salinomycin to bind IA/IB metal ions was accessed using the Gibbs free energy of the competition reaction between SalNa (taken as a reference) and its rival ions: [M+-solution] + [SalNa] → [SalM] + [Na+-solution] (M = Li, K, Rb, Cs, Cu, Ag, Au). The DFT/PCM computations revealed that the ionic radius, charge density and accepting ability of the competing metal cations, as well as the dielectric properties of the solvent, have an influence upon the selectivity of salinomycin. The optimized structures of the monovalent metal complexes demonstrate the flexibility of the ionophore, allowing the coordination of one or two water ligands in SalM-W1 and SalM-W2, respectively. The metal cations are responsible for the inner coordination sphere geometry, with coordination numbers spread between 2 (Au+), 4 (Li+ and Cu+), 5/6 (Na+, K+, Ag+), 6/7 (Rb+) and 7/8 (Cs+). The metals' affinity to salinomycin in low-polarity media follows the order of Li+ > Cu+ > Na+ > K+ > Au+ > Ag+ > Rb+ > Cs+, whereas some derangement takes place in high-dielectric environment: Li+ ≥ Na+ > K+ > Cu+ > Au+ > Ag+ > Rb+ > Cs+.

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.

Engineering potassium activation into biosynthetic thiolase.

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

Physiological Roles of the Rapidly Activated Delayed Rectifier K+ Current in Adult Mouse Heart Primary Pacemaker Activity.

Robust, spontaneous pacemaker activity originating in the sinoatrial node (SAN) of the heart is essential for cardiovascular function. Anatomical, electrophysiological, and molecular methods as well as mathematical modeling approaches have quite thoroughly characterized the transmembrane fluxes of Na+, K+ and Ca2+ that produce SAN action potentials (AP) and 'pacemaker depolarizations' in a number of different in vitro adult mammalian heart preparations. Possible ionic mechanisms that are responsible for SAN primary pacemaker activity are described in terms of: (i) a Ca2+-regulated mechanism based on a requirement for phasic release of Ca2+ from intracellular stores and activation of an inward current-mediated by Na+/Ca2+ exchange; (ii) time- and voltage-dependent activation of Na+ or Ca2+ currents, as well as a cyclic nucleotide-activated current, If; and/or (iii) a combination of (i) and (ii). Electrophysiological studies of single spontaneously active SAN myocytes in both adult mouse and rabbit hearts consistently reveal significant expression of a rapidly activating time- and voltage-dependent K+ current, often denoted IKr, that is selectively expressed in the leading or primary pacemaker region of the adult mouse SAN. The main goal of the present study was to examine by combined experimental and simulation approaches the functional or physiological roles of this K+ current in the pacemaker activity. Our patch clamp data of mouse SAN myocytes on the effects of a pharmacological blocker, E4031, revealed that a rapidly activating K+ current is essential for action potential (AP) repolarization, and its deactivation during the pacemaker potential contributes a small but significant component to the pacemaker depolarization. Mathematical simulations using a murine SAN AP model confirm that well known biophysical properties of a delayed rectifier K+ current can contribute to its role in generating spontaneous myogenic activity.

Genome-wide identification, characterization, and expression analysis of the monovalent cation-proton antiporter superfamily in maize, and functional analysis of its role in salt tolerance.

Na+, K+ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs), and 6 K+ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and most of them were localized to plasma membrane or tonoplast. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5, ZmKEA2 in NaCl tolerance. Analysis of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize breeding.

Early-stage dynamics of chloride ion-pumping rhodopsin revealed by a femtosecond X-ray laser.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

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.

In vitro and in vivo assays reveal that cations affect nematocyst discharge in Myxobolus cerebralis (Cnidaria: Myxozoa).

Myxozoans are parasitic, microscopic cnidarians that have retained the phylum-characteristic stinging capsules called nematocysts. Free-living cnidarians, like jellyfish and corals, utilize nematocysts for feeding and defence, with discharge powered by osmotic energy. Myxozoans use nematocysts to anchor to their fish hosts in the first step of infection, however, the discharge mechanism is poorly understood. We used Myxobolus cerebralis, a pathogenic myxozoan parasite of salmonid fishes, and developed two assays to explore the nature of its nematocyst discharge. Using parasite actinospores, the infectious stage to fish, we stimulated discharge of the nematocysts with rainbow trout mucus in vitro, in solutions enriched with chloride salts of Na+, K+, Ca2+ and Gd3+, and quantified discharge using microscopy. We then used quantitative polymerase chain reaction to evaluate the in vivo effects of these treatments, plus Mg2+ and the common aquaculture disinfectant KMnO4, on the ability of M. cerebralis actinospores to infect fish. We found that Mg2+ and Gd3+ reduced infection in vivo, whereas Na+ and K+ over-stimulated nematocyst discharge in vitro and reduced infection in vivo. These findings align with nematocyst discharge behaviour in free-living Cnidaria, and suggest phylum-wide commonalties, which could be exploited to develop novel approaches for controlling myxozoan diseases in aquaculture.

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.

Simulation model of CA1 pyramidal neurons reveal opposing roles for the Na+/Ca2+ exchange current and Ca2+-activated K+ current during spike-timing dependent synaptic plasticity.

Sodium Calcium exchanger (NCX) proteins utilize the electrochemical gradient of Na+ to generate Ca2+ efflux (forward mode) or influx (reverse mode). In mammals, there are three unique NCX encoding genes-NCX1, NCX2, and NCX3, that comprise the SLC8A family, and mRNA from all three exchangers is expressed in hippocampal pyramidal cells. Furthermore, mutant ncx2-/- and ncx3-/- mice have each been shown to exhibit altered long-term potentiation (LTP) in the hippocampal CA1 region due to delayed Ca2+ clearance after depolarization that alters synaptic transmission. In addition to the role of NCX at the synapse of hippocampal subfields required for LTP, the three NCX isoforms have also been shown to localize to the dendrite of hippocampal pyramidal cells. In the case of NCX1, it has been shown to localize throughout the basal and apical dendrite of CA1 neurons where it helps compartmentalize Ca2+ between dendritic shafts and spines. Given the role for NCX and calcium in synaptic plasticity, the capacity of NCX splice-forms to influence backpropagating action potentials has clear consequences for the induction of spike-timing dependent synaptic plasticity (STDP). To explore this, we examined the effect of NCX localization, density, and allosteric activation on forward and back propagating signals and, next employed a STDP paradigm to monitor the effect of NCX on plasticity using back propagating action potentials paired with EPSPs. From our simulation studies we identified a role for the sodium calcium exchange current in normalizing STDP, and demonstrate that NCX is required at the postsynaptic site for this response. We also screened other mechanisms in our model and identified a role for the Ca2+ activated K+ current at the postsynapse in producing STDP responses. Together, our data reveal opposing roles for the Na+/Ca2+ exchanger current and the Ca2+ activated K+ current in setting STDP.

Sodium Fate after Sodium Bicarbonate Infusion: Influence of Altered Acid-Base Status.

BACKGROUND: We have previously investigated the fate of administered bicarbonate infused as a hypertonic solution in animals with each of the 4 chronic acid-base disorders. Those studies did not address the fate of sodium, the coadministered cation. METHODS: We examined baseline total body water (TBW), Na+ space, HCO3- space, and urinary sodium and bicarbonate excretion after acute hypertonic NaHCO3 infusion (1-N solution, 5 mmol/kg body weight) in dogs with each of the 4 chronic acid-base disorders. Observations were made at 30, 60, and 90 min postinfusion. Retained sodium that remains osmotically active distributes in an apparent space that approximates TBW. Na+ space that exceeds TBW uncovers nonosmotic sodium storage. RESULTS: Na+ space approximated TBW at all times in normal and hyperbicarbonatemic animals (metabolic alkalosis and respiratory acidosis), but exceeded TBW by ~30% in hypobicarbonatemic animals (metabolic acidosis and respiratory alkalosis). Such osmotic inactivation was detected at 30 min and remained stable. The pooled data revealed that Na+ space corrected for TBW was independent of the initial blood pH but correlated with initial extracellular bicarbonate concentration (y = -0.01x + 1.4, p= 0.002). The fate of administered sodium and bicarbonate (internal distribution and urinary excretion) was closely linked. CONCLUSIONS: This study demonstrates that hypobicarbonatemic animals have a Na+ space that exceeds TBW after an acute infusion of hypertonic NaHCO3 indicating osmotic inactivation of a fraction of retained sodium. In addition to an expanded Na+ space, these animals have a larger HCO3- space compared with hyperbicarbonatemic animals. Both phenomena appear to reflect the wider range of titration of nonbicarbonate buffers (Δ pH) occurring during NaHCO3- loading whenever initial [HCO3-]e is low. The data indicate that the fate of administered bicarbonate drives the internal distribution and the external disposal of sodium, the co-administered cation, and is responsible for the early, but non-progressive, osmotic inactivation of a fraction of the retained sodium.

Na+-Coupled Nutrient Cotransport Induced Luminal Negative Potential and Claudin-15 Play an Important Role in Paracellular Na+ Recycling in Mouse Small Intestine.

Many nutrients are absorbed via Na+ cotransport systems, and therefore it is predicted that nutrient absorption mechanisms require a large amount of luminal Na+. It is thought that Na+ diffuses back into the lumen via paracellular pathways to support Na+ cotransport absorption. However, direct experimental evidence in support of this mechanism has not been shown. To elucidate this, we took advantage of claudin-15 deficient (cldn15-/-) mice, which have been shown to have decreased paracellular Na+ permeability. We measured glucose-induced currents (ΔIsc) under open- and short-circuit conditions and simultaneously measured changes in unidirectional 22Na+ fluxes (ΔJNa) in Ussing chambers. Under short-circuit conditions, application of glucose resulted in an increase in ΔIsc and unidirectional mucosal to serosal 22Na+ (∆JNaMS) flux in both wild-type and cldn15-/- mice. However, under open-circuit conditions, ΔIsc was observed but ∆JNaMS was strongly inhibited in wild-type but not in cldn15-/- mice. In addition, in the duodenum of mice treated with cholera toxin, paracellular Na+ conductance was decreased and glucose-induced ∆JNaMS increment was observed under open-circuit conditions. We concluded that the Na+ which is absorbed by Na+-dependent glucose cotransport is recycled back into the lumen via paracellular Na+ conductance through claudin-15, which is driven by Na+ cotransport induced luminal negativity.

Effect of potassium ions at the different concentration on the interaction between AmB and the lipid monolayer containing cholesterol or ergosterol.

AmB is an antifungal drug of polyene. Although it is prone to nephrotoxicity, it is still the gold standard in the clinical treatment of fungal infection. Sterol plays a decisive role in the drug activity of AmB. The antifungal activity of AmB depends on ergosterol in fungal membranes, and its toxicity is related to cholesterol in mammalian membranes. At the same time, AmB interacts with biofilms, leading to a significant loss of potassium ions and affecting the transport of potassium ions across membranes. Meanwhile, metal cation may also affect AmB molecules' aggregation on the membrane. This paper mainly studied the effects of different concentrations of potassium ions on the interactions between AmB and lipid monolayers containing cholesterol or ergosterol and explored the differences in the impact of varying potassium ions on the drug activity of AmB on monolayers rich in these two kinds of sterols. The results show that potassium ions caused the collapse of lipid monolayer and lipid-AmB monolayer to disappear. The limiting molecular area of these monolayers also increased due to potassium ions. The limiting molecular area of the monolayer in the presence of ergosterol has a great difference in the different concentration of potassium ions, which is different from that in the presence of cholesterol. The presence of potassium ions, regardless of the intensity of K+ ions, increased the maximum elastic modulus of the lipid/sterol monolayer with and without AmB. The presence of potassium ions reduced the influence of AmB on the stability of the lipid monolayer containing cholesterol. The impact of AmB on the stability of the lipid monolayer containing ergosterol was related to the concentration of potassium ions. The potassium ions increased the area of the ordered "island" region on the lipid-AmB monolayer containing cholesterol, and the boundary of the microregion produced different degrees of curvature. However, on the lipid/ergosterol monolayer, 5 mM and 10 mM potassium ions made the holes caused by AmB more denser, and the diameter of holes become larger. These results can help to improve the effect of potassium ions on the transmembrane transport of substances affected by AmB. The results will provide a basis for further exploration of the effect mechanism of metal ions on the antifungal activity of polyene drugs.

Identification of Na+-Pumping Cytochrome Oxidase in the Membranes of Extremely Alkaliphilic Thioalkalivibrio Bacteria.

For the first time, the functioning of the oxygen reductase Na+-pump (Na+-pumping cytochrome c oxidase of the cbb3-type) was demonstrated by examining the respiratory chain of the extremely alkaliphilic bacterium Thioalkalivibrio versutus [Muntyan, M. S., et al. (2015) Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase, Proc. Natl. Acad. Sci. USA, 112, 7695-7700], a product of the ccoNOQP operon. In this study, we detected and identified this enzyme using rabbit polyclonal antibody against the predicted C-terminal amino acid sequence of its catalytic subunit. We found that this cbb3-type oxidase is synthesized in bacterial cells, where it is located in the membranes. The 48-kDa oxidase subunit (CcoN) is catalytic, while subunits CcoO and CcoP with molecular masses of 29 and 34 kDa, respectively, are cytochromes c. The theoretical pI values of the CcoN, CcoO, and CcoP subunits were determined. It was shown that parts of the CcoO and CcoP subunits exposed to the aqueous phase on the cytoplasmic membrane P-side are enriched with negatively charged amino acid residues, in contrast to the parts of the integral subunit CcoN adjacent to the aqueous phase. Thus, the Na+-pumping cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions.