Mutation in pore-helix modulates interplay between filter gate and Ba2+ block in a Kcv channel pore.

The selectivity filter of K+ channels catalyzes a rapid and highly selective transport of K+ while serving as a gate. To understand the control of this filter gate, we use the pore-only K+ channel KcvNTS in which gating is exclusively determined by the activity of the filter gate. It has been previously shown that a mutation at the C-terminus of the pore-helix (S42T) increases K+ permeability and introduces distinct voltage-dependent and K+-sensitive channel closures at depolarizing voltages. Here, we report that the latter are not generated by intrinsic conformational changes of the filter gate but by a voltage-dependent block caused by nanomolar trace contaminations of Ba2+ in the KCl solution. Channel closures can be alleviated by extreme positive voltages and they can be completely abolished by the high-affinity Ba2+ chelator 18C6TA. By contrast, the same channel closures can be augmented by adding Ba2+ at submicromolar concentrations to the cytosolic buffer. These data suggest that a conservative exchange of Ser for Thr in a crucial position of the filter gate increases the affinity of the filter for Ba2+ by >200-fold at positive voltages. While Ba2+ ions apparently remain only for a short time in the filter-binding sites of the WT channel before passing the pore, they remain much longer in the mutant channel. Our findings suggest that the dwell times of permeating and blocking ions in the filter-binding sites are tightly controlled by interactions between the pore-helix and the selectivity filter.

Effect of Ba2+ on the biomineralization of Ca2+ and Mg2+ ions induced by Bacillus licheniformis.

The effect of barium ions on the biomineralization of calcium and magnesium ions is often overlooked when utilizing microbial-induced carbonate precipitation technology for removing barium, calcium, and magnesium ions from oilfield wastewater. In this study, Bacillus licheniformis was used to bio-precipitate calcium, magnesium, and barium ions. The effects of barium ions on the physiological and biochemical characteristics of bacteria, as well as the components of extracellular polymers and mineral characteristics, were also studied in systems containing coexisting barium, calcium, and magnesium ions. The results show that the increasing concentrations of barium ions decreased pH, carbonic anhydrase activity, and concentrations of bicarbonate and carbonate ions, while it increased the contents of humic acids, proteins, polysaccharides, and DNA in extracellular polymers in the systems containing all three types of ions. With increasing concentrations of barium ions, the content of magnesium within magnesium-rich calcite and the size of minerals precipitated decreased, while the full width at half maximum of magnesium-rich calcite, the content of O-C=O and N-C=O, and the diversity of protein secondary structures in the minerals increased in systems containing all three coexisting ions. Barium ions does inhibit the precipitation of calcium and magnesium ions, but the immobilized bacteria can mitigate the inhibitory effect. The precipitation ratios of calcium, magnesium, and barium ions reached 81-94%, 68-82%, and 90-97%. This research provides insights into the formation of barium-enriched carbonate minerals and offers improvements for treating oilfield wastewater.

Urinary metals and maternal circulating extracellular vesicle microRNA in the MADRES pregnancy cohort.

Exposure to metals increases risk for pregnancy complications. Extracellular vesicle (EV) miRNA contribute to maternal-foetal communication and are dysregulated in pregnancy complications. However, metal impacts on maternal circulating EV miRNA during pregnancy are unknown. Our objective was to investigate the impact of multiple metal exposures on EV miRNA in maternal circulation during pregnancy in the MADRES Study. Associations between urinary concentrations of nine metals and 106 EV miRNA in maternal plasma during pregnancy were investigated using robust linear regression (N = 231). Primary analyses focused on metal-miRNA associations in early pregnancy (median: 12.3 weeks gestation). In secondary analyses, we investigated associations with late pregnancy miRNA counts (median: 31.8 weeks gestation) in a subset of participants (N = 184) with paired measures. MiRNA associated with three or more metals (PFDR<0.05) were further investigated using Bayesian Kernel Machine Regression (BKMR), an environmental mixture method. Thirty-five miRNA were associated (PFDR<0.05) with at least one metal in early pregnancy. One association (an inverse association between cobalt and miR-150-5p) remained statistically significant when evaluating late pregnancy miRNA counts. Eight miRNA (miR-302b-3p, miR-199a-5p, miR-188-5p, miR-138-5p, miR-212-3p, miR-608, miR-1272, miR-19b-3p) were associated with three metals (barium, mercury, and thallium) in early pregnancy, and their predicted target genes were enriched in pathways important for placental development. Results were consistent when using BKMR. Early pregnancy exposure to barium, mercury, and thallium may have short-term impacts on a common set of EV miRNA which target pathways important for placental development.

Oesophageal IGRT considerations for SBRT of LA-NSCLC: barium-enhanced CBCT and interfraction motion.

BACKGROUND: To determine the optimal volume of barium for oesophageal localisation on cone-beam CT (CBCT) for locally-advanced non-small cell lung cancers (NSCLC) and quantify the interfraction oesophageal movement relative to tumour. METHODS: Twenty NSCLC patients with mediastinal and/or hilar disease receiving radical radiotherapy were recruited. The first five patients received 25 ml of barium prior to their planning CT and alternate CBCTs during treatment. Subsequent five patient cohorts, received 15 ml, 10 ml and 5 ml. Six observers contoured the oesophagus on each of the 107 datasets and consensus contours were created. Overall 642 observer contours were generated and interobserver contouring reproducibility was assessed. The kappa statistic, dice coefficient and Hausdorff Distance (HD) were used to compare barium-enhanced CBCTs and non-enhanced CBCTs. Oesophageal displacement was assessed using the HD between consensus contours of barium-enhanced CBCTs and planning CTs. RESULTS: Interobserver contouring reproducibility was significantly improved in barium-enhanced CBCTs compared to non-contrast CBCTs with minimal difference between barium dose levels. Only 10 mL produced a significantly higher kappa (0.814, p = 0.008) and dice (0.895, p = 0.001). The poorer the reproducibility without barium, the greater the improvement barium provided. The median interfraction HD between consensus contours was 4 mm, with 95% of the oesophageal displacement within 15 mm. CONCLUSIONS: 10 mL of barium significantly improves oesophageal localisation on CBCT with minimal image artifact. The oesophagus moves substantially and unpredictably over a course of treatment, requiring close daily monitoring in the context of hypofractionation.

Splice-variant specific effects of a CACNA1H mutation associated with writer's cramp.

The CACNA1H gene encodes the α1 subunit of the low voltage-activated Cav3.2 T-type calcium channel, an important regulator of neuronal excitability. Alternative mRNA splicing can generate multiple channel variants with distinct biophysical properties and expression patterns. Two major splice variants, containing or lacking exon 26 (± 26) have been found in different human tissues. In this study, we report splice variant specific effects of a Cav3.2 mutation found in patients with autosomal dominant writer's cramp, a specific type of focal dystonia. We had previously reported that the R481C missense mutation caused a gain of function effect when expressed in Cav3.2 (+ 26) by accelerating its recovery from inactivation. Here, we show that when the mutation is expressed in the short variant of the channel (- 26), we observe a significant increase in current density when compared to wild-type Cav3.2 (- 26) but the effect on the recovery from inactivation is lost. Our data add to growing evidence that the functional expression of calcium channel mutations depends on which splice variant is being examined.

In situ barium phytoremediation in flooded soil using Typha domingensis under different planting densities.

The management of initial planting density can be a strategy to increase barium phytoextraction from soil, reducing the time required for soil decontamination. To delimit the ideal planting density for barium (Ba) phytoremediation using Typha domingensis, we conducted a 300-day experiment in an area accidentally contaminated with barite. Four initial planting densities were tested: 4, 8, 12, and 16 plantsm-2 (D4, D8, D12, and D16 treatments, respectively). Plant development was evaluated periodically, and the phytoextraction efficiency was determined at the end of the trial. The initial planting density affected Ba phytoremediation by T. domingensis monoculture. Phytoextraction potential was better represented by the mass-based translocation factor (mTF) than the concentration-based translocation factor. D16 promoted the highest final number of plants and biomass production, but the mass of Ba in the aerial part did not differ among D8, D12, and D16. D4 resulted in more Ba accumulated belowground than aboveground (6.3 times higher), whereas D12 and D16 achieved the greatest mTFs. Higher absorption of Ba from soil can be achieved using less T. domingensis individuals at the beginning of the treatment (D4 and D8) but with high accumulation in belowground tissues. We conclude that the D8 density is considered the most appropriate if considering the phytoextraction potential and field management facilitated using fewer plants.

Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions.

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca2+, Cd2+, Zn2+, and Ba2+ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca2+ and Ba2+ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5-2 kV/cm, much lower than >7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba2+ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing.

The Importance of Extensional Rheology in Bolus Control during Swallowing.

Thickened fluids are commonly used in the medical management of individuals who suffer swallowing difficulty (known as dysphagia). Previous studies have shown that the rheological properties of a liquid affect the flow behavior of the bolus in swallowing, such as pharyngeal transit time. While there is no doubt that shear rheology is a highly important factor for bolus flow, it is suspected that extensional properties of a liquid bolus also plays an important role in swallowing, due to elongation of the bolus as it flows through the oropharynx. Our aim in this work was to observe the effect of extensional viscosity on pharyngeal transit time and elongation of the bolus during swallowing. Eight samples of thickened liquid barium that were shear-controlled, but varied in extensional viscosity and two samples that were extensional-controlled, but varied in shear viscosity were swallowed by eight healthy individuals. Data were collected under lateral view of videofluoroscopy swallow study (VFSS); measures of pharyngeal transit time and the ratio of the length to the width of the bolus on the frame of Upper Esophageal Sphincter (UES) opening were taken from the VFSS recordings. It was observed that the pharyngeal transit time generally increases when the fluids are thickened to higher IDDSI consistency. Additionally, higher extensional viscosity fluids reduced the elongation of the bolus during swallowing, thus potentially reducing the risk of post-swallow residue due to bolus breakage. This study confirmed the relevance of the extensional viscosity of the bolus in swallowing.

Simple Fluorescence Turn-On Chemosensor for Selective Detection of Ba2+ Ion and Its Live Cell Imaging.

A phenoxazine-based fluorescence chemosensor 4PB [(4-(tert-butyl)-N-(4-((4-((5-oxo-5H-benzo[a]phenoxazin-6-yl)amino)phenyl)sulfonyl)phenyl)benzamide)] was designed and synthesized by a simple synthetic methods. The 4PB fluorescence chemosensor selectively detects Ba2+ in the existence of other alkaline metal ions. In addition, 4PB showed high selectivity and sensitivity for Ba2+ detection. The detection limit of 4PB was 0.282 µM and the binding constant was 1.0 × 106 M-1 in CH3CN/H2O (97.5:2.5 v/v, HEPES = 1.25 mM, pH 7.3) medium. This chemosensor functioned through the intramolecular charge transfer (ICT) mechanism, which was further confirmed by DFT studies. Live cell imaging in MCF-7 cells confirmed the cell permeability of 4PB and its capability for specific detection of Ba2+ in living cells.

Interactive effects of strontium and barium water concentration on otolith incorporation in juvenile flounder Paralichthys olivaceus.

Although the relationship between the incorporation of an element into otoliths and the concentration of the element in water has been extensively investigated in many fish species, the interactive effects of multiple elements in water on the otolith incorporation of an element are not adequately explored or well understood. In this study, 16 treatments in triplicate using strontium (Sr; 1, 2, 3 and 4 times the ambient baseline, 6.5 mg l-1) and barium (Ba; 1, 2, 4 and 6 times the ambient baseline, 40 µg l-1) as categorical variables in an orthogonal design were established to evaluate the relative or interactive effects of water elements on otolith elemental incorporation in juvenile flounder Paralichthys olivaceus (from 15 to 116 days post hatching). The results revealed that otolith incorporation (Me:CaOtolith) of Sr and Ba were positively dependent on the concentrations of the elements in water (Me:CaWater). Overall, Sr was incorporated into otoliths more efficiently than was Ba, and the partition efficiency (DMe) of both elements decreased with increasing water elemental concentrations. Increasing Sr concentrations in water appeared to negatively affect the uptake of Ba into otoliths rather than facilitate it, as previously reported in fish reared in freshwater and brackish water, or showed no effect on fish in seawater. Conversely, the Ba concentration in water did not influence the otolith uptake of Sr, which agrees with the findings for other fish species. When applying otolith microchemistry to fish ecology studies, it is essential to cautiously address the interactive effects of multiple elements in the environment on otolith elemental incorporation.

Biodistribution of strontium and barium in the developing and mature skeleton of rats.

Bone acts as a reservoir for many trace elements. Understanding the extent and pattern of elemental accumulation in the skeleton is important from diagnostic, therapeutic, and toxicological perspectives. Some elements are simply adsorbed to bone surfaces by electric force and are buried under bone mineral, while others can replace calcium atoms in the hydroxyapatite structure. In this article, we investigated the extent and pattern of skeletal uptake of barium and strontium in two different age groups, growing, and skeletally mature, in healthy rats. Animals were dosed orally for 4 weeks with either strontium chloride or barium chloride or combined. The distribution of trace elements was imaged in 3D using synchrotron K-edge subtraction micro-CT at 13.5 µm resolution and 2D electron probe microanalysis (EPMA). Bulk concentration of the elements in serum and bone (tibiae) was also measured by mass spectrometry to study the extent of uptake. Toxicological evaluation did not show any cardiotoxicity or nephrotoxicity. Both elements were primarily deposited in the areas of active bone turnover such as growth plates and trabecular bone. Barium and strontium concentration in the bones of juvenile rats was 2.3 times higher, while serum levels were 1.4 and 1.5 times lower than adults. In all treatment and age groups, strontium was preferred to barium even though equal molar concentrations were dosed. This study displayed spatial co-localization of barium and strontium in bone for the first time. Barium and strontium can be used as surrogates for calcium to study the pathological changes in animal models of bone disease and to study the effects of pharmaceutical compounds on bone micro-architecture and bone remodeling in high spatial sensitivity and precision.

Association between concentrations of barium and aluminum in placental tissues and risk for orofacial clefts.

Natural exposure to and increasing use of barium and aluminum in various products, such as plastics, rubber, and food additives, raise concerns for their potential health impacts on pregnant women and vulnerable fetuses. We investigated whether there are associations between barium and aluminum concentrations in placental tissues and the risk for orofacial clefts (OFCs) in offspring. In this case-control study, we recruited 103 women with OFC-affected pregnancies and 206 women who delivered healthy newborns. Concentrations of barium and aluminum in placental tissues were measured using inductively coupled plasma-mass spectrometry. Information on maternal sociodemographic characteristics and diet was collected via face-to-face interviews using a structured questionnaire. Aluminum concentrations in placental tissues were not associated with OFC risk. However, a higher concentration of barium in placental tissues was associated with an increased risk for OFCs, with an adjusted odds ratio (OR) of 2.42 (95% confidence interval [95% CI] 1.34-4.40) for total cleft lip with or without cleft palate (CL ±â€¯P), and 1.90 (95% CI 1.03-3.50) for isolated CL ±â€¯P. There was a positive dose-response relationship between placental barium concentrations and OFC risk. Maternal exposure to barium may increase the risk for OFCs in offspring.

REPORT- Role of metal ions on the catalytic efficiency of dextran hydrolyzing biocatalyst.

Hydrothermal spring isolate Bacillus megaterium KIBGE-IB31was utilized to produce dextranase. Enzyme was partially purified up to 11.8 fold after dialysis. Different metals ions were tested to explore their behavior with dextranase. It was noticed that cobalt (Co+2), copper (Cu+2), magnesium (Mg+2), manganese (Mn+2), nickle (Ni+2) and zinc (Zn+2) act as activator whilst potassium (K+), sodium (Na+), barium (Ba+), calcium (Ca+), mercury (Hg+), vanadium (V+2), aluminum (Al+3) and ferric (Fe+3) ions display inhibitory action.

Elements of time and place: manganese and barium in shark vertebrae reflect age and upwelling histories.

As upper-level predators, sharks are important for maintaining marine food web structure, but populations are threatened by fishery exploitation. Sustainable management of shark populations requires improved understanding of migration patterns and population demographics, which has traditionally been sought through physical and/or electronic tagging studies. The application of natural tags such as elemental variations in mineralized band pairs of elasmobranch vertebrae cartilage could also reveal endogenous and exogenous processes experienced by sharks throughout their life histories. Here, elemental profiles were characterized in vertebrae encompassing complete life histories (birth-to-death) of shortfin mako (Isurus oxyrinchus), common thresher (Alopias vulpinus) and blue shark (Prionace glauca) of known tag and recapture locations in the eastern North Pacific Ocean. All sharks were injected with oxytetracycline at initial capture, released and subsequently recaptured, with individual liberty times ranging from 215 days to 6 years. Vertebral band pairs forming over the liberty intervals were verified by counting the number of band pairs deposited since the oxytetracycline band. Regular oscillations in vertebrae manganese (Mn) content corresponded well with the number of validated band pairs, suggesting that Mn variation could be used to age sharks. Increases in vertebrae barium concentration were correlated with times when individuals occupied areas with high coastal upwelling indices, the timing and spatial intensity of which varied from year to year. Interspecific relationships were probably influenced by behavioural differences in horizontal and vertical habitat use, feeding habits and thermoregulatory physiology. These results indicate that vertebral sclerochronology has the potential to advance our knowledge of elasmobranch life history including age and growth estimation and environmental reconstruction.

An African loss-of-function CACNA1C variant p.T1787M associated with a risk of ventricular fibrillation.

Calcium regulation plays a central role in cardiac function. Several variants in the calcium channel Cav1.2 have been implicated in arrhythmic syndromes. We screened patients with Brugada syndrome, short QT syndrome, early repolarisation syndrome, and idiopathic ventricular fibrillation to determine the frequency and pathogenicity of Cav1.2 variants. Cav1.2 related genes, CACNA1C, CACNB2 and CACNA2D1, were screened in 65 probands. Missense variants were introduced in the Cav1.2 alpha subunit plasmid by mutagenesis to assess their pathogenicity using patch clamp approaches. Six missense variants were identified in CACNA1C in five individuals. Five of them, A1648T, A1689T, G1795R, R1973Q, C1992F, showed no major alterations of the channel function. The sixth C-terminal variant, Cavα1c-T1787M, present mostly in the African population, was identified in two patients with resuscitated cardiac arrest. The first patient originated from Cameroon and the second was an inhabitant of La Reunion Island with idiopathic ventricular fibrillation originating from Purkinje tissues. Patch-clamp analysis revealed that Cavα1c-T1787M reduces the calcium and barium currents by increasing the auto-inhibition mediated by the C-terminal part and increases the voltage-dependent inhibition. We identified a loss-of-function variant, Cavα1c-T1787M, present in 0.8% of the African population, as a new risk factor for ventricular arrhythmia.

Growth hormone secretagogue receptor constitutive activity impairs voltage-gated calcium channel-dependent inhibitory neurotransmission in hippocampal neurons.

KEY POINTS: Presynaptic CaV 2 voltage-gated calcium channels link action potentials arriving at the presynaptic terminal to neurotransmitter release. Hence, their regulation is essential to fine tune brain circuitry. CaV 2 channels are highly sensitive to G protein-coupled receptor (GPCR) modulation. Our previous data indicated that growth hormone secretagogue receptor (GHSR) constitutive activity impairs CaV 2 channels by decreasing their surface density. We present compelling support for the impact of CaV 2.2 channel inhibition by agonist-independent GHSR activity exclusively on GABA release in hippocampal cultures. We found that this selectivity arises from a high reliance of GABA release on CaV 2.2 rather than on CaV 2.1 channels. Our data provide new information on the effects of the ghrelin-GHSR system on synaptic transmission, suggesting a putative physiological role of the constitutive signalling of a GPCR that is expressed at high levels in brain areas with restricted access to its natural agonist. ABSTRACT: Growth hormone secretagogue receptor (GHSR) displays high constitutive activity, independent of its endogenous ligand, ghrelin. Unlike ghrelin-induced GHSR activity, the physiological role of GHSR constitutive activity and the mechanisms that underlie GHSR neuronal modulation remain elusive. We previously demonstrated that GHSR constitutive activity modulates presynaptic CaV 2 voltage-gated calcium channels. Here we postulate that GHSR constitutive activity-mediated modulation of CaV 2 channels could be relevant in the hippocampus since this brain area has high GHSR expression but restricted access to ghrelin. We performed whole-cell patch-clamp in hippocampal primary cultures from E16- to E18-day-old C57BL6 wild-type and GHSR-deficient mice after manipulating GHSR expression with lentiviral transduction. We found that GHSR constitutive activity impairs CaV 2.1 and CaV 2.2 native calcium currents and that CaV 2.2 basal impairment leads to a decrease in GABA but not glutamate release. We postulated that this selective effect is related to a higher CaV 2.2 over CaV 2.1 contribution to GABA release (∼40% for CaV 2.2 in wild-type vs. ∼20% in wild-type GHSR-overexpressing cultures). This effect of GHSR constitutive activity is conserved in hippocampal brain slices, where GHSR constitutive activity reduces local GABAergic transmission of the granule cell layer (intra-granule cell inhibitory postsynaptic current (IPSC) size ∼-67 pA in wild-type vs. ∼-100 pA in GHSR-deficient mice), whereas the glutamatergic output from the dentate gyrus to CA3 remains unchanged. In summary, we found that GHSR constitutive activity impairs IPSCs both in hippocampal primary cultures and in brain slices through a CaV 2-dependent mechanism without affecting glutamatergic transmission.

Biomineralization of strontium and barium contributes to detoxification in the freshwater alga Micrasterias.

The unicellular model alga Micrasterias denticulata inhabits acid peat bogs that are highly endangered by pollutants due to their high humidity. As it was known from earlier studies that algae like Micrasterias are capable of storing barium naturally in form of BaSO4 crystals, it was interesting to experimentally investigate distribution and sequestration of barium and the chemically similar alkaline earth metal strontium. Additionally, we intended to analyze whether biomineralization by crystal formation contributes to diminution of the generally toxic effects of these minerals to physiology and structure of this alga which is closely related to higher plants. The results show that depending on the treatment differently shaped crystals are formed in BaCl2 and Cl2Sr exposed Micrasterias cells. Modern microscopic techniques such as analytical TEM by electron energy loss spectroscopy and Raman microscopy provide evidence for the chemical composition of these crystals. It is shown that barium treatment results in the formation of insoluble BaSO4 crystals that develop within distinct compartments. During strontium exposure long rod-like crystals are formed and are surrounded by membranes. Based on the Raman signature of these crystals their composition is attributed to strontium citrate. These crystals are instable and are dissolved during cell death. During strontium as well as barium treatment cell division rates and photosynthetic oxygen production decreased in dependence of the concentration, whereas cell vitality was reduced only slightly. Together with the fact that TEM analyses revealed only minor ultrastructural alterations as consequence of relatively high concentrated BaCl2 and Cl2Sr exposure, this indicates that biomineralization of Sr and Ba protects the cells from severe damage or cell death at least within a particular concentration range and time period. In the case of Sr treatment where ROS levels were found to be elevated, hallmarks for autophagy of single organelles were observed by TEM, indicating beginning degradation processes.

A disadvantageous effect of adsorption of barium by melanin on transforming activity.

At present, beneficial effects of melanin and harmful effects of barium have been reported. However, little is known about the adsorption of barium, and even less is known about the biological significance of adsorption of barium by melanin. In this study, we showed that there was a strong correlation between the digitalized level of skin pigmentation and barium level in murine skin compared to the correlations between skin pigmentation level and levels of homologous elements of barium (magnesium, calcium and strontium). The concentration of subcutaneously injected barium in skin with a high level of pigmentation was higher than that in skin with a low level of pigmentation. Our cell-free experiment using the Langmuir isotherm for adsorption of barium in synthetic melanin also provided direct evidence of adsorption of barium by melanin. We then investigated the biological significance of melanin-mediated barium adsorption. We found barium-mediated increase in transforming activity in pigmented melanocytes (melan-a) but not in unpigmented melanocytes (melan-c) after confirming that the barium level in melan-a melanocytes was 3.4-fold higher than that in melan-c melanocytes after culture of 5 µM barium for 24 h. Taken together, our results not only indicate adsorption of barium by melanin in mice, cells and cell-free systems but also suggest a disadvantageous effect of adsorption of barium by melanin on transforming activity in cultured cells.

Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes.

The mitochondrial calcium uniporter is a multisubunit Ca2+ channel that mediates mitochondrial Ca2+ uptake, a cellular process crucial for the regulation of oxidative phosphorylation, intracellular Ca2+ signaling, and apoptosis. In the last few years, genes encoding uniporter proteins have been identified, but a lack of efficient tools for electrophysiological recordings has hindered quantitative analysis required to determine functional mechanisms of this channel complex. Here, we redirected Ca2+-conducting subunits (MCU and EMRE) of the human uniporter to the plasma membrane of Xenopus oocytes. Two-electrode voltage clamp reveals inwardly rectifying Ca2+ currents blocked by a potent inhibitor, Ru360 (half maximal inhibitory concentration, ~4 nM), with a divalent cation conductivity of Ca2+ > Sr2+ > Ba2+, Mn2+, and Mg2+ Patch clamp recordings further reveal macroscopic and single-channel Ca2+ currents sensitive to Ru360. These electrical phenomena were abolished by mutations that perturb MCU-EMRE interactions or disrupt a Ca2+-binding site in the pore. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter using classical strategies in ion channel electrophysiology.

TRESK background potassium channel is not gated at the helix bundle crossing near the cytoplasmic end of the pore.

Two-pore domain K+ channels (K2P) are responsible for background K+ currents and regulate the resting membrane potential and cellular excitability. Their activity is controlled by a large variety of physicochemical factors and intracellular signaling pathways. The majority of these effects converge on the intracellular C-terminus of the channels, resulting in the modification of the gating at the selectivity filter. Another gating mechanism, the activation gate at the helix bundle crossing is also well documented in other K+ channel families, however, it remains uncertain whether this type of gating is functional in K2P channels. The regulation of TWIK-related spinal cord K+ channel (TRESK) is different from the other K2P channels. Regulatory factors acting via the C-terminus are not known, instead channel activity is modified by the phosphorylation/dephosphorylation of the unusually long intracellular loop between the 2nd and 3rd transmembrane segments. These unique structural elements of the regulation lead us to examine channel gating at the bundle crossing region. Ba2+ was applied to the intracellular side of excised membrane patches and the characteristics of the channel block were determined. We compared the kinetics of the development of Ba2+ block when the channels were phosphorylated (inhibited) or dephosphorylated (activated) and also in different mutants mimicking the two functional states. Neither the phosphorylation/dephosphorylation nor the point mutations influenced the development of Ba2+ block, suggesting that the conformational changes of the bundle crossing region do not contribute to the phosphorylation-dependent gating of TRESK.