Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.     

Effect of Zinc (II) on the interactions of bovine serum albumin with flavonols bearing different number of hydroxyl substituent on B-ring

The impact of Zn²⁺ ion on interactions of flavonols galangin (Gal), kaempferol (Kae), quercetin (Que) and myricetin (Myr) with bovine serum albumin (BSA) in aqueous solution were studied by fluorescence quenching technique. The results exhibited that Zn²⁺ ion affected significantly the interactions and the effect was distinct for the flavonol bearing different number of B-ring hydroxyl. Each flavonol can quench the fluorescence of BSA, displaying a quenching extent of Myr > Que > Kae > Gal, which is in good agreement with the number variation of the B-ring hydroxyl. The presence of Zn²⁺ ion promoted the quenching for the flavonols, exhibiting an extent of Que > Myr > Kae > Gal. The values of K_a for Kae, Que and Myr decreased whereas K_SV and k_q for Gal, Kae and Que increased with the number of B-ring hydroxyl. The type of BSA fluorescence quenching for Gal, Kae and Que hardly changed but the preference of static quenching increased. The values of K_SV and k_q for Myr remarkably decreased and the fluorescence quenching of BSA alternatively occurred via both static and dynamic type instead of only one (static or dynamic). The results suggest the key role of the B-ring hydroxyl and the distinct effect of its number in the interactions. Each flavonol may capture the BSA-bound Zn^II in the solution, forming Zn^II-flavonol complex that is possibly responsible for BSA fluorescence quenching. The B-ring hydroxyl could establish hydrogen bonds with BSA in the absence of Zn²⁺ and act as donors for chelating in the presence of Zn²⁺. The formation of dinuclear Zn^II-Myr complex together with the hydrogen bonds between the free B-ring hydroxyl and BSA may contribute to the exceptional behavior of Myr.     

A micro-scale method for determining relative metal-binding affinities of proteins

This article describes a quick and easy method for determining relative binding affinities between proteins and metal ions. The method is based on separating unbound metal ions from metal ions bound to protein by ultrafiltration using microcentrifuge ultrafiltration units. Bovine serum albumin (BSA) was used as the test protein and the relative affinity towards divalent metal ions was found to be Cu²⁺>Zn²⁺>Cd²⁺>Pb²⁺>Ni²⁺>Co²⁺, which corresponds to the relative orders reported in the literature.     

Inhibition of proton-transfer steps in transhydrogenase by transition metal ions

Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a “cyclic reaction” was stimulated. Of metal ions tested they were effective in the order Pb²⁺ > Cu²⁺ > Zn²⁺ = Cd²⁺ > Ni²⁺ > Co²⁺. The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn²⁺-binding site(s). A mutant in which βHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn²⁺. Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn²⁺-induced FTIR difference spectra of the βHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that βHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.     

Metal complexes of modified cyclen as catalysts for hydrolytic restriction of plasmid DNA

Simple and novel nuclease models have been synthesized. These involve metal-binding ligand 1,4,7,10-tetraazlcyclododecane (cyclen) tethered to an acridine ring (a DNA-binding group) by amide linkers of various lengths. Binding of these probes to DNA was studied by monitoring changes in their UV-visible spectra affected by the presence of DNA. Titration of these compounds with increasing amounts of pBR322 DNA caused hypochromic effects and shifted the acridine absorption at 360 nm to a longer wavelength. Under biologically relevant conditions (37 °C and pH 7.4), specific transition metal complexes of these compounds are found to be highly effective catalysts toward the hydrolysis of plasmid DNA. This is demonstrated by their ability to convert the super-coiled DNA (form I) to open-circular DNA (form II). Structure-activity correlation studies show that hydrolytic activity depends on both the structure of ligand (L₁ > L₂ > L₃) and the nature of metal ion cofactor (Co³⁺ > Zn²⁺ > Cr²⁺ > Ni²⁺ > Cu²⁺ > Fe³⁺).     

Mechanism of inhibition of arginine kinase by flavonoids consistent with thermodynamics of docking simulation

Arginine kinase plays a vital role in invertebrate homeostasis by buffering ATP concentrations. Arginine kinase might serve as a target for environmentally friendly insect-selective pesticides, because it differs notably from its counterpart in vertebrates, creatine kinase. In this study, two members of the flavonoid family, quercetin (QU) and luteolin (LU), were identified as novel noncompetitive inhibitors of locust arginine kinase. They were found to have inhibition parameters (K_i) of 11.2 and 23.9 μM, respectively. By comparing changes in the activity and intrinsic fluorescence of AK, the inhibition mechanisms of these flavonoids were found to involve binding to Trp residues in the active site. This was determined by examination of the static quenching parameter K_sv. The main binding forces between flavonoids and AK were found to be hydrophobic based on the thermodynamic parameters of changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG) and on docking simulation results. Molecular docking analyses also suggested that flavonoids could bind to the active site of AK and were close to the Trp 221 in active site. Molecular simulation results mimic the experimental results, indicated that QU had a lower binding energy and a stronger inhibitory effect on AK than LU, suggesting that the extra hydroxyl group in QU might increase binding ability.     

Phagocytic activity,respiratory burst,cytoplasmic free-Ca concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress

The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24 h, 48 h, 72 h and 96 h LC₅₀ (median lethal concentration) of Cu²⁺ on P. monodon (11.63 ± 1.14 g) were found to be 3.49, 1.54, 0.73 and 0.40 mg L^− 1, respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca²⁺ (cf-Ca²⁺) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu²⁺ (0, 0.05, 0.5, 1.5 and 3.5 mg L^− 1) for 0, 6, 12, 24 and 48 h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05 mg L^− 1 Cu²⁺. THC decreased after Cu-exposure to 0.5 mg L^− 1 for 48 h, 1.5 mg L^− 1 for 24 h and 3.5 mg L^− 1 for 12 h. Phagocytic activity decreased in P. monodon following 48 h exposure to 3.5 mg L^− 1 Cu²⁺. RB was induced after 6 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. cf-Ca²⁺ concentration increased after 48 h exposure to 0.5 mg L^− 1 Cu²⁺, and 12 h exposure to 1.5 and 3.5 mg L^− 1 Cu²⁺. The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. These results indicate that Cu can induce oxidative stress, elevation of cf-Ca²⁺ and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu²⁺ to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis.     

Copper(II)-induced secondary structure changes and reduced folding stability of the prion protein

The cellular isoform of the prion protein PrP^C is a Cu²⁺-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP^C conformation are intimately linked with disease pathogenesis, the effect of Cu²⁺ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu²⁺ ions destabilise the native fold of PrP^C. The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu²⁺. This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol^− 1 at the midpoint of unfolding). We relate Cu²⁺-induced changes in secondary structure for full-length PrP(23-231) to smaller Cu²⁺ binding fragments. In particular, Cu²⁺-induced structural changes can directly be attributed to Cu²⁺ binding to the octarepeat region of PrP^C. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90-126) is due only to local Cu²⁺ coordination to the individual binding sites centred at His95 and His110. Cu²⁺ binding does not directly generate a β-sheet conformation within PrP^C; however, Cu²⁺ ions do destabilise the native fold of PrP^C and may make the transition to a misfolded state more favourable.     

A rhodamine-based "turn-on" fluorescent chemodosimeter for Cu2+ and its application in living cell imaging

A new fluorescent probe 1, N-(Rhodamine-6G)lactam-hydrazinecarbothioamide, was synthesized as a fluorescent and colorimetric chemodosimeter in aqueous solution for Cu²⁺. Following Cu²⁺-promoted ring opening, redox and hydrolysis reactions, comparable amplifications of absorption and fluorescence signals were observed upon addition of Cu²⁺; this suggests that chemodosimeter 1 effectively avoided the fluorescence quenching caused by the paramagnetic nature of Cu²⁺. Importantly, 1 can selectively recognize Cu²⁺ in aqueous media in the presence of other trace metal ions in organisms, abundant cellular cations and the prevalent toxic metal ions in the environment with high sensitivity (detection limit < 3 ppb) and a rapid response time (< 2 min). In addition, the biological imaging study has demonstrated that 1 can detect Cu²⁺ in the living cells.     

DNA photocleavage activity of cobalt(III) polypyridyl complexes containing dpq ligand

Four cobalt(III) polypyridyl complexes, [Co(phen)_3−n(dpq)_n]³⁺ (phen = 1,10-phenanthroline, dpq = dipyrido[3,2-f:2′,3′-h]-quinoxaline) (n = 0, 1, 2, and 3) were synthesized and the influences of the dpq ligand on the photophysical properties, electrochemical properties, DNA binding affinities, as well as photonuclease activities of the complexes, were examined in detail. The presence of dpq ligand increases the DNA binding affinities of the corresponding complexes remarkably with respect to [Co(phen)₃]³⁺. With the sequential substitution of phen ligand by dpq ligand, the ¹O₂ quantum yields of the corresponding complexes are enhanced greatly. As a result, the photonuclease activities follow the order of [Co(dpq)₃]³⁺ > [Co(phen)(dpq)₂]³⁺ > [Co(phen)₂(dpq)]³⁺ ? [Co(phen)₃]³⁺. It was found all the examined complexes can generate OH upon UV irradiation, and OH is also involved in DNA photocleavage as reactive oxygen species.     

Interactions of KChIP4a and its mutants with Ca or Kv4.3 N-terminus by affinity capillary electrophoresis

The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca²⁺ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (K_b). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 10⁶ L mol^–1 and (5.26 ± 0.71) × 10⁶ L mol^–1, respectively. In addition, in the presence of calcium (10 μmol L^–1), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 10⁷ L mol^–1. In addition, the binding constant of KChIP4a with Ca²⁺ was (7.1 ± 1.5) × 10⁷ L mol^–1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca²⁺, and the integrity of the molecular structure of KChIP4a was important for Ca²⁺ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

Fasciola gigantica: purification and characterization of adenosine deaminase

Nucleotidase cascades (apyrase, 5′ nucleotidase, and adenosine deaminase (ADA) were investigated in the parasitic trematode Fasciola gigantica. ADA had the highest activity in the nucleotidase cascades. Adenosine deaminase was purified from F. gigantica through acetone precipitation and chromatography on CM-cellulose. Two forms of enzyme (ADAI, ADAII) were separated. ADAII was purified to homogeneity after chromatography on Sephacryl S-200. The molecular mass was 29 KDa for the native and denatured enzyme using gel filtration and SDS-PAGE, respectively. The enzyme (ADAII) had a pH optimum at 7.5 and a K_m 1.0 mM adenosine, a temperature optimum at 40 °C and heat stability up to 40 °C. The order of effectiveness of metals as inhibitors was found to be Hg²⁺ > Mn²⁺ > Cu²⁺ > Ca²⁺ > Zn²⁺ > Ni²⁺ > Ba²⁺.     

Modeling the Zn and Cd metalation mechanism in mammalian metallothionein 1a

Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn²⁺ or Cd²⁺ ions into two distinct domains: an N-terminal β-domain that binds 3 Zn²⁺ or Cd²⁺, and a C-terminal α-domain that binds 4 Zn²⁺ or Cd²⁺. Although stepwise metalation to the saturated M₇-MT (where M = Zn²⁺ or Cd²⁺) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (M_n-MT, n = 0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K₁ < K₂ < K₃ < ··· < K₇), weakly cooperative (where K₁ = K₂ = K₃ = ··· = K₇), and noncooperative, (where K₁ > K₂ > K₃ > ··· > K₇). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn²⁺ and Cd²⁺ are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn²⁺ and Cd²⁺ binding to rhMT 1a.     

A ditopic receptor for cation binding and facilitated transport through a supported liquid membrane

The binding properties of an artificial receptor towards a series of cations including Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Fe²⁺ and Al³⁺ in acetonitrile are described. The receptor comprises a photo-responsive pyrene unit connected via a short spacer to a 2,2′:6′,2″-terpyridine metal ion binding site. Interaction of cations with the receptor was monitored by changes in absorption profile and the association constants calculated for 1:1 and 1:2 cation:ligand binding fall within the range log β = 3-12. The receptor is highly fluorescent and quenching of the emission is observed upon cation binding. The potassium picrate transport properties of the membrane-bound receptor are also described. This receptor when immobilised in a polymer support, which separates two aqueous solutions, has been shown to transport potassium ions in the dark with a flux rate of 1.5 × 10⁸ mol/s m². In contrast, when the membrane-bound receptor is selectively illuminated with light (λ > 400 nm), the flux increases to 2.0 × 10⁸ mol/s m². The transport efficiency depends on the nature of the trap used in the receiver phase.     

Study on the interactions of mapenterol with serum albumins using multi‐spectroscopy and molecular docking

The interactions of mapenterol with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated systematically using fluorescence spectroscopy, absorption spectroscopy, circular dichroism (CD) and molecular docking techniques. Mapenterol has a strong ability to quench the intrinsic fluorescence of BSA and HSA through static quenching procedures. At 291 K, the binding constants, K_a, were 1.93 × 10³ and 2.73 × 10³ L/mol for mapenterol–BSA and mapenterol–HAS, respectively. Electrostatic forces and hydrophobic interactions played important roles in stabilizing the mapenterol–BSA/has complex. Using site marker competitive studies, mapenterol was found to bind at Sudlow site I on BSA/HSA. There was little effect of K⁺, Ca²⁺, Cu²⁺, Zn²⁺ and Fe³⁺ on the binding. The conformation of BSA/HSA was changed by mapenterol, as seen from the synchronous fluorescence spectra. The CD spectra showed that the binding of mapenterol to BSA/HSA changed the secondary structure of BSA/HSA. Molecular docking further confirmed that mapenterol could bind to Sudlow site I of BSA/HSA. According to Förster non‐radiative energy transfer theory (FRET), the distances r₀ between the donor and acceptor were calculated as 3.18 and 2.75 nm for mapenterol–BSA and mapenterol–HAS, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Copper·Lys-Gly-His-Lys mediated cleavage of tRNA: Studies of reaction mechanism and cleavage specificity

The reactivity of [Cu²⁺·Lys-Gly-His-Lys-NH₂]²⁺ and [Cu²⁺·Lys-Gly-His-Lys]⁺ toward tRNA^Phe has been evaluated. The amidated and carboxylate forms of the copper peptides display complex binding behavior with strong and weak sites evident (, for the amide form; and , for the carboxylate form), while Cu²⁺(aq) yielded and . The time-dependence of the reaction of [Cu²⁺·Lys-Gly-His-Lys]⁺ and [Cu²⁺·Lys-Gly-His-Lys-NH₂]²⁺ with tRNA^Phe yielded k_obs ∼ 0.075 h⁻¹ for both complexes. HPLC analysis of the reaction products demonstrated guanine as the sole base product. Mass spectrometric data shows a limited number of cleavage fragments with product peak masses consistent with chemistry occurring at a discrete site defined by the structurally contiguous D and TΨC loops, and in a domain where high affinity magnesium centers have previously been observed to promote hydrolysis of the tRNA^Phe backbone. This cleavage pattern is more selective than that previously observed by Long and coworkers for nickel complexes of a series of C-terminally amidated peptides (Gly-Gly-His, Lys-Gly-His, and Arg-Gly-His), and may reflect variations in structural recognition and a distinct reaction path by the nickel derivatives. The data emphasizes the optimal positioning of the metal-associated reactive oxygen species, relative to scissile bonds, as a major criterion for development of efficient catalytic nucleases or therapeutics.     

Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes

Aims

Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated (a) the pathogenic role of CaM in the channel disorder in CPVT and (b) the possibility of correcting the CPVT-linked channel disorder, using knock-in (KI) mouse model with CPVT-associated RyR2 mutation (R2474S).

Methods and results

Transmembrane potentials were recorded in whole cell current mode before and after pacing (1–5 Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with HiLyte Fluor® in saponin-permeabilized myocytes. In the presence of cAMP (1 μM) the apparent affinity of CaM binding to the RyR decreased in KI cells (Kd: 140–400 nM), but not in WT cells (Kd: 110–120 nM). Gly-Ser-His-CaM (GSH-CaM that has much higher RyR-binding than CaM) restored normal binding to the RyR of cAMP-treated KI cells (140 nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5 Hz pacing, whereas both DAD and TA were observed in 20% and 12% of KI cells, respectively. In response to 10 nM isoproterenol, only DAD (but not TA) was observed in 11% of WT cells, whereas in KI cells the incidence of DAD and TA further increased to 60% and 38% of cells, respectively. Addition of GSH-CaM (100 nM) to KI cells decreased both DADs and TA (DAD: 38% of cells; TA: 10% of cells), whereas CaM (100 nM) had no appreciable effect. Addition of GSH-CaM to saponin-permeabilized KI cells decreased Ca²⁺ spark frequency (+33% of WT cells), which otherwise markedly increased without GSH-CaM (+100% of WT cells), whereas CaM revealed much less effect on the Ca²⁺ spark frequency (+76% of WT cells). Then, by incorporating CaM or GSH-CaM to intact cells (with protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM] = ∼240 nM, cytosolic [GSH-CaM] = ∼230 nM) on the frequency of spontaneous Ca²⁺ transient (sCaT, % of total cells). Addition of 10 nM isoproterenol to KI cells increased sCaT after transient 5 Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) (P < 0.01 vs CaM).

Conclusions

Several disorders in the RyR channel function characteristic of the CPVT-mutant cells (increased spontaneous Ca²⁺ leak, delayed afterdepolarization, triggered activity, Ca²⁺ spark frequency, spontaneous Ca²⁺ transients) can be corrected to a normal function by increasing the affinity of CaM binding to the RyR.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

A membrane-free baffled microbial fuel cell for cathodic reduction of Cu(II) with electricity generation

A membrane-free baffled microbial fuel cell (MFC) was developed to treat synthetic Cu(II) sulfate containing wastewater in cathode chamber and synthetic glucose-containing wastewater fed to anode chamber. Maximum power density of 314 mW/m³ with columbic efficiency of 5.3% was obtained using initial Cu²⁺ concentration of 6400 mg/L. Higher current density favored the cathodic reduction of Cu²⁺, and removal of Cu²⁺ by 70% was observed within 144 h using initial concentration of 500 mg/L. Powder X-ray diffraction (XRD) analysis indicated that the Cu²⁺ was reduced to Cu₂O or Cu₂O plus Cu which deposited on the cathode, and the deficient cathodic reducibility resulted in the formation of Cu₄(OH)₆SO₄ at high initial Cu²⁺ concentration (500-6400 mg/L). This study suggested a novel low-cost approach to remove and recover Cu(II) from Cu²⁺-containing wastewater using MFC-type reactor.