A new mitochondrial fluorescent zinc sensor

A novel cationic fluorescent zinc (Zn2+) indicator (RhodZin-3) with nanomolar affinity for Zn2+ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca2+ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn2+ ([Zn2+]m).     

Analysis of neurotransmitter release mechanisms by photolysis of caged Ca²⁺ in an autaptic neuron culture system

Neurotransmitter release is triggered by membrane depolarization, Ca(2+) influx and Ca(2+) sensing by the release machinery, causing synaptic vesicle (SV) fusion with the plasma membrane. Interlinked is a complex membrane cycle in which vesicles are tethered to the release site, primed, fused and recycled. As many of these processes are Ca(2+) dependent and simultaneously occurring, it is difficult to dissect them experimentally. This problem can be partially circumvented by controlling synaptic Ca(2+) concentrations via UV photolysis of caged Ca(2+). We developed a culture protocol for Ca(2+) uncaging in small synapses on the basis of the generation of small glia cell islands with single neurons on top, which are sufficiently small to be covered with a UV-light flash. Neurons are loaded with the photolabile Ca(2+)-chelator nitrophenyl-EGTA and Ca(2+) indicators, and a UV flash is used to trigger Ca(2+)-uncaging and SV fusion. The protocol takes three weeks to complete and provides unprecedented insights into the mechanisms of transmitter release.     

Zinc influx and physiological consequences in the beta-insulinoma cell line, Min6

In the mammalian pancreas, high concentrations of Zn(2+) are co-secreted with insulin, which may then permeate via abundant L-type Ca(2+) channels (LTCC) present on the beta-cells. Neither the mechanisms utilized by these cells to lower cytosolic Zn(2+) nor the implications of increased intracellular Zn(2+) on beta-cell survival are well understood. To address this, we employed cell imaging of Zn(2+) and Ca(2+) in the beta-insulinoma cell line, Min6. Depolarization induced an intense zinc influx that was blocked by nifedipine and verapamil, indicating that Zn(2+) permeates via the LTCC. Both Ca(2+) and Zn(2+) permeated concomitantly, yet while Ca(2+) was subsequently removed from the cytosol, Zn(2+) was retained in the cells. Fluorescent staining of vesicular Zn(2+) using ZP1 demonstrated that Zn(2+) could be slowly sequestered following a brief exposure to low concentration of Zn(2+). In contrast, cells were unable to sequester Zn(2+) following application of high concentrations, which was followed by massive cell death. Our results demonstrate homeostatic crosstalk between the plasma membrane and intracellular zinc transporters and suggest that attenuating zinc influx may enhance beta-cell survival.     

Carbonic anhydrase II-based metal ion sensing: Advances and new perspectives

Carbonic anhydrases are archetypical zinc metalloenzymes and as such, they have been developed as the recognition element of a family of fluorescent indicators (sensors) to detect metal ions, particularly Zn²⁺ and Cu²⁺. Subtle modification of the structure of human carbonic anhydrase II isozyme (CAII) alters the selectivity, sensitivity, and response time for these sensors. Sensors using CAII variants coupled with zinc-dependent fluorescent ligands demonstrate picomolar sensitivity, unmatched selectivity, ratiometric fluorescence signal, and near diffusion-controlled response times. Recently, these sensors have been applied to measuring the readily exchangeable concentrations of zinc in the cytosol and nucleus of mammalian tissue culture cells and concentrations of free Cu²⁺ in seawater.     

Loading Drosophila nerve terminals with calcium indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca(2+)-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca(2+) changes in nerve terminals. However, these indicators have limited sensitivity to Ca(2+) and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca(2+) changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca(2+) indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca(2+) to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca(2+)-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca(2+) dynamics. Dextran-conjugation helps prevent Ca(2+) indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.     

Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes

Cytosolic alterations of calcium ion concentrations are an integral part of signal transduction. Similar functions have been hypothesized for other metal ions, in particular zinc (Zn(2+)), but this still awaits experimental verification. Zn(2+) is important for multiple cellular functions, especially in the immune system. Among other effects, it influences formation and secretion of pro-inflammatory cytokines, including TNF-alpha. Here we demonstrate that these effects are due to a physiological signaling system involving intracellular Zn(2+) signals. An increase of the intracellular zinc ion concentration occurs upon stimulation of human leukocytes with Escherichia coli, LPS, Pam(3)CSK(4), TNF-alpha, or insulin, predominantly in monocytes. Chelating this zinc signal with the membrane permeable zinc-specific chelator TPEN (N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine) completely blocks activation of LPS-induced signaling pathways involving p38 MAPK, ERK1/2, and NF-kappaB, and abrogates the release of proinflammatory cytokines, including TNF-alpha. This function of Zn(2+) is not limited to monocytes or even the immune system, but seems to be another generalized signaling system based on intracellular fluctuations of metal ion concentrations, acting parallel to Ca(2+).     

Zinc-induced decrease of the thermal stability and regeneration of rhodopsin

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn(2+) on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn(2+) concentrations (0-50 microm Zn(2+)). The thermal bleaching process is accelerated in the presence of Zn(2+) with k rate constants, at 55 degrees C, of 0.028 +/- 0.002 min(-1) (0 microm Zn(2+)) and 0.056 +/- 0.003 min(-1) (50 microm Zn(2+)), corresponding to t(12) values of 24.4 +/- 1.6 min and 11.8 +/- 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E(a) increase at 50 microm Zn(2+) for the process, with deltaG++ decrease and increase in deltaH++ and deltaS++ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn(2+) in normal and pathological retinal function.     

Design, synthesis, and evaluation of peptidyl fluorescent probe for Zn2+ in aqueous solution

A new fluorescent peptide probe for the detection of Zn(2+) was synthesized on the basis of zinc binding ligands in zinc enzymes. The peptide that has a unique amino acid sequence displayed a great selectivity for Zn(2+) in the presence of several transition metal ions in aqueous solution. The reversibility, binding stoichiometry, binding affinity, and pH sensitivity of the sensor were studied. Further, on-bead application of the peptide as chemosensors was demonstrated.     

Zinc binding of Tim10: evidence for existence of an unstructured binding intermediate for a zinc finger protein

Zinc-finger proteins are among the most abundant proteins in eukaryotic genomes. Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX(3)C zinc-finger motif. Zn(2+) can bind to the reduced Tim10, but not disulphide bonded (oxidized) protein. However, the zinc-binding reaction of Tim10 and of zinc-finger proteins, in general, is ill-defined. In this study, the thermodynamic and kinetic properties of zinc-binding to reduced Tim10 were investigated using circular dichroism (CD), fluorescence spectrometry, and stopped-flow fluorescence techniques. At equilibrium, coupled with the use of protein fluorescence and metal chelators, the zinc-binding affinity was determined for Tim10 to be about 8 x 10(-10)M. Then, far UV CD was used to investigate the secondary structure change upon zinc-binding of the same set of protein samples at various free Zn(2+) concentrations. Comparison between the results of CD and fluorescence studies showed that the zinc-binding reaction is not a simple one-step process. It involves formation of a binding intermediate that is structurally as unfolded as the apoTim10; subsequently, a degree of folding is induced at increased zinc concentrations in the final complex. Next, the stopped-flow fluorescence technique was used to investigate the kinetic process of the binding reaction. Data analysis shows that the reaction has a single kinetic phase at a low free Zn(2+) concentration ( approximately 1 nM), and a double kinetic phase at a high free Zn(2+) concentration. The kinetic result is consistent with that of the studies at equilibrium. Therefore, a two-step reaction model mechanism is proposed, in which zinc-binding is regulated by the initial selective-binding of Zn(2+) to Cys followed by folding. Implication of the two-step zinc-binding mechanism for Zn(2+) trafficking in the cell is discussed.     

A transporter in the endoplasmic reticulum of Schizosaccharomyces pombe cells mediates zinc storage and differentially affects transition metal tolerance

The cation diffusion facilitator (CDF) family represents a class of ubiquitous metal transporters. Inactivation of a CDF in Schizosaccharomyces pombe, Zhf, causes drastically different effects on the tolerance toward various metals. A deletion mutant is Zn(2+)/Co(2+)-hypersensitive yet displays significantly enhanced Cd(2+) and Ni(2+) tolerance. Accumulation of zinc, cobalt, and cadmium is reduced in mutant cells. Non-vacuolar zinc content, as measured by analytical electron microscopy, is lower in zhf(-) cells compared with wild-type cells in the presence of elevated Zn(2+) concentrations. The protective effect against cadmium toxicity is independent of the phytochelatin detoxification pathway. Phytochelatin synthase-deficient cells show extremely enhanced (about 200-fold) cadmium tolerance when zhf is disrupted. Immunogold labeling indicates endoplasmic reticulum (ER) localization of Zhf. Electron spectroscopic imaging shows that accumulation of zinc coincides with Zhf localization, demonstrating a major role of the ER for metal storage and the involvement of Zhf in cellular zinc homeostasis. Also, these observations indicate that Cd(2+) ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol.     

Tuning magnesium sensitivity of BK channels by mutations

Intracellular Mg(2+) at physiological concentrations activates mSlo1 BK channels by binding to a metal-binding site in the cytosolic domain. Previous studies suggest that residues E374, Q397, and E399 are important in Mg(2+) binding. In the present study, we show that mutations of E374 or E399 to other amino acids, except for Asp, abolish Mg(2+) sensitivity. These results further support that the side chains of E374 and E399 are essential for Mg(2+) coordination. To the contrary, none of the Q397 mutations abolishes Mg(2+) sensitivity, suggesting that its side chain may not coordinate to Mg(2+). However, because Q397 is spatially close to E374 and E399, its mutations affect the Mg(2+) sensitivity of channel gating by either reducing or increasing the Mg(2+) binding affinity. The pattern of mutational effects and the effect of chemical modification of Q397C indicate that Q397 is involved in the Mg(2+)-dependent activation of BK channels and that mutations of Q397 alter Mg(2+) sensitivity by affecting the conformation of the Mg(2+) binding site as well as by electrostatic interactions with the bound Mg(2+) ion.     

Biochemical characterization of the histidine triad protein PhtD as a cell surface zinc-binding protein of pneumococcus

Zinc homeostasis is critical for pathogen host colonization. Indeed, during invasion, Streptococcus pneumoniae has to finely regulate zinc transport to cope with a wide range of Zn(2+) concentrations within the various host niches. AdcAII was identified as a pneumococcal Zn(2+)-binding protein; its gene is present in an operon together with the phtD gene. PhtD belongs to the histidine triad protein family, but to date, its function has not been clarified. Using several complementary biochemical methods, we provide evidence that like AdcAII, PhtD is a metal-binding protein specific for zinc. When Zn(2+) binds (K(d) = 131 ± 10 nM), the protein displays substantial thermal stabilization. We also present the first direct evidence of a joint function of AdcAII and PhtD by demonstrating that their expression is corepressed by Zn(2+), that they interact directly in vitro, and that they are colocalized at the bacterial surface. These results suggest the common involvement of the AdcAII-PhtD system in pneumococcal zinc homeostasis.     

Membrane potential-controlled inhibition of cytochrome c oxidase by zinc

Like many voltage-sensitive ion pumps, cytochrome c oxidase is inhibited by zinc. Binding of zinc to the outside surface of Rhodobacter sphaeroides cytochrome c oxidase inhibits the enzyme with a K(I) of < or = 5 microm when the enzyme is reconstituted into phospholipid vesicles in the presence of a membrane potential. In the absence of a membrane potential and a pH gradient, millimolar concentrations of zinc are required to inhibit. This differential inhibition causes a dramatic increase in the respiratory control ratio from 6 to 40 for wild-type oxidase. The external zinc inhibition is removed by EDTA and is not competitive with cytochrome c binding but is competitive with protons. Only Cd(2+) of the many metals tested (Mg(2+), Mn(2+), Ca(2+), Ba(2+), Li(2+), Cs(2+), Hg(2+), Ni(2+), Co(2+), Cu(2+) Tb(3+), Tm(3+)) showed inhibitory effects similar to Zn(2+). Proton pumping is slower and less efficient with zinc. The results suggest that zinc inhibits proton movement through a proton exit path, which can allow proton back-leak at high membrane potentials. The physiological and mechanistic significance of proton movement in the exit pathway and its blockage by zinc is discussed in terms of regulation of the efficiency of energy transduction.     

Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins

Edema factor (EF) and CyaA are calmodulin (CaM)-activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance spectroscopy analyses, we show that low Ca(2+) concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca(2+) concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca(2+) concentrations. Despite the similarity in Ca(2+) sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100-fold higher affinity for CaM than EF. CaM has N- and C-terminal globular domains, each binding two Ca(2+) ions. CyaA can be fully activated by CaM mutants with one defective C-terminal Ca(2+)-binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca(2+)-CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.     

Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.     

Structural transition from the random coil to quadruplex of AG(3)(T(2)AG(3))(3) induced by Zn(2+)

Structures of G-quadruplex DNAs can be typically stabilized by monovalent cations such as K(+), Na(+). Some divalent and trivalent cations, such as Sr(2+), Pb(2+), Tb(3+) and Eu(3+), can also induce the formation of G-quadruplex DNA. Here we show that Zn(2+) can induce the human telomeric sequence AG(3)(T(2)AG(3))(3) to fold the G-quadruplex structure by UV absorbance difference spectra and circular dichroism (CD) spectroscopy. At micromolar concentrations, the Zn(2+)-induced changes in the UV absorbance difference spectra and CD spectra are the characteristics of antiparallel G-quadruplexes although the long wavelength CD maximum is around 285 nm rather than the typical value of 295 nm. The binding stoichometry of Zn(2+) per one AG(3)(T(2)AG(3))(3) molecule is four. To our knowledge, the structural transition of human telomeric sequence induced by Zn(2+) was observed for the first time.     

Regulation of ryanodine receptor opening by lumenal Ca(2+) underlies quantal Ca(2+) release in PC12 cells

Graded or "quantal" Ca(2+) release from intracellular stores has been observed in various cell types following activation of either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP(3)R). The mechanism causing the release of Ca(2+) stores in direct proportion to the strength of stimulation is unresolved. We investigated the properties of quantal Ca(2+) release evoked by activation of RyR in PC12 cells, and in particular whether the sensitivity of RyR to the agonist caffeine was altered by lumenal Ca(2+). Quantal Ca(2+) release was observed in cells stimulated with 1 to 40 mM caffeine, a range of caffeine concentrations giving a >10-fold change in lumenal Ca(2+) content. The Ca(2+) load of the caffeine-sensitive stores was modulated by allowing them to refill for varying times after complete discharge with maximal caffeine, or by depolarizing the cells with K(+) to enhance their normal steady-state loading. The threshold for RyR activation was sensitized approximately 10-fold as the Ca(2+) load increased from a minimal to a maximal loading. In addition, the fraction of Ca(2+) released by low caffeine concentrations increased. Our data suggest that RyR are sensitive to lumenal Ca(2+) over the full range of Ca(2+) loads that can be achieved in an intact PC12 cell, and that changes in RyR sensitivity may be responsible for the termination of Ca(2+) release underlying the quantal effect.     

Effects of zinc and other metal ions on the stability and activity of Escherichia coli alkaline phosphatase

Measurement of the ultraviolet circular dichroism of apo-(alkaline phosphatase) in urea solutions showed substantial denaturation in 3m-urea. A zinc-deficient mutant alkaline phosphatase behaved similarly. The stability of the enzyme in 6m-urea was followed as a function of its zinc content and was found to be dependent on the first two of the four zinc atoms bound by apoenzyme. Phosphatase activity was mostly dependent on a second pair of zinc atoms. Mn(2+), Co(2+), Cu(2+) or Cd(2+) also restored structural stability. Sedimentation-velocity and -equilibrium experiments revealed that dissociation of the dimer accompanied apoenzyme denaturation in urea concentrations of 1m or higher, without treatment with disulphide-reducing agent.     

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn(2+) ions (10nM to 50 microM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn(2+) (10 microM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn(2+) (5 microM and lower), the enzymatic activity was enhanced.