Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.
AuthorsBerlin JR,Konishi M
JournalBiophysical journal
PubMed ID8274651
Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes ... More
Matrix free Mg2+ changes with metabolic state in isolated heart mitochondria.
AuthorsJung DW, Apel L, Brierley GP
JournalBiochemistry
PubMed ID2361136
'The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored by using the fluorescent probe furaptra (mag-fura-2). Beef heart mitochondria respiring in a KCl medium in the absence of external Mg2+ maintain free matrix Mg2+ near 0.50 mM. Addition of Pi under these conditions decreases ... More
Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra.
AuthorsTursun P, Tashiro M, Konishi M
JournalBiophys J
PubMed ID15626700
'The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, ... More
Mg2+ buffering in cultured chick ventricular myocytes: quantitation and modulation by Ca2+.
AuthorsKoss KL, Putnam RW, Grubbs RD
JournalAm J Physiol
PubMed ID8498485
'To characterize the Mg2+ buffering of cultured chick ventricular myocytes, cytosolic Mg2+ was increased by liberating Mg2+ normally chelated by ATP upon total depletion of ATP content. Because the total Mg content and cell volume remained constant during this time, the difference between the amount of Mg2+ liberated (2.7 mM) ... More
Capacitative Ca2+ entry is closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+].
AuthorsHofer AM, Fasolato C, Pozzan T
JournalJ Cell Biol
PubMed ID9442108
'ICRAC (the best characterized Ca2+ current activated by store depletion) was monitored concurrently for the first time with [Ca2+] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca2+] within stores of intact cells at any ... More
The binding of calcium to the B-repeat segment of SdrD, a cell surface protein of Staphylococcus aureus.
AuthorsJosefsson E, O'Connell D, Foster TJ, Durussel I, Cox JA
JournalJ Biol Chem
PubMed ID9813018
'In the Sdr family of Staphylococcus aureus cell surface proteins, three recently cloned members (Josefsson, E., McCrea, K., Ni Eidhin, D., O''Connell, D., Cox, J. A., Hook, M., and Foster, T. (1998) Microbiology, in press) display variable numbers of B-repeats, i.e. segments of 110-113 residues that probably make up one ... More
Calcium signalling in sarcoplasmic reticulum, cytoplasm and mitochondria during activation of rabbit aorta myocytes.
AuthorsGurney AM, Drummond RM, Fay FS
JournalCell Calcium
PubMed ID11013464
'This study investigated the relationship between cytoplasmic, mitochondrial, and sarcoplasmic reticulum (SR) [Ca(2+)] in rabbit aorta smooth muscle cells, following cell activation. Smooth muscle cells were loaded with the Ca(2+)-sensitive fluorescent indicator Mag-Fura-2-AM, and then either permeabilized by exposure to saponin, or dialyzed with a patch pipette in the whole-cell ... More
Fluorescence signals from the Mg2+/Ca2+ indicator furaptra in frog skeletal muscle fibers.
AuthorsKonishi M, Suda N, Kurihara S
JournalBiophys J
PubMed ID8431543
'The fluorescent Mg2+/Ca2+ indicator, furaptra, was injected into single frog skeletal muscle fibers, and the indicator''s fluorescence signals were measured and analyzed with particular interest in the free Mg2+ concentration ([Mg2+]) in resting muscle. Based on the fluorescence excitation spectrum of furaptra, the calibrated myoplasmic [Mg2+] level averaged 0.54 mM, ... More
Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.
AuthorsRupnik M, Kreft M, Sikdar SK, Grilc S, Romih R, Zupancic G, Martin TF, Zorec R
JournalProc Natl Acad Sci U S A
PubMed ID10792045
'Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, ... More
Membrane binding of phospholipases C-beta 1 and C-beta 2 is independent of phosphatidylinositol 4,5-bisphosphate and the alpha and beta gamma subunits of G proteins.
AuthorsRunnels LW, Jenco J, Morris A, Scarlata S
JournalBiochemistry
PubMed ID8988021
'We have measured the membrane binding affinities of purified phosphatidylinositol-specific phospholipases C-beta 1 and C-beta 2 to membranes of varying lipid composition using fluorescence methods. Our studies show that these proteins bind with affinities of 10(-5)-10(-4) M, with a small dependence on lipid type. Binding was relatively insensitive to the ... More
Ca transients in cardiac myocytes measured with a low affinity fluorescent indicator, furaptra.
AuthorsKonishi M, Berlin JR
JournalBiophys J
PubMed ID8494988
'Intracellular calcium ion ([Ca2+]i) transients were measured in single rat ventricular myocytes with the fluorescent indicator furaptra. Cells were voltage clamped with a single patch electrode containing the K+ salt of furaptra and fluorescence at 500 nm was measured during illumination with 350 and 370 nm light. Depolarizing voltage-clamp pulses ... More
Fast activation and inactivation of inositol trisphosphate-evoked Ca2+ release in rat cerebellar Purkinje neurones.
AuthorsKhodakhah K, Ogden D
JournalJ Physiol
PubMed ID8558468
'1. Calcium release from stores via inositol trisphosphate (InsP3) activation of intracellular Ca2+ receptor-channels is thought to have a role in regulating the excitability of cerebellar Purkinje neurones. The kinetic characteristics of InsP3 receptor activation in Purkinje neurones are reported here. 2. InsP3 was applied by flash photolysis of caged ... More
A fluorescent indicator for measuring cytosolic free magnesium.
AuthorsRaju B, Murphy E, Levy LA, Hall RD, London RE
JournalAm J Physiol
PubMed ID2923192
'The previously developed chelator O-aminophenol-N,N,O-triacetic acid (APTRA) (L. A. Levy, E. Murphy, B. Raju, and R. E. London. Biochemistry 27: 4041-4048, 1988) has been modified to yield a fluorescent analogue which can be utilized as an intracellular probe for ionized Mg2+. The fluorescent analogue, FURAPTRA, with a magnesium dissociation constant ... More
Competition between Li+ and Mg2+ for the phosphate groups in the human erythrocyte membrane and ATP: an NMR and fluorescence study.
AuthorsMota de Freitas D, Amari L, Srinivasan C, Rong Q, Ramasamy R, Abraha A, Geraldes CF, Boyd MK
JournalBiochemistry
PubMed ID8155627
'We investigated the mechanism of competition between Li+ and Mg2+ in Li(+)-loaded human red blood cells (RBCs) by making 7Li and 31P NMR and fluorescence measurements. We used 7Li NMR relaxation times to probe Li+ binding to the human RBC membrane and ATP; an increase in Mg2+ concentration caused an ... More
Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle.
AuthorsWesterblad H, Allen DG
JournalJ Physiol
PubMed ID1464836
'1. The role of the myoplasmic free Mg2+ concentration ([Mg2+]i) in fatigue was studied in intact single fibres isolated from mouse skeletal muscle. Fatigue was produced by repeated tetanic stimulation. The fluorescent Mg2+ indicator furaptra was pressure injected into fibres. In vivo calibrations were performed to convert fluorescence signals into ... More
Presynaptic calcium dynamics and transmitter release evoked by single action potentials at mammalian central synapses.
AuthorsSinha SR, Wu LG, Saggau P
JournalBiophys J
PubMed ID9017193
'The relationship between presynaptic calcium transients ([Ca2+]t) and transmitter release evoked by a single stimulus was both investigated experimentally and modeled at a mammalian central synapse, the CA3 to CA1 pyramidal cell synapse in guinea pig hippocampal slices. In the present study, we compared the low-affinity calcium indicator furaptra with ... More
Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC.
AuthorsHyrc KL, Bownik JM, Goldberg MP
JournalCell Calcium
PubMed ID10756974
'Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ ... More
Assessment of intra-SR free [Ca] and buffering in rat heart.
AuthorsShannon TR, Bers DM
JournalBiophys J
PubMed ID9284319
'To measure the free intrasarcoplasmic reticulum [Ca] ([Ca]SR) in isolated rat cardiac microsomes, ventricular tissue was homogenized in the presence of the low-affinity Ca indicator furaptra. Stepwise increases in cuvette [Ca] ([Ca]c) in the presence of ATP caused progressive increases in steady-state intravesicular fluorescence ratio to a maximum (Rmax). Steady-state ... More
Calcium sensitivity of glutamate release in a calyx-type terminal.
AuthorsBollmann JH, Sakmann B, Borst JG
JournalScience
PubMed ID10937999
'Synaptic efficacy critically depends on the presynaptic intracellular calcium concentration ([Ca2+]i). We measured the calcium sensitivity of glutamate release in a rat auditory brainstem synapse by laser photolysis of caged calcium. A rise in [Ca2+]i to 1 micromolar readily evoked release. An increase to >30 micromolar depleted the releasable vesicle ... More
Measurement and simulation of myoplasmic calcium transients in mouse slow-twitch muscle fibres.
AuthorsHollingworth S, Kim MM, Baylor SM,
JournalJ Physiol
PubMed ID22124146
'Bundles of intact fibres from soleus muscles of adult mice were isolated by dissection and one fibre within a bundle was micro-injected with either furaptra or mag-fluo-4, two low-affinity rapidly responding Ca(2+) indicators. Fibres were activated by action potentials to elicit changes in indicator fluorescence (?F), a monitor of the ... More
The whistle and the rattle: the design of sound producing muscles.
AuthorsRome LC, Syme DA, Hollingworth S, Lindstedt SL, Baylor SM
JournalProc Natl Acad Sci U S A
PubMed ID8755609
'Vertebrate sound producing muscles often operate at frequencies exceeding 100 Hz, making them the fastest vertebrate muscles. Like other vertebrate muscle, these sonic muscles are "synchronous," necessitating that calcium be released and resequestered by the sarcoplasmic reticulum during each contraction cycle. Thus to operate at such high frequencies, vertebrate sonic ... More
Cytosolic free magnesium in cardiac myocytes: identification of a Mg2+ influx pathway.
AuthorsQuamme GA, Rabkin SW
JournalBiochem Biophys Res Commun
PubMed ID2322282
'Regulation of intracellular Mg2+ activity in the heart is not well characterized. Cardiac myocytes were prepared as primary cultures from 7 day old chick embryo hearts and intracellular Mg2+ concentration [( Mg2+]i) was determined in single ventricular cells with mag-fura-2. Basal [Mg2+]i was 0.48 +/- 0.03 mM in normal culture ... More
Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells.
AuthorsXu T, Naraghi M, Kang H, Neher E
JournalBiophys J
PubMed ID9199815
'The Ca2+ binding kinetics of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer, which determine the time course of Ca2+ changes after photolysis of DM-nitrophen, were studied in bovine chromaffin cells. The in vivo Ca2+ association rate constants of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer were measured to be 5.17 ... More
High magnesium concentration inhibits ligand-stimulated calcium influx and hormone secretion in rat pituitary lactotropes with involvement of intracellular free magnesium.
AuthorsKasahara K, Tasaka K, Masumoto N, Nishizaki T, Mizuki J, Tahara M, Miyake A, Tanizawa O
JournalBiochem Biophys Res Commun
PubMed ID8250951
'The effects of extracellular magnesium concentration ([Mg2+]ex) on thyrotropin-releasing hormone (TRH)-stimulated intracellular free calcium mobilization and prolactin secretion were investigated concomitantly with measurement of the intracellular free magnesium concentration ([Mg2+]i). TRH-stimulated intracellular free calcium mobilization was significantly inhibited when the medium was replaced by high Mg2+ medium ([Mg2+]ex = 10 ... More
On the relationship between matrix free Mg2+ concentration and total Mg2+ in heart mitochondria.
AuthorsJung DW, Panzeter E, Baysal K, Brierley GP
JournalBiochim Biophys Acta
PubMed ID9230923
'The matrix free magnesium ion concentration, [Mg2+]m, estimated using the fluorescent probe furaptra, averaged 0.67 mM in 15 preparations of beef heart mitochondria containing an average of 21 nmol total Mg2+ per mg protein. [Mg2+]m was compared with total Mg2+ during respiration-dependent uptake and efflux of Mg2+ and during osmotic ... More
Rapid mobilization of intracellular Mg2+ by bombesin in Swiss 3T3 cells: mobilization through external Ca(2+)- and tyrosine kinase-dependent mechanisms.
AuthorsIshijima S, Tatibana M
JournalJ Biochem (Tokyo)
PubMed ID8089090
'We examined the effects of growth factors on intracellular free Mg2+ concentrations ([Mg2+]i) in single Swiss 3T3 fibroblasts, using microfluorometry of a Mg(2+)-sensitive dye mag-fura-2. We had already noted an increase in [Mg2+]i after exposure to bombesin for 30-60 min [Ishijima, S., Sonoda, T., & Tatibana, M. (1991) Am. J. ... More
Measurement of free Zn2+ ion concentration with the fluorescent probe mag-fura-2 (furaptra).
AuthorsSimons TJ
JournalJ Biochem Biophys Methods
PubMed ID8409208
'The fluorescent probe mag-fura-2, previously used to measure [Mg2+], can also be used to measure [Zn2+]. The peak in the excitation spectrum occurs at 323 nm for Zn2+, compared with 335 nm for Ca2+ and Mg2+. This allows simultaneous measurements of [Zn2+] and either [Ca2+] or [Mg2+], by using 3 ... More
Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis.
AuthorsXu T, Rammner B, Margittai M, Artalejo AR, Neher E, Jahn R
JournalCell
PubMed ID10619425
'In chromaffin cells, an increase in intracellular Ca2+ leads to an exocytotic burst followed by sustained secretion. The burst can be further resolved into two kinetically distinct components, which suggests the presence of two separate pools of vesicles. To investigate how these components relate to SNARE complex formation, we introduced ... More
Multiple calcium-dependent processes related to secretion in bovine chromaffin cells.
AuthorsNeher E, Zucker RS
JournalNeuron
PubMed ID8427700
'We have used the caged calcium compound DM-nitrophen to investigate the kinetics of calcium-dependent secretion in bovine chromaffin cells. Perfusion with partially calcium-loaded nitrophen often caused a loading transient--slow secretion for up to 1 min due to displacement of Ca2+ by cytoplasmic Mg2+. Flash photolysis elicited 100 microM [Ca2+]i steps ... More
Millisecond studies of secretion in single rat pituitary cells stimulated by flash photolysis of caged Ca2+.
AuthorsThomas P, Wong JG, Almers W
JournalEMBO J
PubMed ID8428588
'To study the final steps in the secretory pathway of rat pituitary melanotrophs, we have monitored changes in cell surface area due to exocytosis after flash photolysis of caged Ca2+. A step increase in cytosolic [Ca2+] to 45-125 microM triggers three phases of exocytic secretion. A small cohort of a ... More
Determination of cytosolic Mg2+ activity and buffering in BC3H-1 cells with mag-fura-2.
AuthorsGrubbs RD, Walter A
JournalMol Cell Biochem
PubMed ID7854327
'The magnesium buffer coefficient (BMg) was calculated for BC3H-1 cells from the rise in cytosolic Mg2+ activity observed when magnesium was released from ATP after iodoacetate (IAA) and NaCN treatment. The basal cytosolic Mg2+ activity (0.54 +/- 0.1 mM) measured with mag-fura-2 doubled when 4.54 mM magnesium was liberated from ... More
The zinc metalloregulatory protein Synechococcus PCC7942 SmtB binds a single zinc ion per monomer with high affinity in a tetrahedral coordination geometry.
AuthorsVanZile ML, Cosper NJ, Scott RA, Giedroc DP
JournalBiochemistry
PubMed ID10995250
'The Synechococcus PCC7942 SmtB is a zinc-responsive transcriptional repressor and a member of the ArsR superfamily of prokaryotic metalloregulatory transcription factors. The mechanism of negative regulation by Zn(II) and other metals as well as the coordination chemistry (stoichiometry, affinity, and specificity) of SmtB is poorly understood. In contrast to previous ... More
Methods for measurement of intracellular magnesium: NMR and fluorescence.
AuthorsLondon RE
JournalAnnu Rev Physiol
PubMed ID2042961
Interactions of polyamines in the measurement of free magnesium concentration by mag-fura-2 and 31P-NMR.
AuthorsGünther T, Vormann J, Konstanczak P, Schäfer A
JournalBiochim Biophys Acta
PubMed ID8018709
Polyamines, particularly spermine, in physiological concentrations interact with mag-fura-2 and the mag-fura-2/Mg2+ complex, resulting in reduced values of free Mg2+ concentration. Similarly, polyamines interact with ATP and MgATP. Thus, free Mg2+ concentration, as measured by 31P-NMR or mag-fura-2, is underestimated in the presence of polyamines, particularly of spermine. ... More
The effect of pH on rate constants, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators.
AuthorsLattanzio FA, Bartschat DK
JournalBiochem Biophys Res Commun
PubMed ID2043105
Fluorescent calcium indicators fluo-3, fura-2 and indo-1, and fluorescent magnesium indicators mag-fura-2 (FURAPTRA) and mag-indo-1 were evaluated for the effects of pH on their association and dissociation rates, ion selectivity and thermodynamic properties. Calcium indicator affinities for Ca and Mg were reduced and the discrimination between Ca and Mg decreased ... More
Concentration, compartmentation and metabolic function of intracellular free Mg2+.
AuthorsGünther T,
JournalMagnes Res
PubMed ID17402290
Intracellular total Mg2+ and free Mg2+ are compartmentalized between cell organelles and within the cytosol. Different values of [Mg2+]i in the cytosol of the same cell type were measured by various investigators. A main reason for the differences is the uncertainty of the dissociation constants used for the Mg furaptra ... More
A triggered mechanism retrieves membrane in seconds after Ca(2+)-stimulated exocytosis in single pituitary cells.
AuthorsThomas P, Lee AK, Wong JG, Almers W
JournalJ Cell Biol
PubMed ID8120090
In neuroendocrine cells, cytosolic Ca2+ triggers exocytosis in tens of milliseconds, yet known pathways of endocytic membrane retrieval take minutes. To test for faster retrieval mechanisms, we have triggered short bursts of exocytosis by flash photolysis of caged Ca2+, and have tracked subsequent retrieval by measuring the plasma membrane capacitance. ... More
Cytosolic Ni(II) sensor in cyanobacterium: nickel detection follows nickel affinity across four families of metal sensors.
Efflux of surplus Ni(II) across the outer and inner membranes of Synechocystis PCC 6803 is mediated by the Nrs system under the control of a sensor of periplasmic Ni(II), NrsS. Here, we show that the product of ORF sll0176, which encodes a CsoR/RcnR-like protein now designated InrS (for internal nickel-responsive ... More
Analysis of neurotransmitter release mechanisms by photolysis of caged Ca²? in an autaptic neuron culture system.
AuthorsBurgalossi A, Jung S, Man KN, Nair R, Jockusch WJ, Wojcik SM, Brose N, Rhee JS,
JournalNat Protoc
PubMed ID22722370
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 ... More
Low-affinity Ca2+ indicators compared in measurements of skeletal muscle Ca2+ transients.
AuthorsHollingworth S, Gee KR, Baylor SM,
JournalBiophys J
PubMed ID19804716
The low-affinity fluorescent Ca(2+) indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca(2+) transient (Delta[Ca(2+)]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, furaptra (previously shown to rapidly ... More
Munc18-1 promotes large dense-core vesicle docking.
AuthorsVoets T, Toonen RF, Brian EC, de Wit H, Moser T, Rettig J, Südhof TC, Neher E, Verhage M
JournalNeuron
PubMed ID11545717
Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. ... More
Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse.
AuthorsBeutner D, Voets T, Neher E, Moser T
JournalNeuron
PubMed ID11301027
Release of neurotransmitter at the inner hair cell (IHC) afferent synapse is a fundamental step in translating sound into auditory nerve excitation. To study the Ca2+ dependence of the underlying vesicle fusion and subsequent endocytosis, we combined Ca2+ uncaging with membrane capacitance measurements in mouse IHCs. Rapid elevations in [Ca2+]i ... More
Ca2+/calmodulin stimulates GTP binding to the ras-related protein ral-A.
AuthorsWang KL, Roufogalis BD
JournalJ Biol Chem
PubMed ID10329639
Ral-A is a Ras-related GTP-binding protein that has been suggested to be the downstream target of Ras proteins and is involved in the tyrosine kinase-mediated, Ras-dependent activation of phospholipase D. We reported recently that Ral-A purified from human erythrocyte membrane binds to calmodulin in a Ca2+-dependent manner at a calmodulin ... More
Synergistic release of Ca2+ from IP3-sensitive stores evoked by synaptic activation of mGluRs paired with backpropagating action potentials.
AuthorsNakamura T, Barbara JG, Nakamura K, Ross WN
JournalNeuron
PubMed ID10595522
Increases in postsynaptic [Ca2+]i can result from Ca2+ entry through ligand-gated channels or voltage-gated Ca2+ channels, or through release from intracellular stores. Most attention has focused on entry through the N-methyl-D-aspartate (NMDA) receptor in causing [Ca2+]i increases since this pathway requires both presynaptic stimulation and postsynaptic depolarization, making it a ... More
Calcium control of transmitter release at a cerebellar synapse.
AuthorsMintz IM, Sabatini BL, Regehr WG
JournalNeuron
PubMed ID7546746
The manner in which presynaptic Ca2+ influx controls the release of neurotransmitter was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory postsynaptic currents were measured using whole-cell voltage clamp, and changes in presynaptic Ca2+ influx were determined with the Ca(2+)-sensitive dye furaptra. We manipulated ... More
Intracellular calibration of the fluorescent Mg2+ indicator furaptra in rat ventricular myocytes.
AuthorsWatanabe M, Konishi M
JournalPflugers Arch
PubMed ID11374066
Single ventricular myocytes enzymatically isolated from rat hearts were loaded with the Mg2+ indicator furaptra, and the relationship between the fluorescence ratio signal (R) and the intracellular free concentration of Mg2+ ([Mg2+]i) was studied in situ at 25 degrees C. After the application of ionophores (ionomycin, monensin, nigericin and valinomycin), ... More
Monitoring cytosolic free magnesium in cultured chicken heart cells by use of the fluorescent indicator Furaptra.
AuthorsMurphy E, Freudenrich CC, Levy LA, London RE, Lieberman M
JournalProc Natl Acad Sci U S A
PubMed ID2704755
Cytosolic free magnesium concentration [Mg2+]i and its regulation were studied in cultured embryonic chicken heart cells by use of the fluorescent indicator 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacet ic acid (Furaptra). The intracellular location of Furaptra was confirmed by its complete release from cells upon addition of saponin. The basal [Mg2+]i, which averaged 0.48 +/- ... More
Insulin secretagogues induce Ca(2+)-like changes in cytoplasmic Mg2+ in pancreatic beta-cells.
AuthorsGylfe E
JournalBiochim Biophys Acta
PubMed ID2223873
The effects of insulin secretagogues on the cytoplasmic Mg2+ concentration ([Mg2+]i) of pancreatic beta-cells were studied in suspensions and in individual beta-cells using dual-wavelength fluorometry and the indicator mag-fura-2. Average [Mg2+]i was in the 800-900 microM range in a medium containing 3 mM glucose. When the sugar concentration was raised ... More
Parvalbumin relaxes frog skeletal muscle when sarcoplasmic reticulum Ca(2+)-ATPase is inhibited.
AuthorsJiang Y, Johnson JD, Rall JA
JournalAm J Physiol
PubMed ID8779902
Inhibition of sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ) in frog skeletal muscle fibers at 10 degrees C prolonged the half time of the fall of the Ca2+ transient by 62% and twitch force by 100% and increased peak force by 120% without increasing the amplitude of the Ca2+ ... More
Analogue computation of transient changes of intracellular free Ca2+ concentration with the low affinity Ca2+ indicator furaptra during whole-cell patch-clamp recording.
AuthorsOgden D, Khodakhah K, Carter T, Thomas M, Capiod T
JournalPflugers Arch
PubMed ID7617450
The measurement of cytosolic free Ca2+ ion concentration ([Ca2+]) with low affinity Ca2+ indicators has advantages for kinetic studies of cytosolic [Ca2+] transients when compared with more commonly used high affinity Ca2+ indicators. Their dynamic range and linearity are better suited to measurement of high localised transient concentration changes that ... More
Caffeine interaction with fluorescent calcium indicator dyes.
AuthorsMuschol M, Dasgupta BR, Salzberg BM
JournalBiophys J
PubMed ID10388782
We report that caffeine, in millimolar concentrations, interacts strongly with four common calcium indicator dyes: mag-fura-2, magnesium green, fura-2, and fluo-3. Fluorescence intensities are either noticeably enhanced (mag-fura-2, fura-2) or diminished (magnesium green, fluo-3). The caffeine-induced changes in the fluorescence spectra are clearly distinct from those of metal ion binding ... More
T-jump study of calcium binding kinetics of calcium chelators.
AuthorsNaraghi M
JournalCell Calcium
PubMed ID9481476
Recent experimental studies have investigated the kinetic competition between calcium chelators and the secretion apparatus at a fast central synapse. Simultaneously, mathematical modelling studies indicate the importance of a quantitative knowledge of the binding kinetics of the chelators in studying fast physiological processes operating on a millisecond time scale. Using ... More
Regulation of intracellular Mg2+ by superoxide in amnion cells.
AuthorsMasumoto N, Tasaka K, Mizuki J, Miyake A, Tanizawa O
JournalBiochem Biophys Res Commun
PubMed ID1310402
Changes of intracellular free Mg2+ concentration ([Mg2+]i) in human amnion cells induced by superoxide anion were determined using a highly Mg(2+)-sensitive fluorescent dye Mg(2+)-fura2 or Mg(2+)-indol. Superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, induced decrease of [Mg2+]i. The decrease was significantly inhibited by an anion channel blocker, ... More
Competition between Li+ and Mg2+ in neuroblastoma SH-SY5Y cells: a fluorescence and 31P NMR study.
AuthorsAmari L, Layden B, Nikolakopoulos J, Rong Q, Mota de Freitas D, Baltazar G, Castro MM, Geraldes CF
JournalBiophys J
PubMed ID10354421
Because Mg2+ and Li+ ions have similar chemical properties, we have hypothesized that Li+/Mg2+ competition for Mg2+ binding sites is the molecular basis for the therapeutic action of lithium in manic-depressive illness. By fluorescence spectroscopy with furaptra-loaded cells, the free intracellular Mg2+ concentration within the intact neuroblastoma cells was found ... More
Calcium dynamics associated with a single action potential in a CNS presynaptic terminal.
AuthorsHelmchen F, Borst JG, Sakmann B
JournalBiophys J
PubMed ID9138591
Calcium dynamics associated with a single action potential were studied quantitatively in the calyx of Held, a large presynaptic terminal in the rat brainstem. Terminals were loaded with different concentrations of high- or low-affinity Ca2+ indicators via patch pipettes. Spatially averaged Ca2+ signals were measured fluorometrically and analyzed on the ... More
Comparison of fluorescence, (31)P NMR, and (7)Li NMR spectroscopic methods for investigating Li(+)/Mg(2+) competition for biomolecules.
AuthorsAmari L, Layden B, Rong Q, Geraldes CF, Mota de Freitas D
JournalAnal Biochem
PubMed ID10405286
The biochemical action of lithium in the treatment of manic-depressive illness is still unknown. One hypothesis is that Li(+) competes for Mg(2+)-binding sites in biomolecules. We report here our studies on metal ion competition by three distinct methods: fluorescence, (31)P NMR, and (7)Li NMR spectroscopy, using ATP as a model ... More
A reevaluation of neuronal zinc measurements: artifacts associated with high intracellular dye concentration.
AuthorsDineley KE, Malaiyandi LM, Reynolds IJ
JournalMol Pharmacol
PubMed ID12181438
The emergence of zinc as a potent neurotoxin has prompted the development of techniques suitable for the measurement of intracellular free zinc ([Zn(2+)](i)) in cultured cells. Accordingly, a new family of Zn(2+)-sensitive fluorophores has become available. Using ionophore-induced elevations of [Zn(2+)](i) in cultured neurons, we measured [Zn(2+)](i)-induced changes in the ... More
Basal intracellular free Mg2+ concentration in smooth muscle cells of guinea pig tenia cecum: intracellular calibration of the fluorescent indicator furaptra.
AuthorsTashiro M, Konishi M
JournalBiophys J
PubMed ID9414246
Longitudinal muscle strips dissected from tenia cecum of guinea pig were loaded with the Mg2+ indicator, furaptra, and the relation between the fluorescent ratio signal (R) and cytoplasmic free Mg2+ concentration ([Mg2+]i) was studied in smooth muscle cells at 25 degrees C. After the application of ionophores (4-bromo-A23187, monensin, and ... More
Intracellular Mg2+ regulation in voltage-clamped Helix aspersa neurones measured with mag-fura-2 and Mg(2+)-sensitive microelectrodes.
AuthorsKennedy HJ
JournalExp Physiol
PubMed ID9717067
The extrusion mechanism for intracellular Mg2+ was investigated in voltage-clamped snail neurones using Mg(2+)-sensitive microelectrodes and mag-fura-2. The intracellular free magnesium ion concentration ([Mg2+]1) of snail neurones voltage clamped to -60 mV was estimated to be 0.57 +/- 0.06 mM (mean +/- S.E.M.; n = 12) using Mg(2+)-sensitive microelectrodes and ... More
Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies.
A method is described to introduce by electroporation membrane-impermeant molecules into adherent living cells with little perturbation. The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrier-based dyes (e.g., fluorescein-labeled dextran), large macromolecules (e.g., antibodies), and metabolic precursors (e.g., 32P-ATP) with high efficiency. Conditions are relatively ... More
Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals.
Authorsvon Gersdorff H, Matthews G
JournalNature
PubMed ID7906397
Communication among neurons occurs at specialized synaptic junctions, where neurotransmitter is released via calcium-dependent exocytosis from the synaptic terminal of the presynaptic cell onto the postsynaptic target neuron. Here we exploit the unique properties of giant synaptic terminals of bipolar neurons from goldfish retina to establish the kinetics and calcium-dependence ... More
Acetylcholine-stimulated changes of membrane potential and intracellular Ca2+ concentration recorded in endothelial cells in situ in the isolated rat aorta.
AuthorsCarter TD, Ogden D
JournalPflugers Arch
PubMed ID7838669
The intracellular free Ca2+ concentration and membrane potential changes evoked by acetylcholine were recorded from whole-cell patch-clamped endothelial cells in situ in the isolated rat aorta. The endothelium had a resting membrane potential of -52 +/- 3 mV (SEM, range -35 mV to -76 mV n = 34) and a ... More
Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers.
AuthorsZhao M, Hollingworth S, Baylor SM
JournalBiophys J
PubMed ID8789107
Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators ... More
The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis.
AuthorsSørensen JB, Matti U, Wei SH, Nehring RB, Voets T, Ashery U, Binz T, Neher E, Rettig J
JournalProc Natl Acad Sci U S A
PubMed ID11830673
Synchronous neurotransmission depends on the tight coupling between Ca(2+) influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. ... More
NIPA1(SPG6), the basis for autosomal dominant form of hereditary spastic paraplegia, encodes a functional Mg2+ transporter.
AuthorsGoytain A, Hines RM, El-Husseini A, Quamme GA
JournalJ Biol Chem
PubMed ID17166836
Mutations in the NIPA1(SPG6) gene, named for "nonimprinted in Prader-Willi/Angelman" has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression ... More
Characterization of a range of fura dyes with two-photon excitation.
AuthorsWokosin DL, Loughrey CM, Smith GL
JournalBiophys J
PubMed ID14990500
Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of ... More
Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2.
AuthorsHurley TW, Ryan MP, Brinck RW
JournalAm J Physiol
PubMed ID1514577
In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are ... More
Magnesium-dependent stimulation of protein synthesis by the insulin mimic, pervanadate.
AuthorsBarnes DM, Sykes DB, Smith JJ, Miller DS
JournalJ Cell Physiol
PubMed ID7542661
The insulin mimic, peroxide of vanadate (pervanadate), stimulated 35S-methionine incorporation into Xenopus oocyte protein in a Mg(2+)-dependent manner. Reducing the extracellular Mg2+ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg2+ or lower, the pervanadate-stimulated component was abolished. In addition, reducing ... More
The effects of sulfur, thiol, and thiol inhibitor compounds on arsine-induced toxicity in the human erythrocyte membrane.
AuthorsRael LT, Ayala-Fierro F, Carter DE
JournalToxicol Sci
PubMed ID10828280
The mechanism of arsine (AsH(3)) toxicity is not completely understood. The first cytotoxic effect of AsH(3) is disruption of ion homeostasis, with a subsequent hemolytic action. The only accepted treatment for AsH(3) toxicity is exchange transfusion of the blood. In this study the effect of sulfur, sulfur compounds, thiol-containing compounds, ... More
Millisecond studies of calcium-dependent exocytosis in pituitary melanotrophs: comparison of the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen.
AuthorsParsons TD, Ellis-Davies GC, Almers W
JournalCell Calcium
PubMed ID8732258
DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca]i) to trigger exocytosis. ... More
Reciprocal changes in intracellular and extracellular magnesium in rat pancreatic acinar cells in response to different secretagogues.
AuthorsGonzález A, Pariente JA, Salido GM, Wisdom D, Singh J
JournalMagnes Res
PubMed ID8845285
This study employs the fluorescent bioprobe Mag-fura-2 to measure the mobilisation of magnesium (Mg2+) in rat pancreatic acinar cells at rest and during stimulation with either cholecystokinin-octapeptide (CCK-8; 10(-8) M), acetylcholine (ACh; 10(-5) M) or histamine (10(-4) M). In Mag-fura-2 loaded acini suspended in normal Krebs-Ringer-Hepes (KRH) solution containing (mMol/litre) ... More
Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra.
AuthorsKonishi M, Hollingworth S, Harkins AB, Baylor SM
JournalJ Gen Physiol
PubMed ID2016581
Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations ... More
Kinetics of Ca2+ release by InsP3 in pig single aortic endothelial cells: evidence for an inhibitory role of cytosolic Ca2+ in regulating hormonally evoked Ca2+ spikes.
AuthorsCarter TD, Ogden D
JournalJ Physiol
PubMed ID9350614
1. The role of the InsP3 receptor and its interaction with Ca2+ in shaping endothelial Ca2+ spikes was investigated by comparing InsP3-evoked intracellular Ca2+ release with hormonally evoked Ca2+ spikes in single endothelial cells. 2. InsP3 was generated by flash photolysis of intracellular caged InsP3. InsP3 at 0.2 microM or ... More
Calcium concentration dynamics produced by synaptic activation of CA1 hippocampal pyramidal cells.
AuthorsRegehr WG, Tank DW
JournalJ Neurosci
PubMed ID1359030
The spatial and temporal dynamics of many electrophysiological and biochemical processes in nerve cells are in turn dependent on the concentration dynamics of the second messenger calcium. We have used microfluorimetry of the calcium indicator fura-2 (Grynkiewicz et al., 1985) to measure and characterize synaptically activated calcium changes in individual ... More
Mechanisms of glutamate-stimulated Mg2+ influx and subsequent Mg2+ efflux in rat forebrain neurones in culture.
AuthorsStout AK, Li-Smerin Y, Johnson JW, Reynolds IJ
JournalJ Physiol
PubMed ID8734978
1. Mag-fura-2 fluorescence microscopy and whole-cell patch-clamp recordings were used to measure glutamate-induced changes in the intracellular free Mg2+ concentration ([Mg2+]i) and Mg2+ currents, respectively, in cultured forebrain neurones from fetal rats in the absence of extracellular Na+ (Nao+) and Ca2+ (Cao2+). 2. Increasing the extracellular Mg2+ concentration ([Mg2+]o) from ... More
RNA catalysis in model protocell vesicles.
AuthorsChen IA, Salehi-Ashtiani K, Szostak JW
JournalJ Am Chem Soc
PubMed ID16173749
We are engaged in a long-term effort to synthesize chemical systems capable of Darwinian evolution, based on the encapsulation of self-replicating nucleic acids in self-replicating membrane vesicles. Here, we address the issue of the compatibility of these two replicating systems. Fatty acids form vesicles that are able to grow and ... More
Metal-binding affinity of the transmembrane site in ZntA: implications for metal selectivity.
AuthorsLiu J, Dutta SJ, Stemmler AJ, Mitra B
JournalBiochemistry
PubMed ID16411752
ZntA, a P1B-type ATPase, confers resistance specifically to Pb2+, Zn2+, and Cd2 in Escherichia coli. Inductively coupled plasma mass spectrometry measurements show that ZntA binds two metal ions with high affinity, one in the N-terminal domain and another in the transmembrane domain. Both sites can bind monovalent and divalent metal ... More
Characterizing the response of calcium signal transducers to generated calcium transients.
AuthorsDavis JP, Tikunova SB, Walsh MP, Johnson JD
JournalBiochemistry
PubMed ID10194340
Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological phenomena as diverse as muscle contraction, neurosecretion, and cell division. When Ca2+ is rapidly mixed with slow Ca2+ chelators, EGTA, or Mg2+/EDTA, artificial Ca2+ transients (ACTs) of varying duration (0.1-50 ms half-widths (hws)) and amplitude can be generated. We have exposed several ... More
Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: effect on metal selectivity and activity.
AuthorsDutta SJ, Liu J, Stemmler AJ, Mitra B
JournalBiochemistry
PubMed ID17326661
ZntA from Escherichia coli belongs to the P1B-ATPase transporter family and mediates resistance to toxic levels of selected divalent metal ions. P1B-type ATPases can be divided into subgroups based on substrate cation selectivity. ZntA has the highest selectivity for Pb2+, followed by Zn2+ and Cd2+; it also shows low levels ... More
Dendritic Ca2+ accumulations and metabotropic glutamate receptor activation associated with an N-methyl-D-aspartate receptor-independent long-term potentiation in hippocampal CA1 neurons.
AuthorsPetrozzino JJ, Connor JA
JournalHippocampus
PubMed ID7889125
Bathing hippocampal slices in the potassium channel blocker tetraethylammonium (TEA), while stimulating the Schaffer collaterals at a low frequency, induces Ca(2+)-dependent, N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation of synaptic transmission (LTPk) in CA1 neurons. We have combined ratio imaging of fura-2 and mag-fura-5 in hippocampal CA1 neurons with intracellular and field ... More
Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming.