Presynaptic Ca(2+) influx at the inhibitor of the crayfish neuromuscular junction: a photometric study at a high time resolution.
AuthorsVyshedskiy A, Lin JW
JournalJ Neurophysiol
PubMed ID10634895
'Presynaptic calcium influx at the inhibitor of the crayfish neuromuscular junction was investigated by measuring fluorescence transients generated by calcium-sensitive dyes. This approach allowed us to correlate presynaptic calcium influx with transmitter release at a high time resolution. Systematic testing of the calcium indicators showed that only low-affinity dyes, with ... More
Ensemble and single-molecule fluorescence spectroscopy of a calcium-ion indicator dye.
AuthorsBagh S, Paige MF,
JournalJ Phys Chem A
PubMed ID16737253
'The spectroscopic properties of Calcium Green 2 (CG-2), a dual-fluorophore Ca(2+) indicator dye, were characterized by a combination of steady state and time-resolved ensemble spectroscopic measurements, molecular mechanics calculations and single-molecule fluorescence spectroscopy. It was found that in Ca(2+) free solutions, CG-2 exists primarily as a highly quenched intramolecular dimer, ... More
Localized sampling of cytoplasm from Xenopus oocytes for capillary electrophoresis.
AuthorsLuzzi V, Lee CL, Allbritton NL
JournalAnal Chem
PubMed ID9406526
'Continued progress in cellular physiology requires new measurement strategies which can be applied to solitary cells. Since many cellular signaling pathways act on time scales of a few seconds, there is a critical need for single-cell techniques with subsecond time resolution. Capillary electrophoresis shows great promise as a tool for ... More
Ca(2+) measurements in skinned cardiac fibers: effects of Mg(2+) on Ca(2+) activation of force and fiber ATPase.
AuthorsAllen K, Xu YY, Kerrick WG
JournalJ Appl Physiol
PubMed ID10642379
'In contrast to previous studies, a new fluorescent method was used to accurately determine the Ca(2+) concentration in test solutions used to activate skinned rat cardiac cells. This method used the calcium green-2 fluorescent indicator, which is shown to change its fluorescence over the Ca(2+) range responsible for Ca(2+) activation ... More
Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.
AuthorsEscobar AL, Velez P, Kim AM, Cifuentes F, Fill M, Vergara JL
JournalPflugers Arch
PubMed ID9242727
'We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca2+ indicators in response to large (50-200 microM), short duration (< 100 ns), free [Ca2+] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca2+). The equilibrium and kinetic ... More
Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate.
AuthorsParker I, Yao Y, Ilyin V
JournalBiophys J
PubMed ID8770200
'Inositol 1,4,5-trisphosphate (InsP3) acts on intracellular receptors to cause liberation of Ca2+ ions into the cytosol as repetitive spikes and propagating waves. We studied the processes underlying this regenerative release of Ca2+ by monitoring with high resolution the kinetics of Ca2+ flux evoked in Xenopus oocytes by flash photolysis of ... More
A method for recording intracellular [Ca2+] transients in cardiac myocytes using calcium green-2.
AuthorsSpencer CI, Berlin JR
JournalPflugers Arch
PubMed ID7491285
Calcium green-2 (Ca green) is a non-ratiometric fluorescent Ca2+ indicator with an affinity for Ca2+ (dissociation constant Kd = 3 microM) that is lower than more commonly used indicators such as fura-2 and fluo-3. This low Ca2+ affinity, coupled with a high quantum yield, allows cells to be loaded with ... More
An ensemble and single-molecule fluorescence spectroscopy investigation of Calcium Green 1, a calcium-ion sensor.
AuthorsLu Y, Paige MF,
JournalJ Fluoresc
PubMed ID17638063
The calcium-ion indicator dye, Calcium Green 1 (CG-1), has been characterized using a combination of ensemble and single-molecule optical spectroscopy measurements. In terms of ensemble measurements, CG-1 demonstrated a strong increase in fluorescence emission as a function of increasing [Ca(2+)]. This was accompanied by a change in the relative proportions ... More
Impact of mitochondrial Ca2+ cycling on pattern formation and stability.
AuthorsFalcke M, Hudson JL, Camacho P, Lechleiter JD
JournalBiophys J
PubMed ID10388738
Energization of mitochondria significantly alters the pattern of Ca2+ wave activity mediated by activation of the inositol (1,4,5) trisphosphate (IP3) receptor (IP3R) in Xenopus oocytes. The number of pulsatile foci is reduced and spiral Ca2+ waves are no longer observed. Rather, target patterns of Ca2+ release predominate, and when fragmented, ... More
Confocal imaging of calcium microdomains and calcium extrusion in turtle hair cells.
AuthorsTucker T, Fettiplace R
JournalNeuron
PubMed ID8845156
We have studied spatial Ca2+ distribution in hair cells filled with the low affinity fluorescent indicator Calcium Green 5N using real-time confocal microscopy and whole-cell recording. During depolarizations lasting several hundred milliseconds, Ca2+ fluorescence increased at a number of hotspots around the base of the cell but changed little near ... More
Confocal imaging and local photolysis of caged compounds: dual probes of synaptic function.
AuthorsWang SS, Augustine GJ
JournalNeuron
PubMed ID7576625
Chemical signals generated at synapses are highly limited in both spatial range and time course, so that experiments studying such signals must measure and manipulate them in both these dimensions. We describe an optical system that combines confocal laser scanning microscopy, to measure such signals, with focal photolysis of caged ... More
Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.
AuthorsKubitscheck U, Pratsch L, Passow H, Peters R
JournalBiophys J
PubMed ID7669907
The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ... More
Two dye two wavelength excitation calcium imaging: results from bovine adrenal chromaffin cells.
AuthorsOheim M, Naraghi M, Müller TH, Neher E
JournalCell Calcium
PubMed ID9793690
We tested a mixture of Calcium-Green-1 (CG-1) and Brilliantsulfaflavine (BS) for dual excitation ratiometric measurements of the intracellular free calcium concentration ([Ca2+]i) in bovine adrenal chromaffin cells. Dyes were coloaded (without being molecularly linked to each other) in the whole-cell configuration of the patch clamp technique. We compared the loading ... More
Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.
AuthorsMiller T, Szczesna D, Housmans PR, Zhao J, de Freitas F, Gomes AV, Culbreath L, McCue J, Wang Y, Xu Y, Kerrick WG, Potter JD
JournalJ Biol Chem
PubMed ID11060294
This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine alpha-myosin heavy chain promoter. Extensive ... More
Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.
AuthorsIsenberg G, Etter EF, Wendt-Gallitelli MF, Schiefer A, Carrington WA, Tuft RA, Fay FS
JournalProc Natl Acad Sci U S A
PubMed ID8643589
Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that ... 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
High Throughput Screening with Multiphoton Excitation.
AuthorsLakowicz JR, Gryczynski I, Gryczynski Z
JournalJ Biomol Screen
PubMed ID10838432
Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons ... More
Microdomain Ca2+ activation during exocytosis in Paramecium cells. Superposition of local subplasmalemmal calcium store activation by local Ca2+ influx.
AuthorsErxleben C, Klauke N, Flötenmeyer M, Blanchard MP, Braun C, Plattner H
JournalJ Cell Biol
PubMed ID9024690
In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben. C., and H. Plattner. 1994. J. Cell Biol. 127:935-945). We now show by voltage clamp and extracellular recordings that the product of current x time (As) closely parallels the number of exocytotic ... More
Stimulation-induced changes in [Ca2+] in lizard motor nerve terminals.
AuthorsDavid G, Barrett JN, Barrett EF
JournalJ Physiol
PubMed ID9350620
1. Motor axons were injected ionophoretically with one of five Ca(2+)-sensitive dyes (fluo-3, Calcium Green-2, Calcium Green-5N, fluo-3FF and Oregon Green BAPTA-5N). Changes in fluorescence (delta F/Frest) within motor terminal boutons following a single action potential and brief stimulus trains were monitored with high temporal resolution using a confocal microscope. ... More
Calcium transients in cerebellar granule cell presynaptic terminals.
AuthorsRegehr WG, Atluri PP
JournalBiophys J
PubMed ID7612860
Calcium ions act presynaptically to modulate synaptic strength and to trigger neurotransmitter release. Here we detect stimulus-evoked changes in residual free calcium ([Ca2+]i) in rat cerebellar granule cell presynaptic terminals. Granule cell axons, known as parallel fibers, and their associated boutons, were labeled with several calcium indicators. When parallel fibers ... More
Synaptic differentiation of a single motor neuron: conjoint definition of transmitter release, presynaptic calcium signals, and ultrastructure.
AuthorsCooper RL, Marin L, Atwood HL
JournalJ Neurosci
PubMed ID7790906
The opener muscle in the walking legs of the crayfish (Procambarus clarkii) is innervated by only one excitatory motor neuron, yet excitatory postsynaptic potentials (EPSPs) of proximal fibers are eightfold larger than those of central muscle fibers at low frequencies of activation, due in large measure to differences in presynaptic ... More
The endoplasmic reticulum in PC12 cells. Evidence for a mosaic of domains differently specialized in Ca2+ handling.
AuthorsRooney E, Meldolesi J
JournalJ Biol Chem
PubMed ID8910591
Velocity and isopycnic gradient centrifugation were employed to fractionate post-nuclear supernatants rapidly prepared from PC12 cells in order to characterize areas of the endoplasmic reticulum involved in various aspects of intracellular Ca2+ homeostasis. The endoplasmic reticulum Ca2+ pumping activity, defined by three properties studied in parallel in the isolated fractions; ... More