The effect of pH on rate constants, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators

doi:10.1016/0006-291X(91)91966-G

Biochemical and Biophysical Research Communications

Volume 177, Issue 1, 31 May 1991, Pages 184-191

https://doi.org/10.1016/0006-291X(91)91966-G Get rights and content

Abstract

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 in fura-2 and indo-1 at acidic pH. Alterations in apparent dissociation constants were caused primarily by reduced association rates. Magnesium indicators did not show these changes. The enthalphies of the calcium indicators' Ca complex were 1–3 kcal/mole and magnesium indicators' Mg complex were 7–9 kcal/mole. The potential effects of a biexponential dissociation rate of fluo-3 and of Ca interactions with magnesium indicators were examined.

References (15)

L.A. Blatter et al.
Biophysical J
(1990)
M. Konishi et al.
Biophysical J
(1988)
F. Lattanzio
Biochem. Biophys. Res. Comm
(1990)
J.P.Y. Kao et al.
Biophysical J
(1988)
G. Grynkiewicz et al.
J. Biol. Chem
(1985)
T.R. Hesketh et al.
J. Biol. Chem
(1983)
M. Eberhard et al.
Biochem. Biophys. Res. Comm
(1989)

There are more references available in the full text version of this article.

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2021, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
The in situ Kd may also be extrapolated among laboratories because it mostly depends on the properties of the dye inside the cytoplasmic environment, not on the properties of the measurement devices. Finally, considering our thermodynamic results, and those previously reported [37], it seems that the changes in the reaction enthalpies and entropies of several families of Ca2+ indicators are similar, suggesting a common mechanism of reaction involving changes in the hydration status (i.e. release of water molecules) of the dye and the metal ion upon binding. Determining the precise cytoplasmic [Ca2+] have implications for our understanding of many physiological phenomena, as well as in the development and evaluation of new therapies, for conditions that involve alterations in cells specialized in mobilizing Ca2+, such as skeletal, cardiac, or smooth muscle, neurons or endocrine.
Mag-Fluo-4 is increasingly employed for studying Ca²⁺ signaling in skeletal muscle; however, the lack of information on the Ca²⁺-Mag-Fluo-4 reaction limits its wider usage.
Fluorescence and isothermal titration calorimetry (ITC) experiments were performed to determine the binding stoichiometry (n) and thermodynamics (enthalpy (ΔH) and entropy (ΔS) changes), as well as the in vitro and in situ K_d of the Ca²⁺-Mag-Fluo-4 reaction. Rate constants (k_on, k_off), fluorescence maximum (F_max), minimum (F_min), and the dye compartmentalization were also estimated. Experiments in cells used enzymatically dissociated flexor digitorum brevis fibres of C57BL6, adult mice, loaded at room temperature for 8 min, with 6 μM Mag-Fluo-4, AM, and permeabilized with saponin or ionomycin. All measurements were done at 20 °C.
The in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca²⁺/Mag-Fluo-4 (n = 5) reaction. ITC results (n = 3) provided ΔH and ΔS values of 2.3 (0.7) kJ/mol and 97.8 (5.9) J/mol.K, respectively. The in situ K_d was 1.652 × 10⁵μM²(n = 58 fibres, R² = 0.99). With an F_max of 150.9 (8.8) A.U. (n = 8), F_min of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca²⁺ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca²⁺]_peak reached 22.5 (7.8) μM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10).
CONCLUSIONS: Two Mag-Fluo-4 molecules coalesce around one Ca²⁺ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca²⁺ transients.
Our results may be relevant to the quantitative study of Ca²⁺ kinetics in many other cell types.
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Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague–Dawley rats were established. P2Y₂ receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Ca²⁺, whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y₂R-PLC-IP3 pathway to evoke Ca²⁺ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca²⁺]_I; this effect was partly inhibited by P2Y₂R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca²⁺]_i was significantly lower than that after treatment with P2Y₂R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca²⁺ triggers Ca²⁺ release from ER Ca²⁺ stores. Baseline [Ca²⁺]_i declined after treatment with the Ca²⁺ chelator 50 μM bis-(aminophenolxy) ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca²⁺ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca²⁺ triggers Ca²⁺ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca²⁺ signals indirectly via the P2Y₂R-PLC-IP3 pathway.
Measuring Ca2 + inside intracellular organelles with luminescent and fluorescent aequorin-based sensors
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GFP-Aequorin Protein (GAP) can be used to measure [Ca^2 +] inside intracellular organelles, both by luminescence and by fluorescence. The low-affinity variant GAP3 is adequate for ratiometric imaging in the endoplasmic reticulum and Golgi apparatus, and it can be combined with conventional synthetic indicators for simultaneous measurements of cytosolic Ca^2 +. GAP is bioorthogonal as it does not have mammalian homologues, and it is robust and functionally expressed in transgenic flies and mice, where it can be used for Ca^2 + measurements ex vivo and in vivo to explore animal models of health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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2016, Trends in Biochemical Sciences
Citation Excerpt :
Besides the existence and specificity of a Ca2+ endolysosome release factor, the release of Ca2+ by lysosomes requires high [Ca2+] in the organelle. The measurement of Ca2+ inside acidic stores has been challenging because many fluorescent probes for measuring calcium are sensitive to pH [94]. pH-corrected measurements showed that, in macrophages, lysosomes contain high [Ca2+], ranging from 400 to 600 μM and are highly sensitive to pH [91,95,96], although these measurements are far from being conclusive.
In recent years, rapid discoveries have been made relating to Ca²⁺ handling at specific organelles that have important implications for whole-cell Ca²⁺ homeostasis. In particular, the structures of the endoplasmic reticulum (ER) Ca²⁺ channels revealed by electron cryomicroscopy (cryo-EM), continuous updates on the structure, regulation, and role of the mitochondrial calcium uniporter (MCU) complex, and the analysis of lysosomal Ca²⁺ signaling are milestones on the route towards a deeper comprehension of the complexity of global Ca²⁺ signaling. In this review we summarize recent discoveries on the regulation of interorganellar Ca²⁺ homeostasis and its role in pathophysiology.
Activity-dependent gating of calcium spikes by A-type K+ channels controls climbing fiber signaling in purkinje cell dendrites
2014, Neuron
Citation Excerpt :
Electrophysiological techniques fail to provide accurate measure of the time course of fast regenerative events in dendrites, due to filtering and dampening by leak, pipette access resistance, and capacitive load. The temporal resolution of optical recordings of calcium transients is defined by the time constant of calcium binding to the dye, which is approximately 2 μs for 500 μM Fluo5F, assuming a kon of 109 M−1 s−1 (Lattanzio and Bartschat, 1991). The stimulus-evoked change in fluorescence is linearly related to the cumulative Ca influx up to the dye concentration (Higley and Sabatini, 2008).
In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules.
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Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca²⁺) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca²⁺ probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca²⁺ probes. In response to this demand more red-emitting chemical and FP-based Ca²⁺-sensitive indicators have been developed since 2009 than in the thirty years before.
In this topical review, we survey the physicochemical properties of these red-emitting Ca²⁺ probes and discuss their utility for biological Ca²⁺ imaging. Using the spectral separability index X_ijk (Oheim M., 2010. Methods in Molecular Biology 591: 3–16) we evaluate their performance for multi-color excitation/emission experiments, involving the identification of morphological landmarks with GFP/YFP and detecting Ca²⁺-dependent fluorescence in the red spectral band. We also establish a catalog of criteria for evaluating Ca²⁺ indicators that ideally should be made available for each probe. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

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^☆: This research was supported by grants from the Eastern Virginia Medical School and NIH grant NS 21758 (DKB).

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Biochemical and Biophysical Research Communications

Abstract

References (15)

Biophysical J

Biophysical J

Biochem. Biophys. Res. Comm

Biophysical J

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Comm

Cited by (140)

Calibration of mammalian skeletal muscle Ca<sup>2+</sup> transients recorded with the fast Ca<sup>2+</sup> dye Mag-Fluo-4

Lysosomal exocytosis of ATP is coupled to P2Y <inf>2</inf> receptor in marginal cells in the stria vascular in neonatal rats

Measuring Ca<sup>2 +</sup> inside intracellular organelles with luminescent and fluorescent aequorin-based sensors

Calcium at the Center of Cell Signaling: Interplay between Endoplasmic Reticulum, Mitochondria, and Lysosomes

Activity-dependent gating of calcium spikes by A-type K+ channels controls climbing fiber signaling in purkinje cell dendrites

New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging

The effect of pH on rate constants, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators☆

Abstract

Biophysical J

Biophysical J

Biochem. Biophys. Res. Comm

Biophysical J

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Comm