A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells

Localized subcellular changes in Ca²⁺ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca²⁺ signaling have been greatly facilitated by the availability of fluorescent Ca²⁺ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca²⁺ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca²⁺ signals is lacking. Here, we evaluated several fluorescent Ca²⁺ indicators in the context of local Ca²⁺ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP₃) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca²⁺ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca²⁺-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca²⁺ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca²⁺ signals.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.

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 was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

The slowing of Ca2+ signals by Ca2+ indicators in cardiac muscle.

The use of high-affinity fluorescent probes for monitoring intracellular free Ca2+ in cardiac muscle is now widespread. We have investigated the consequences of introducing intracellular buffers with the properties of Fura-2 or Indo-1 on the action potential, Ca2+ transient and contractile activity of the myocardium. Our theoretical results suggest that, at the high intracellular concentrations of these fluorescent probes used on occasion to improve the signal-to-noise ratio of the emitted fluorescence, modulation of action potential profile and attenuation of the amplitudes of the Ca2+ transient and contraction can occur, together with subtle changes in the kinetics of these events.     

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading.     

A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes

The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.     

Internalization of metallochromic Ca2+ indicators in mammalian cells

Two new techniques for internalizing metallochromic indicators into the cytosol of mammalian cells are described. One method consists of hypertonically treating the cells in the presence of the indicator, followed by a hypoosmotic treatment. The second method consists of incubating the cells at high density in a concentrated indicator solution in physiological saline. Using either method, arsenazo III or antipyrylazo III was internalized into Ehrlich Ascites tumor (EAT) cells at concentrations yielding measurable differential absorbance changes which correspond to changes in the intracellular Ca2+ concentration. In the case of antipyrylazo III, the amount of indicator internalized ranged between 140 and 350 microM, and was dependent on the metabolic state of the cell during loading. Control and loaded cells possessed virtually identical ATP/ADP ratios, as measured by high performance liquid chromatography (HPLC) in cell extracts. Antipyrylazo III was also internalized by rat hepatocytes without detectable cell damage. Treatment of metabolically active EAT cells with the calcium ionophore A23187 results in only a slight increase in the intracellular free Ca2+ concentration, [Ca2+]i, whereas treatment with the calcium ionophore ionomycin induces a substantial but transient increase in the [Ca2+]i. In contrast, metabolically inhibited EAT cells show a large rise in the [Ca2+]i upon addition of A23187. Thus, these techniques offer another way of measuring intracellular free Ca2+ changes in mammalian cells and may prove useful, especially where concentrations of free cytosolic Ca2+ larger than 1 microM are expected.     

Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.

Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities to Ca2+ concentration. The relative intensity of Ca2+-triggered luminescence of the photoproteins ranged from 0.01 to 190 when compared with natural aequorin (relative intensity 1.0) at pCa 6 for the cases where the relative intensity is less than 1 and at pCa 7 for the cases where the relative intensity is higher than 1. Eight of the semi-synthetic aequorins belonged to the class of e-aequorin. With two of those photoproteins, the degree of dependence of the luminescence intensity ratio I400/I465 on pCa was greater than that with e-aequorin, suggesting that these two photoproteins are possibly superior to e-aequorin in measuring Ca2+ concentration by the ratio method.     

Structural properties of sarcoplasmic reticulum Ca2+-ATPase as studied by intrinsic protein fluorescence

1. From the intrinsic fluorescence spectral properties and fluorescence quenching experiments done with acrylamide and iodide, using native sarcoplasmic reticulum vesicles, purified ATPase and ATPase solubilized with 1% Triton X-100, it is deduced that practically all the fluorescent tryptophanyl residues of this protein belong to a single population showing similar hydrophobic microenvironments. 2. Both acrylamide and iodide seem to be able to penetrate through the sarcoplasmic reticulum membrane. 3. The intrinsic fluorescence of the Ca2+-ATPase due to tryptophan residues probably buried inside the membrane is used as a tool to follow thermotropic changes in membrane fluidity of reconstituted systems.     

X537A:A Ca2+ ionophore with a polarity-dependent and complexation-dependent fluorescence signal

Summary The Ca²⁺ ionophore X537 A has the characteristics required of a hydrophobic fluorescent probe. The quantum yield of the uncomplexed anion varies from 0.021 in water to 1.0 in dioxane, increasing with decreasing solvent polarity. Complexation with K⁺, Ca²⁺ or Ba²⁺ serves to increase the fluorescence signal insolvents of high polarity. In solvents of low polarity decreases in fluorescence upon complexation have been found. The ionophore X537A^– binds to dimyristoyl--lecithin membranes with a quantum yield of 0.4, and evidence is given that the ionophore is situated on the membrane surface. The fluorescent signal of X537A^– on the membrane increases with cation complexation and values are reported for the complexation constants of X537A^– with several monovalent and divalent cations on the membrane. The use of the fluorescent signal of X537A in the study of the mechanism of cation transport facilitated by this ionophore is discussed.     

Iminocoumarin-based low affinity fluorescent Ca2+ indicators excited with visible light.

A series of iminocoumarin-based fluorescent Ca2+ indicators were synthesized and the spectral profiles of their free and Ca2+ bound forms were studied. The newly-synthesized compounds incorporate the Ca2+ chelating structure of BAPTA. The chromophore moieties are iminocoumarins substituted at the 3-position with benzothiazolyl, benzoxazolyl and benzimidazolyl groups. These compounds are excited with visible light and their Ca2+ dissociation constants range from 5.4 to 27.5 microM. Fluorescence spectra studies of these probes indicated a clear shift in their excitation wavelength maxima upon Ca2+ binding along with changes in fluorescence intensity that enable the compounds to be used as low Ca2+ affinity, visible excitable probes.     

Free cytosolic Ca2+ measurements with fluorine labelled indicators using 19FNMR

Characterisation by 19F NMR of fluorine-labelled indicators of cytosolic free Ca2+ concentration (by 5FBAPTA) and pH (by Fquene) is described, together with the techniques used to load the cell suspensions with the indicators for NMR spectroscopy. Useful features of the 19F NMR indicators include direct identification of the intracellular cation bound to the indicators, internal calibration of [Ca]i and pHi from the spectra, and simultaneous measurements of two or more indicators in the same cell suspension. Perturbations of cellular functions by 5FBAPTA and quin 2 are very similar, but vary widely in different cell systems. The [Ca]i and pHi responses of normal and transformed cells to mitogens and growth factors in serum are compared with data from similar experiments using fluorescence indicators. The only major discrepancy in [Ca]i measurements using the two independent assays was observed in Ehrlich ascites tumour cells. These cells have a high intracellular Zn2+ content which substantially quenches the quin 2 fluorescence, but does not affect [Ca]i measurements by 5FBAPTA. The Zn2+ present in the cells is detected as a separate response in the 5FBAPTA spectrum. The time course of the Ca signal in 2H3 cells stimulated by antigen to release histamine by exocytosis has been defined using 5FBAPTA and quin 2. Extension of the 19F NMR technique to [Ca] i and pHi measurements in perfused organs is illustrated in rat heart and responses to pharmacological agents are demonstrated. Developments in prospect to improve sensitivity and to measure [Na]i with a new family of indicators are outlined.     

Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1

Cells of Saccharomyces cerevisiae were loaded with indo-1, by incubation in a medium of pH 4.5, which contained penta-potassium indo-1. Cells were then washed and resuspended in a buffer of pH 4.0. The emission fluorescence spectra were recorded between 390 and 500 nm (excitation at 355 nm) and the autofluorescent spectra of the matched controls were subtracted. A 19-fold cellular accumulation of indo-1 was achieved. By permeabilization of plasma membranes, leaving the vacuolar membrane intact, it was proved that indo-1 was accumulated in the cytosol. It was also shown that intracellular indo-1 did not leak out of the cells and was not modified by cellular metabolism. Using the emission fluorescence ratio at 410/480 nm, the concentration of a free cytosolic Ca2+ was found to be 346 nM. Vacuolar Ca2+ concentration, calculated from indo-1 fluorescence after lysis of vacuolar and cellular membranes, was found to be 1.3 mM.     

Ca2+ regulation of thyroid NADPH-dependent H2O2 generation

A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s).