The development of a new family of intracellular calcium probes

doi:10.1016/0143-4160(94)90058-2

Cell Calcium

Volume 15, Issue 2, February 1994, Pages 190-198

https://doi.org/10.1016/0143-4160(94)90058-2 Get rights and content

Abstract

Four new potential fluorescent probes for Ca²⁺ using the coumarin moiety as a fluorophore have been synthesized and their spectral properties and binding affinities for Ca²⁺ determined. The most promising derivative for intracellular use, BTC, exhibits an excitation wavelength shift from 462 nm to 401 nm on binding Ca²⁺, with an emission wavelength of 530 nm. The quantum yield of this probe increases from 0.07 as the free indicator to 0.12 on binding Ca²⁺. BTC, loaded as its tetraacetoxymethyl ester (AM ester) into mouse myeloma P3X cells, responded only when cytoplasmic Ca²⁺ exceeded typical intracellular calcium concentrations. The dye, therefore, appears to be useful in excitatory cells or extracellular spaces with intracellular calcium concentrations high enough to saturate typical excitation ratio Ca²⁺ indicators such as Fura-2.

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However, in contrast to the vehicle control (DMSO), both Mtb extract and capsaicin triggered a significant increase in intracellular [Ca2+] (Figures 2A and 2B). While Fluo-4 allows for a semiquantitative determination of intracellular [Ca2+], the ratiometric dye Fura-2 allows quantification of intracellular [Ca2+] by determining the ratio of Fura-2 excitation at 340 nm and 380 nM (Grynkiewicz et al., 1985; Iatridou et al., 1994; Kao et al., 2010). Thus, to confirm our results with Fluo-4, we exposed Fura-2-loaded MED17.11 neurons to DMSO, the Mtb extract, and capsaicin and monitored intracellular [Ca2+] over time for individual cells.
Pulmonary tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb), manifests with a persistent cough as both a primary symptom and mechanism of transmission. The cough reflex can be triggered by nociceptive neurons innervating the lungs, and some bacteria produce neuron-targeting molecules. However, how pulmonary Mtb infection causes cough remains undefined, and whether Mtb produces a neuron-activating, cough-inducing molecule is unknown. Here, we show that an Mtb organic extract activates nociceptive neurons in vitro and identify the Mtb glycolipid sulfolipid-1 (SL-1) as the nociceptive molecule. Mtb organic extracts from mutants lacking SL-1 synthesis cannot activate neurons in vitro or induce cough in a guinea pig model. Finally, Mtb-infected guinea pigs cough in a manner dependent on SL-1 synthesis. Thus, we demonstrate a heretofore unknown molecular mechanism for cough induction by a virulent human pathogen via its production of a complex lipid.
Synthesis and properties of Asante Calcium Red-A novel family of long excitation wavelength calcium indicators
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Citation Excerpt :
Since the development of the first modern UV-excitable indicators, fura-2 and indo-1 [4] over a quarter century ago, a number of new indicators with different excitation/emission wavelengths and distinct affinities for calcium, have been synthesized [5–10]. While some of them including fura-2 [4] and fluo-3 [5], enjoy undying popularity, others like BTC [6] and Calcium Crimson [11] have attracted only a limited and short-lived interest. The analysis of their features and almost three decades of experience allows for the creation of a “perfect” fluorescent indicator profile.
Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca²⁺ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca²⁺-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca²⁺]_i measurement in GFP expressing cells. The indicators bind Ca²⁺ with either high (K_d = 0.49 ± 0.07 μM; ACR-1) or low affinity (K_d = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn²⁺ (K_d = 0.38 ± 0.02 nM) or Mg²⁺ (K_d ∼ 5 mM) slightly raises and binding Co²⁺ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pK_a = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.
Novel Ca<sup>2+</sup>-selective merocyanine-type chromoionophore derived from calix[4]arene-diamide
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The synthesis and calcium-selective chromogenic ion binding properties of benzothiazolium derivative of calix[4]arene are described. The merocyanine-type ionophore derived from calix[4]arene-diamide showed selective chromogenic properties toward Ca²⁺ ions in aqueous methanol. The compound showed significant Ca²⁺ ion dependent changes in UV–vis spectral properties in the presence of other physiologically important metal ions (Na⁺, K⁺, and Mg²⁺). The pH sensitive chromogenic behavior also suggests that the compound can be used as a sensitive pH indicator around pH 6.5.
Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2
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The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K_d values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP–Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.
Fluorescence-sensing methods
2003, Methods in Enzymology
Novel approaches to sensor design, based on the use of an internal standard with appropriate spectral properties, provide new possibilities for designing simple devices for fluorescence sensing. Detection of combined emission from the reference and an analyte-sensitive fluorophore has been achieved in numerous measurements in cuvettes,^7–10,42,43 tissues,^46,47 and high-throughput formats.⁴⁸ These methods have been used with a long-lifetime reference to measure pH, O₂, pCO₂, glucose, and calcium^8,9 by means of modulation-sensing methods as well as by the use of oriented films as the reference for polarization sensing of glucose, pH, oxygen, and lactate.^21–23 Polarization sensing has also been developed with visual detection to measure the concentration of rhodamine B and pH.²⁴
Modulation and polarization sensing was found to be effective in highly scattering media such as Intralipid or tissue. The applicability of these technologies to transdermal diagnostics depends on the availability of red fluorophores that can be used in vivo. One dye that could possibly be used is indocyanine green (IcG), which absorbs and emits at wavelengths above 700 nm. Furthermore, IcG has already been approved for use in humans for monitoring burn severity⁴⁹ and it has been detected through the skin.⁵⁰
It appears likely that modern optics and electronic technology will allow the development of practical devices for biomedical use as shown in Scheme 1.
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Two new potential near-membrane iminocoumarin-based fluorescent Ca²⁺ indicators were synthesized and the spectral profiles of their free and Ca²⁺ bound forms were studied. The probes incorporate in their BAPTA-related structures, the 3-(benzimidazolyl)iminocoumarin or the 3-(benzothiazolyl)iminocoumarin moiety, substituted at the imino nitrogen with an n-dodecyl lipophilic chain. The compounds are excited with visible light and have Ca²⁺ dissociation constant values of 5.50 and 4.49 μM, respectively, the highest reported to date in the literature. Fluorescence spectra studies 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 ratiometric near-membrane, low Ca²⁺ affinity probes.

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The development of a new family of intracellular calcium probes

Abstract

Progr. Biophys. Mol. Biol.

J. Biol. Chem.

Cell Calcium

Trends Neurosci.

J. Biol. Chem.

Calcium action in synaptic transmitter release

Annu. Rev. Neurosci.

Measurement of free Ca2+ in cytoplasm

A non disruptive technique for loading calcium buffers and indicators into cells

Nature

Measurement of cytosolic free Ca2+ with quin-2: Practical aspects

Meth. Enzymol.

Measurement of free Ca²⁺ in cytoplasm

Measurement of cytosolic free Ca²⁺ with quin-2: Practical aspects