Synthesis and properties of Asante Calcium Red-A novel family of long excitation wavelength calcium indicators

Krzysztof L. Hyrc; Akwasi Minta; P. Rogelio Escamilla; Patrick P.L. Chan; Xenia A. Meshik; Mark P. Goldberg

doi:10.1016/j.ceca.2013.08.001

Synthesis and properties of Asante Calcium Red-A novel family of long excitation wavelength calcium indicators

Krzysztof L. Hyrc, Akwasi Minta, P. Rogelio Escamilla, Patrick P.L. Chan, Xenia A. Meshik, Mark P. Goldberg

Research output: Contribution to journal › Article › peer-review

12 Scopus citations

Abstract

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 (450-540nm) excitation with deep-red fluorescence (640nm). 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 (640nm) and quasi-independent (530nm) emission when using visible (<490nm) or multiphoton (~780nm) 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±0.07μM; ACR-1) or low affinity (K_d=6.65±0.13μM; ACR-1-LA). Chelating Zn²⁺ (K_d=0.38±0.02nM) or Mg²⁺ (K_d~5mM) 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.

Original language	English
Pages (from-to)	320-333
Number of pages	14
Journal	Cell Calcium
Volume	54
Issue number	4
DOIs	https://doi.org/10.1016/j.ceca.2013.08.001
State	Published - Oct 2013

Keywords

Confocal microscopy
Dissociation constant
Emission spectra
Excitation spectra
Fluorescence
Intracellular free calcium concentration
Magnesium ions
Multiphoton microscopy
Neurons
Ratio imaging
Zinc ions

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10.1016/j.ceca.2013.08.001

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@article{88269e8f914f4b99970917b66558eb55,

title = "Synthesis and properties of Asante Calcium Red-A novel family of long excitation wavelength calcium indicators",

abstract = "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 (450-540nm) excitation with deep-red fluorescence (640nm). 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 (640nm) and quasi-independent (530nm) emission when using visible (<490nm) or multiphoton (~780nm) 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 (Kd=0.49±0.07μM; ACR-1) or low affinity (Kd=6.65±0.13μM; ACR-1-LA). Chelating Zn2+ (Kd=0.38±0.02nM) or Mg2+ (Kd~5mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa=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.",

keywords = "Confocal microscopy, Dissociation constant, Emission spectra, Excitation spectra, Fluorescence, Intracellular free calcium concentration, Magnesium ions, Multiphoton microscopy, Neurons, Ratio imaging, Zinc ions",

author = "Hyrc, {Krzysztof L.} and Akwasi Minta and Escamilla, {P. Rogelio} and Chan, {Patrick P.L.} and Meshik, {Xenia A.} and Goldberg, {Mark P.}",

note = "Funding Information: The authors thank Drs. S. Achilefu, M. Berezin and C. Frieden of Washington University School of Medicine (WUSM) and members of their laboratories for their help with spectrofluorometry. We are very grateful to Dr. S. Mennerick and members of his laboratory at WUSM, Ms. A. Benz and Dr. D. Crawford, for providing the primary cultures of hippocampal neurons and transfecting them with GFP, respectively. We appreciate our WUSM colleagues for their generous gifts of GFP transfected HEK293T (Ms. N. Husic), HT22 (Dr. B. Wice) and N2A neuroblastoma (Dr. Q. Xiao) cells. We thank Mr. Gary London for his help in preparing the manuscript. This work was supported by NIH Neuroscience Blueprint Center Core Grant P30 NS057105 to Washington University, NIH grants P01 NS032636 and R01 NS36265 (to M.P.G.), NIH R01 MH078823 (to Dr. S. Mennerick), and funds from the Center for Investigation of Membrane Excitability Diseases (CIMED) at Washington University. ",

year = "2013",

month = oct,

doi = "10.1016/j.ceca.2013.08.001",

language = "English",

volume = "54",

pages = "320--333",

journal = "Cell Calcium",

issn = "0143-4160",

number = "4",

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TY - JOUR

T1 - Synthesis and properties of Asante Calcium Red-A novel family of long excitation wavelength calcium indicators

AU - Hyrc, Krzysztof L.

AU - Minta, Akwasi

AU - Escamilla, P. Rogelio

AU - Chan, Patrick P.L.

AU - Meshik, Xenia A.

AU - Goldberg, Mark P.

N1 - Funding Information: The authors thank Drs. S. Achilefu, M. Berezin and C. Frieden of Washington University School of Medicine (WUSM) and members of their laboratories for their help with spectrofluorometry. We are very grateful to Dr. S. Mennerick and members of his laboratory at WUSM, Ms. A. Benz and Dr. D. Crawford, for providing the primary cultures of hippocampal neurons and transfecting them with GFP, respectively. We appreciate our WUSM colleagues for their generous gifts of GFP transfected HEK293T (Ms. N. Husic), HT22 (Dr. B. Wice) and N2A neuroblastoma (Dr. Q. Xiao) cells. We thank Mr. Gary London for his help in preparing the manuscript. This work was supported by NIH Neuroscience Blueprint Center Core Grant P30 NS057105 to Washington University, NIH grants P01 NS032636 and R01 NS36265 (to M.P.G.), NIH R01 MH078823 (to Dr. S. Mennerick), and funds from the Center for Investigation of Membrane Excitability Diseases (CIMED) at Washington University.

PY - 2013/10

Y1 - 2013/10

N2 - 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 (450-540nm) excitation with deep-red fluorescence (640nm). 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 (640nm) and quasi-independent (530nm) emission when using visible (<490nm) or multiphoton (~780nm) 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 (Kd=0.49±0.07μM; ACR-1) or low affinity (Kd=6.65±0.13μM; ACR-1-LA). Chelating Zn2+ (Kd=0.38±0.02nM) or Mg2+ (Kd~5mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa=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.

AB - 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 (450-540nm) excitation with deep-red fluorescence (640nm). 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 (640nm) and quasi-independent (530nm) emission when using visible (<490nm) or multiphoton (~780nm) 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 (Kd=0.49±0.07μM; ACR-1) or low affinity (Kd=6.65±0.13μM; ACR-1-LA). Chelating Zn2+ (Kd=0.38±0.02nM) or Mg2+ (Kd~5mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa=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.

KW - Confocal microscopy

KW - Dissociation constant

KW - Emission spectra

KW - Excitation spectra

KW - Fluorescence

KW - Intracellular free calcium concentration

KW - Magnesium ions

KW - Multiphoton microscopy

KW - Neurons

KW - Ratio imaging

KW - Zinc ions

UR - http://www.scopus.com/inward/record.url?scp=84884280124&partnerID=8YFLogxK

U2 - 10.1016/j.ceca.2013.08.001

DO - 10.1016/j.ceca.2013.08.001

M3 - Article

C2 - 24017967

AN - SCOPUS:84884280124

SN - 0143-4160

VL - 54

SP - 320

EP - 333

JO - Cell Calcium

JF - Cell Calcium

IS - 4

ER -

Synthesis and properties of Asante Calcium Red-A novel family of long excitation wavelength calcium indicators

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Keywords

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