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 (450-540 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 Ca(2+)-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 (Kd = 0.49 ± 0.07 μM; ACR-1) or low affinity (Kd = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn2+ (Kd = 0.38 ± 0.02 nM) 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.
authors.affiliation: The Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA; Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, MO 63110, USA; Alafi Neuroimaging Laboratory, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: hyrck@neuro.wustl.edu.
authors.forename: Krzysztof L
authors.initials: KL
authors.lastname: Hyrc
authors.forename: Akwasi
authors.initials: A
authors.lastname: Minta
authors.forename: P Rogelio
authors.initials: PR
authors.lastname: Escamilla
authors.forename: Patrick P L
authors.initials: PP
authors.lastname: Chan
authors.forename: Xenia A
authors.initials: XA
authors.lastname: Meshik
authors.forename: Mark P
authors.initials: MP
authors.lastname: Goldberg
mesh_terms: D000814:Aniline Compounds / Q000138:chemical synthesis* / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002138:Calibration; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000138:chemical synthesis* / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry; D007477:Ions; D008027:Light; D051379:Mice; D018613:Microscopy, Confocal; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence
chemical_list: D000814:Aniline Compounds; D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings; D007202:Indicators and Reagents; D007477:Ions; C000588550:asante calcium red-1; D049452:Green Fluorescent Proteins; D002118:Calcium
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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