Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC.
Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.
Hyrc
K L
KL
Department of Neurology, Washington University School of Medicine, St. Louis, Missouri, USA.
Bownik
J M
JM
Goldberg
M P
MP
D000818:Animals; D052160:Benzothiazoles; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D002614:Chelating Agents / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D052160:Benzothiazoles; D002412:Cations; D002614:Chelating Agents; D003374:Coumarins; D005456:Fluorescent Dyes; C116748:N-(3-(2-benzothiazolyl)-6-(2-(2-(bis(carboxymethyl)amino)-5-methylphenoxy)ethoxy)-2-oxo-2H-1-benzopyran-7-yl)-N-(carboxymethyl)glycine; D002118:Calcium; D005998:Glycine; D016257:Fura-2