Mag-Fura-2 (Furaptra) exhibits both low (microM) and high (nM) affinity for Ca2+.
Based on studies using high-affinity Ca2+ probes (dissociation constant (Kd) = 0.15-0.3 microM), steady-state [Ca2+]in is believed to be in the nanomolar range in most cells. However, probes with lower affinity indicate that [Ca2+]in may increase to micromolar levels during activation of specific cell functions, e.g., contraction. These conclusions rely on accurate knowledge of the Kd of the dyes for Ca2+. Mag-Fura-2 (also known as Furaptra) is a low-affinity Ca2+ indicator (Kd ca. 50 microM) which has been used for such studies. In the present work, Mag-Fura-2 is shown to respond to changes in cytosolic Ca2+ in the submicromolar range. In vitro, and in situ titration of Mag-Fura-2 in A7r5 cells, demonstrate that Mag-Fura-2 exhibits both high- and low-affinity for Ca2+. Moreover, pH affects both high and low affinity Ca2+ binding site. Since Mag-Fura-2 has been used to study Ca2+ within specific subcellular compartments, the present observations indicate that knowledge of factors such as ambient pH of these compartments is required to accurately interpret Ca2+ responses. Furthermore, the sensitivity of Mag-Fura-2 at submicromolar levels must be considered for accurate determination of Ca2+ in specific compartments believed to exhibit high micromolar levels of Ca2+.
Martínez-Zaguilán
R
R
Department of Physiology, Texas Tech University Health Sciences Center, Lubbock 79430, USA. phyrmz@ttuhsc.edu
Parnami
J
J
Martinez
G M
GM
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D002118:Calcium; D016257:Fura-2