Low-Affinity Ca2+ Indicators Compared in Measurements of Skeletal Muscle Ca2+ Transients

The low-affinity fluorescent Ca2+ indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca2+ transient (Δ[Ca2+]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, furaptra (previously shown to rapidly track Δ[Ca2+]). In response to an action potential, the full duration at half-maximum of the indicator's fluorescence change (ΔF) was found to be larger with OGB-5N, Fluo-5N, fura-5N, and Rhod-5N than with furaptra; thus, these indicators do not track Δ[Ca2+] with kinetic fidelity. In contrast, the ΔF time course of Mag-fluo-4 was identical to furaptra's; thus, Mag-fluo-4 also yields reliable kinetic information about Δ[Ca2+]. Mag-fluo-4's ΔF has a larger signal/noise ratio than furaptra's (for similar indicator concentrations), and should thus be more useful for tracking Δ[Ca2+] in small cell volumes. However, because the resting fluorescence of Mag-fluo-4 probably arises largely from indicator that is bound with Mg2+, the amplitude of the Mag-fluo-4 signal, and its calibration in Δ[Ca2+] units, is likely to be more sensitive to variations in [Mg2+] than furaptra's.