Calibration of mammalian skeletal muscle Ca2+ transients recorded with the fast Ca2+ dye Mag-Fluo-4

BackgroundMag-Fluo-4 is increasingly employed for studying Ca²⁺ signaling in skeletal muscle; however, the lack of information on the Ca²⁺-Mag-Fluo-4 reaction limits its wider usage.MethodsFluorescence and isothermal titration calorimetry (ITC) experiments were performed to determine the binding stoichiometry (n) and thermodynamics (enthalpy (ΔH) and entropy (ΔS) changes), as well as the in vitro and in situ K_d of the Ca²⁺-Mag-Fluo-4 reaction. Rate constants (k_on, k_off), fluorescence maximum (F_max), minimum (F_min), and the dye compartmentalization were also estimated. Experiments in cells used enzymatically dissociated flexor digitorum brevis fibres of C57BL6, adult mice, loaded at room temperature for 8 min, with 6 μM Mag-Fluo-4, AM, and permeabilized with saponin or ionomycin. All measurements were done at 20 °C.ResultsThe in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca²⁺/Mag-Fluo-4 (n = 5) reaction. ITC results (n = 3) provided ΔH and ΔS values of 2.3 (0.7) kJ/mol and 97.8 (5.9) J/mol.K, respectively. The in situ K_d was 1.652 × 10⁵μM²(n = 58 fibres, R² = 0.99). With an F_max of 150.9 (8.8) A.U. (n = 8), F_min of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca²⁺ transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca²⁺]_peak reached 22.5 (7.8) μM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10).CONCLUSIONS: Two Mag-Fluo-4 molecules coalesce around one Ca²⁺ ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca²⁺ transients.General significanceOur results may be relevant to the quantitative study of Ca²⁺ kinetics in many other cell types.