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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1865; no. 9; p. 129939
• Main Authors: Milán, Andrés F., Rincón, Oscar A., Arango, Leidy B., Reutovich, Aliaksandra A., Smith, Gideon L., Giraldo, Marco A., Bou-Abdallah, Fadi, Calderón, Juan C.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.09.2021
• Subjects: adults; ambient temperature; Ca2; calcium; Calibration; calorimetry; Dyes; Enthalpy; Entropy; Fluorescence; Isothermal titration calorimetry; muscles; saponins; Skeletal muscle; Stoichiometry; titration; Stoichiometry; Dyes; Ca2; Enthalpy; Fluorescence; Entropy; Calibration; Isothermal titration calorimetry; Skeletal muscle
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2021.129939

Mag-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. Fluorescence 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 Kd of the Ca2+-Mag-Fluo-4 reaction. Rate constants (kon, koff), fluorescence maximum (Fmax), minimum (Fmin), 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. The 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 Kd was 1.652 × 105μM2(n = 58 fibres, R2 = 0.99). With an Fmax of 150.9 (8.8) A.U. (n = 8), Fmin 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. Our results may be relevant to the quantitative study of Ca2+ kinetics in many other cell types. [Display omitted] •Mag-Fluo-4 is increasingly used to study Ca2+ signaling in skeletal muscle.•A characterization of the in vitro and in situ Ca2+―Mag-Fluo-4 reaction is lacking.•Thermodynamics, affinity, rate constants and fluorescence were determined.•2 Mag-Fluo-4 molecules coalesce around 1 Ca2+ ion, in a very low affinity reaction.•Ca2+ transients from mammalian skeletal muscle fibres gave a [Ca2+]peak of 22.5 μM.