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Calibration of mammalian skeletal muscle Ca 2+ transients recorded with the fast Ca 2+ dye Mag-Fluo-4.
- Source :
-
Biochimica et biophysica acta. General subjects [Biochim Biophys Acta Gen Subj] 2021 Sep; Vol. 1865 (9), pp. 129939. Date of Electronic Publication: 2021 Jun 01. - Publication Year :
- 2021
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Abstract
- Background: Mag-Fluo-4 is increasingly employed for studying Ca <superscript>2+</superscript> signaling in skeletal muscle; however, the lack of information on the Ca <superscript>2+</superscript> -Mag-Fluo-4 reaction limits its wider usage.<br />Methods: 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 K <subscript>d</subscript> of the Ca <superscript>2+</superscript> -Mag-Fluo-4 reaction. Rate constants (k <subscript>on</subscript> , k <subscript>off</subscript> ), fluorescence maximum (F <subscript>max</subscript> ), minimum (F <subscript>min</subscript> ), 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.<br />Results: The in vitro fluorescence assays showed a binding stoichiometry of 0.5 for the Ca <superscript>2+</superscript> /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 <subscript>d</subscript> was 1.652 × 10 <superscript>5</superscript> μM <superscript>2</superscript> (n = 58 fibres, R <superscript>2</superscript>  = 0.99). With an F <subscript>max</subscript> of 150.9 (8.8) A.U. (n = 8), F <subscript>min</subscript> of 0.14 (0.1) A.U. (n = 10), and ΔF of Ca <superscript>2+</superscript> transients of 8.4 (2.5) A.U. (n = 10), the sarcoplasmic [Ca <superscript>2+</superscript> ] <subscript>peak</subscript> reached 22.5 (7.8) μM. Compartmentalized dye amounted to only 1.1 (0.7)% (n = 10).<br />Conclusions: Two Mag-Fluo-4 molecules coalesce around one Ca <superscript>2+</superscript> ion, in an entropy-driven, very low in situ affinity reaction, making it suitable to reliably track the kinetics of rapid muscle Ca <superscript>2+</superscript> transients.<br />General Significance: Our results may be relevant to the quantitative study of Ca <superscript>2+</superscript> kinetics in many other cell types.<br /> (Copyright © 2021 Elsevier B.V. All rights reserved.)
Details
- Language :
- English
- ISSN :
- 1872-8006
- Volume :
- 1865
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Biochimica et biophysica acta. General subjects
- Publication Type :
- Academic Journal
- Accession number :
- 34082059
- Full Text :
- https://doi.org/10.1016/j.bbagen.2021.129939