Background

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.

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_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.

Results

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 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 significance

Our results may be relevant to the quantitative study of Ca²⁺ kinetics in many other cell types.

中文翻译：

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使用快速 Ca2+ 染料 Mag-Fluo-4 记录的哺乳动物骨骼肌 Ca2+ 瞬变的校准

背景

Mag-Fluo-4 越来越多地用于研究骨骼肌中的Ca ²⁺信号；然而，缺乏有关 Ca ²⁺ -Mag-Fluo-4 反应的信息限制了其更广泛的应用。

方法

进行荧光和等温滴定量热 (ITC) 实验以确定结合化学计量 (n) 和热力学（焓 (ΔH) 和熵 (ΔS) 变化），以及Ca ^{2+ 的}体外和原位 K _d -Mag-Fluo-4 反应。速率常数 ( k _on , k _off )、荧光最大值 ( F _max )、最小值 ( F _{min )})，并且还估计了染料区室化。细胞实验使用酶促分离的 C57BL6 屈肌短屈肌纤维，成年小鼠，在室温下加载 8 分钟，使用 6 μM Mag-Fluo-4，AM，并用皂苷或离子霉素进行透化。所有测量均在 20 °C 下进行。

结果

在体外荧光的测定显示出0.5的钙结合化学计量²⁺ / MAG-的Fluo-4（Ñ  = 5）的反应。ITC 结果 ( n  = 3) 提供的 ΔH 和 ΔS 值分别为 2.3 (0.7) kJ/mol 和 97.8 (5.9) J/mol.K。该原位ķ _d为1.652×10 ⁵ μM ²（Ñ  = 58种纤维，R ²  = 0.99）。与˚F_最大的150.9（8.8）AU（Ñ  = 8），˚F_分钟0.14（0.1）AU（Ñ  = 10），和ΔF的Ca ²⁺ 8.4（2.5）AU（瞬变Ñ = 10)，肌浆 [Ca ²⁺ ]_峰达到 22.5 (7.8) μM。分隔染料仅占 1.1 (0.7)% (n = 10)。

结论：在熵驱动的非常低的原位亲和反应中，两个 Mag-Fluo-4 分子在一个 Ca ²⁺离子周围聚结，使其适合可靠地跟踪快速肌肉 Ca ²⁺瞬变的动力学。

一般意义

我们的结果可能与许多其他细胞类型中 Ca ²⁺动力学的定量研究有关。