KU Leuven, Lab. Molecular & Cellular Signaling, Dep. Cellular & Molecular Medicine, Campus Gasthuisberg O/N-I bus 802, Herestraat 49, B-3000 Leuven, Belgium.
KU Leuven, Lab. Molecular & Cellular Signaling, Dep. Cellular & Molecular Medicine, Campus Gasthuisberg O/N-I bus 802, Herestraat 49, B-3000 Leuven, Belgium; KU Leuven, Department of Cellular and Molecular Medicine, Laboratory of Chemical Biology, Herestraat 49 box 901b, B-3000 Leuven, Belgium.
Biochim Biophys Acta Mol Cell Res. 2024 Feb;1871(2):119589. doi: 10.1016/j.bbamcr.2023.119589. Epub 2023 Sep 20.
Intracellular Ca signals play a vital role in a broad range of cell biological and physiological processes in all eukaryotic cell types. Dysregulation of Ca signaling has been implicated in numerous human diseases. Over the past four decades, the understanding of how cells use Ca as a messenger has flourished, largely because of the development of reporters that enable visualization of Ca signals in different cellular compartments, and tools that can modulate cellular Ca signaling. One such tool that is frequently used is BAPTA; a fast, high-affinity Ca-chelating molecule. By making use of a cell-permeable acetoxymethyl ester (AM) variant, BAPTA can be readily loaded into the cytosol of cells (referred to as BAPTAi), where it is trapped and able to buffer changes in cytosolic Ca. Due to the ease of loading of the AM version of BAPTA, this reagent has been used in hundreds of studies to probe the role of Ca signaling in specific processes. As such, for decades, researchers have almost universally attributed changes in biological processes caused by BAPTAi to the involvement of Ca signaling. However, BAPTAi has often been used without any form of control, and in many cases has neither been shown to be retained in cells for the duration of experiments nor to buffer any Ca signals. Moreover, increasing evidence points to off-target cellular effects of BAPTA that are clearly not related to Ca chelation. Here, we briefly introduce Ca signaling and the history of Ca chelators and fluorescent Ca indicators. We highlight Ca-independent effects of BAPTAi on a broad range of molecular targets and describe some of BAPTAi's impacts on cell functions that occur independently of its Ca-chelating properties. Finally, we propose strategies for determining whether Ca chelation, the binding of other metal ions, or off-target interactions with cell components are responsible for BAPTAi's effect on a particular process and suggest some future research directions.
细胞内 Ca 信号在所有真核细胞类型的广泛细胞生物学和生理学过程中起着至关重要的作用。Ca 信号的失调与许多人类疾病有关。在过去的四十年中，人们对细胞如何将 Ca 用作信使的理解有了很大的发展，这在很大程度上要归功于能够可视化不同细胞区室中的 Ca 信号的报告基因的发展，以及能够调节细胞 Ca 信号的工具。其中一种常用的工具是 BAPTA；一种快速、高亲和力的 Ca 螯合剂。通过利用细胞通透性的乙氧羰基甲酯（AM）变体，BAPTA 可以很容易地加载到细胞质中（称为 BAPTAi），在那里它被捕获并能够缓冲细胞质 Ca 的变化。由于 AM 版本的 BAPTA 易于加载，这种试剂已在数百项研究中用于研究 Ca 信号在特定过程中的作用。因此，几十年来，研究人员几乎普遍将 BAPTAi 引起的生物学过程的变化归因于 Ca 信号的参与。然而，BAPTAi 经常在没有任何形式的对照的情况下使用，并且在许多情况下，既没有显示在实验过程中保留在细胞内，也没有缓冲任何 Ca 信号。此外，越来越多的证据表明 BAPTA 具有与 Ca 螯合无关的细胞内非靶向作用。在这里，我们简要介绍 Ca 信号和 Ca 螯合剂和荧光 Ca 指示剂的历史。我们强调 BAPTAi 对广泛的分子靶点的 Ca 独立作用，并描述 BAPTAi 对细胞功能的一些影响，这些影响独立于其 Ca 螯合特性。最后，我们提出了确定 Ca 螯合、其他金属离子的结合或与细胞成分的非靶向相互作用是否是 BAPTAi 对特定过程的影响的策略，并提出了一些未来的研究方向。
