采用体外锌转运测定法鉴定哺乳动物锌转运体。

Department of Life Sciences and National Institute for Biotechnology in the Negev, Ben-Gurion University;

Department of Life Sciences and National Institute for Biotechnology in the Negev, Ben-Gurion University.

J Vis Exp. 2023 Jun 2(196). doi: 10.3791/65217.

Transition metals such as Zn ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn levels. With the development of intracellular Zn sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn, detected using fluorescent probes, with the expression of the Zn transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn by utilizing the Zn ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

过渡金属，如锌离子，由于其细胞毒性，必须受到严格控制。以前，锌转运体的活性是通过在不同浓度的锌下测定转运体的表达水平来间接测量的。这是通过利用免疫组织化学、测量组织中的 mRNA 或测定细胞内的锌水平来实现的。随着细胞内锌传感器的发展，锌转运体的活性目前主要通过将荧光探针检测到的细胞内锌的变化与锌转运体的表达相关联来确定。然而，即使在今天，也只有少数几个实验室监测细胞内锌的动态变化，并使用它来直接测量锌转运体的活性。部分问题在于，锌转运体家族的 10 种锌转运体中，除了 ZnT10（转运锰）外，只有锌转运体 1（ZnT1）定位于质膜。因此，将转运活性与细胞内锌浓度的变化联系起来是很困难的。本文描述了一种使用基于锌特异性荧光染料 FluoZin-3 的测定来直接确定锌转运动力学的方法。该染料以其酯形式被载入哺乳动物细胞中，然后由于细胞中二酯酶的活性而被捕获在细胞质中。细胞通过利用锌离子载体吡咯烷酮被装载锌。通过细胞洗涤后荧光的减少的线性部分评估 ZnT1 的活性。在 470nm 的激发和 520nm 的发射下测量的荧光与游离细胞内锌成正比。选择表达被 mCherry 荧光团标记的 ZnT1 的细胞允许仅监测表达转运体的细胞。该测定用于研究 ZnT1 蛋白的不同结构域对人 ZnT1（一种从细胞中排出多余锌的真核跨膜蛋白）转运机制的贡献。