Dietrich P, Gradogna A, Carpaneto A
Lehrstuhl für Molekulare Pflanzenphysiologie, Department Biologie Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.
Institute of Biophysics, Genoa, Italy.
Handb Exp Pharmacol. 2023;278:235-247. doi: 10.1007/164_2022_604.
Human TPC channels are an emerging family of intracellular proteins fundamental for cell physiology and involved in various severe pathologies. Their localization in the membranes of endo-lysosomes, intracellular compartments of submicrometric dimensions, makes their study difficult with usual electrophysiological techniques. In this work, we show how the plant vacuole, a versatile organelle that can occupy up to 90% of the volume in mature plant cells, can be used as a heterologous system of expression for functional characterization. For this purpose, the use of vacuoles isolated from mesophyll cells of the Arabidopsis thaliana mutant lacking the endogenous TPC avoids unwanted interferences. The patch-clamp technique can be successfully applied to plant vacuoles in all different configuration modes; of note, the whole-vacuole configuration allows to study channel modulation by cytosolic factors. The combination of patch-clamp with fluorescence techniques, for example, by using fluorescent probes sensitive to specific ions of interest, represents a useful extension to investigate the selectivity properties of the channels. Therefore, the plant vacuole, similar to Xenopus oocytes for ion channels and transporters localized in the plasma membrane, has the capability to become a model system for functional studies on intracellular ion channels and transporters.
人类双孔通道蛋白（TPC）是一类新发现的细胞内蛋白家族，对细胞生理功能至关重要，并参与多种严重病理过程。它们定位于内溶酶体膜上，内溶酶体是亚微米级的细胞内区室，这使得用常规电生理技术研究它们变得困难。在这项工作中，我们展示了植物液泡，一种在成熟植物细胞中可占据高达90%体积的多功能细胞器，如何能够用作功能表征的异源表达系统。为此，使用从缺乏内源性TPC的拟南芥突变体叶肉细胞中分离的液泡可避免不必要的干扰。膜片钳技术能够成功应用于处于所有不同配置模式的植物液泡；值得注意的是，全液泡配置模式能够研究胞质因子对通道的调节作用。例如，通过使用对特定目标离子敏感的荧光探针，将膜片钳技术与荧光技术相结合，是研究通道选择性特性的一种有效扩展方法。因此，与用于研究定位在质膜上的离子通道和转运蛋白的非洲爪蟾卵母细胞类似，植物液泡有能力成为细胞内离子通道和转运蛋白功能研究的模型系统。
