Konno Masae, Inoue Keiichi, Kandori Hideki
The Institute for Solid State Physics, The University of Tokyo, Kashiwa, Chiba 277-8581 Japan.
PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
Bio Protoc. 2021 Aug 5;11(15):e4115. doi: 10.21769/BioProtoc.4115.
Microbial rhodopsins have diverse functions, including roles as light-driven ion pumps, light-gated ion channels, photosensors, and light-regulated enzymes. As the number of rhodopsin-like genes identified has increased in recent years, so has the requirement for rapid identification of their functions. The patch-clamp method is often used to investigate the ion transport mechanism of microbial rhodopsins in mammalian cells; however, this requires a dedicated system and advanced techniques. The ion transport assay using the expression system described here evaluates the ion transport capacity by monitoring the pH change in suspensions; if the target rhodopsin has a light-dependent ion transport activity, a light-dependent pH change is observed. The pH increase or decrease corresponds to proton release from the cell or proton uptake into the cell, respectively. This method can be used to evaluate ion transport capacity in a high-throughput manner using a combination of general-purpose equipment and common techniques. Graphic abstract: Schematic diagram of the ion transport assay in rhodopsin-expressing cells.
微生物视紫红质具有多种功能，包括作为光驱动离子泵、光门控离子通道、光传感器和光调节酶。近年来，随着已鉴定出的类视紫红质基因数量的增加，快速鉴定其功能的需求也随之增加。膜片钳方法常用于研究微生物视紫红质在哺乳动物细胞中的离子转运机制；然而，这需要专门的系统和先进的技术。使用此处所述表达系统的离子转运测定通过监测悬浮液中的pH变化来评估离子转运能力；如果目标视紫红质具有光依赖性离子转运活性，则会观察到光依赖性pH变化。pH升高或降低分别对应于质子从细胞释放或质子进入细胞。该方法可结合通用设备和常用技术以高通量方式评估离子转运能力。图形摘要：视紫红质表达细胞中离子转运测定的示意图。
