A new functional analysis of intracellular ion-transporting proteins

基于固体支撑膜(SSM)的一种新的电生理检测技术已被证明可用于通道和转运体的分析。我们试图将这项技术应用于分别从大鼠脑和小鼠肝脏纯化的突触小泡(SVS)和溶酶体膜(LMS)。将三磷酸腺苷应用于SVS传感器，可触发依赖于镁离子且对巴菲罗星A1(Baf)敏感的电流反应，这被认为是V-ATPase特异性电流。另一方面，虽然LMS也嵌入了V-ATPase，但传感器产生的电流响应具有不同的特征。它们不依赖于镁离子，对BaF仅部分敏感，甚至可由ADP触发。这些特征表明，电流不是归因于V-ATPase，而是任何其他溶酶体蛋白。本研究显示了基于SSM的电生理学的实用性和潜力，特别是在分析天然细胞器膜蛋白方面。

A novel electrophysiological assay technique based on solid supported membranes (SSM) has been shown to be useful for analysis of channels and transporters. We tried to apply this technique to synaptic vesicles (SVs) and lysosomal membranes (LMs), purified from rat brains and mouse livers, respectively. Application of ATP to the sensors with SVs triggered current responses dependent on Mg2+ and sensitive to bafilomycin A1 (Baf), which are thought to be V-ATPase specific currents. On the other hand, though LMs are also embedded with V-ATPases, the sensors evoked current responses with different characteristics. They were independent of Mg2+, only partially sensitive to Baf and even triggered by application of ADP. These features suggest that the currents are not attributed to V-ATPases but any other lysosomal protein. The present study shows the utility and potential of SSM-based electrophysiology especially for analyzing native organellar membrane proteins.