Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes.
Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes.
复制标题
使用 SecA 和 Cx26 间隙连接监测单个卵母细胞中脂蛋白体的通道活性。
DOI：
10.1016/j.ab.2015.04.005
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发表时间：
2015
影响因子：
2.9
通讯作者：
Tai,PhangC
中科院分区：
文献类型：
--
作者：
Hsieh,Ying-Hsin;Zou,Juan;Jin,Jin-Shan;Yang,Hsiuchin;Chen,Yanyi;Jiang,Chun;Yang,Jenny;Tai,PhangC
Establishing recordable channels in membranes of oocytes formed by expressing exogenous complementary DNA (cDNA) or messenger RNA (mRNA) has contributed greatly to understanding the molecular mechanisms of channel functions. Here, we report the extension of this semi-physiological system for monitoring the channel activity of preassembled membrane proteins in single cell oocytes by injecting reconstituted proteoliposomes along with substrates or regulatory molecules. We build on the observation that SecA from various bacteria forms active protein-conducting channels with injection of proteoliposomes, protein precursors, and ATP–Mg2+. Such activity was enhanced by reconstituted SecYEG–SecDF•YajC liposome complexes that could be monitored easily and efficiently, providing correlation of in vitro and intact cell functionality. In addition, inserting reconstituted gap junction Cx26 liposomes into the oocytes allowed the demonstration of intracellular/extracellular Ca2+-regulated hemi-channel activities. The channel activities can be detected rapidly after injection, can be monitored for various effectors, and are dependent on specific exogenous lipid compositions. This simple and effective functional system with low endogenous channel activity should have broad applications for monitoring the specific channel activities of complex interactions of purified membrane proteins with their effectors and regulatory molecules.
