Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.
Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.
复制标题
来自含有来自 Torpedo electroplax 的功能性重建氯离子通道的脂质体的分离斑块记录。
DOI：
10.1073/pnas.79.24.7749
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发表时间：
1982
影响因子：
11.1
通讯作者：
Webb,WW
中科院分区：
文献类型：
--
作者：
Tank,DW;Miller,C;Webb,WW
通过洗涤剂/透析方法纯化磷脂形成的小单层囊泡可以通过冷冻和解冻扩大到直径30微米。通过在这些大脂质体表面应用玻璃微移液管形成非常高阻力的密封，并从脂质体表面分离出单层双层膜“斑块”，同时保持对微移液管的密封。外源性通道形成肽gramicidin和alamethicin在电压箝位条件下诱导了这些切除斑块的特征单通道波动行为。大脂质体是由鱼雷电火花质膜囊泡的巧克力提取物制成的小单层囊泡形成的。由这些重建膜形成的孤立斑块由于来自非神经支配面膜的单电压门控Cl-通道而显示电流波动；这些氯离子通道的性质与从天然膜直接插入后在平面双层膜中观察到的相同。这种脂质体贴片方法结合了膜蛋白与脂质体结合的优点和高分辨率电记录方法，可能为研究溶解和重构后的整体膜通道蛋白提供一种普遍适用的方法。
Small unilamellar vesicles formed from purified phospholids by detergent/dialysis methods may be enlarged to 30-microns diameter by freezing and thawing. Very-high-resistance seals were formed by applying a glass micropipette to the surface of these large liposomes, and single bilayer "patches" of membrane were isolated from the liposome surface while remaining sealed to the micropipette. The exogenous channel-forming peptides gramicidin and alamethicin induced characteristic single-channel fluctuation behavior in these excised patches held under voltage-clamp conditions. Large liposomes were formed from the small unilamellar vesicles made from cholate extracts of Torpedo electroplax plasma membrane vesicles. Isolated patches formed from these reconstituted membranes displayed current fluctuations due to single voltage-gated Cl- channels from non-innervated-face membranes; the properties of these Cl- channels are identical to those observed in planar bilayer membranes after direct insertion from native membranes. This liposome-patch method combines the advantages of membrane protein incorporation into liposomes with high-resolution electrical recording methods and may provide a generally applicable approach to the study of integral membrane channel proteins after solubilization and reconstitution.