Tilly B C, Winter M C, Ostedgaard L S, O'Riordan C, Smith A E, Welsh M J
Howard Hughes Medical Institute, Department of Internal Medicine and Physiology, University of Iowa College of Medicine, Iowa City 52242.
J Biol Chem. 1992 May 15;267(14):9470-3.
Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.
从小鼠NIH-3T3成纤维细胞和表达高水平囊性纤维化跨膜传导调节因子（CFTR）的中国仓鼠卵巢细胞制备的膜囊泡，与穆勒-鲁丁平面脂质双层融合。加入环磷酸腺苷依赖性蛋白激酶的催化亚基和ATP后，在16次实验中的10次观察到低电导Cl(-)选择性离子通道。这些通道具有线性电流-电压关系，单位电导约为6.5 pS。这些通道对Cl-的通透性比对I-更高，并且没有明显的时间依赖性电压激活。相比之下，向与从 mock 转染（n = 14）细胞制备的囊泡融合的脂质双层中加入环磷酸腺苷依赖性蛋白激酶和ATP未能激活Cl-通道。这些数据支持CFTR是一种Cl-通道的结论。它们表明它可以在平面脂质双层中重构，并且其生物物理和调节特性与在天然细胞膜中观察到的非常相似。这些数据也反对通道调节或功能需要松散相关因子的观点。
