Goldberg A F, Miller C
Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts.
J Membr Biol. 1991 Dec;124(3):199-206. doi: 10.1007/BF01994354.
Chloride channels were detergent-extracted from Torpedo electroplax plasma membrane vesicles and reconstituted into liposomes by rapid detergent removal and a freeze-thaw-sonication procedure. Concentrative uptake of 36Cl-, driven by a Cl- gradient was used to determine conductance properties of reconstituted channels. Chloride flux assayed by this method is strongly selective for Cl- over cations, is blocked by SCN-, inactivated by treatment with DIDS, and exhibits an anion selectivity sequence Cl- greater than Br- greater than F- greater than SO4(2-), as does the voltage-gated Cl- channel from Torpedo observed in planar lipid bilayers. The channels are localized to the noninnervated face of the electrocyte, and a novel trapped-volume method is used to estimate a channel density on the order of 500 pmol/mg protein. An initial fractionation of the membrane extract by anion exchange chromatography yields fivefold enrichment of the channel activity.
氯离子通道从电鳐电板细胞膜囊泡中经去污剂提取，通过快速去除去污剂以及冻融-超声处理程序重组成脂质体。由氯离子梯度驱动的³⁶Cl⁻的浓缩摄取用于确定重组通道的电导特性。用这种方法测定的氯离子通量对氯离子的选择性远高于阳离子，被硫氰酸盐阻断，经二异丙基氟磷酸（DIDS）处理会失活，并且呈现出阴离子选择性顺序Cl⁻＞Br⁻＞F⁻＞SO₄²⁻，这与在平面脂质双层中观察到的电鳐电压门控氯离子通道相同。这些通道定位于电细胞的非神经支配面，并且使用一种新颖的捕获体积法来估计通道密度约为500 pmol/mg蛋白质。通过阴离子交换色谱对膜提取物进行初步分级分离可使通道活性富集五倍。
