SOLUBILIZATION AND FUNCTIONAL RECONSTITUTION OF A CHLORIDE CHANNEL FROM TORPEDO-CALIFORNICA ELECTROPLAX
SOLUBILIZATION AND FUNCTIONAL RECONSTITUTION OF A CHLORIDE CHANNEL FROM TORPEDO-CALIFORNICA ELECTROPLAX
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DOI：
10.1007/bf01994354
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发表时间：
1991-12-01
影响因子：
2.4
通讯作者：
MILLER, C
中科院分区：
文献类型：
--
作者：
GOLDBERG, AFX;MILLER, C
用洗涤剂从鱼雷电浆膜囊泡中提取氯离子通道，并通过快速去污剂和冷冻-解冻-超声法将其重组为脂质体。用氯-梯度驱动的氯-36-的集中吸收来测定重组通道的电导性质。用这种方法测定的氯离子通量对氯离子具有很强的选择性，被SCN-阻断，用DDS处理使其失活，并显示出阴离子选择性序列Cl->；bR->；F->；SO4(2-)，正如在平面脂质双层中观察到的来自鱼雷的电压门控氯通道一样。通道被定位在细胞的非神经面，并用一种新的捕获体积法估计了约500pmol/mg蛋白质的通道密度。通过阴离子交换层析对膜提取物进行初始分级，可使通道活性增加五倍。
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 Cl-36-, 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- > Br- > F- > 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.