PURIFICATION, RECONSTITUTION, AND SUBUNIT COMPOSITION OF A VOLTAGE-GATED CHLORIDE CHANNEL FROM TORPEDO ELECTROPLAX
PURIFICATION, RECONSTITUTION, AND SUBUNIT COMPOSITION OF A VOLTAGE-GATED CHLORIDE CHANNEL FROM TORPEDO ELECTROPLAX
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DOI：
10.1021/bi00249a005
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发表时间：
1994-11-15
期刊：
影响因子：
2.9
通讯作者：
MILLER, C
中科院分区：
文献类型：
--
作者：
MIDDLETON, RE;PHEASANT, DJ;MILLER, C
电鳐的电压门控氯离子通道的功能形式纯化的免疫亲和程序。通过Cl-36（-）摄取到重构脂质体中以及插入平面脂质双层后直接记录来测定通道活性。纯化的通道显示与天然膜观察到的相同的“双barbridd”门控动力学，以及正确的单通道渗透特性。活性通道的制备物由90-kDa多肽组成，如从已知cDNA序列所预期的。在纯化的材料中不存在相关的亚基。直接蛋白质测序证实了不存在可切割的信号序列，并证明了cDNA衍生序列的Ser-2处的N-末端。这种“ClC-O”蛋白是轻度糖基化的，在用内切糖苷酶H或N-聚糖酶处理时仅损失约2kDa的糖。大多数（如果不是全部的话）这种糖基化在Asn-365上发现。这一结果需要修改目前的跨膜拓扑结构的建议，这已经把这个残基的细胞质侧的膜。在活性保留条件下蔗糖密度梯度中的沉降表明ClC-O通道略大于Na/K-ATP酶α/β-原聚体（类似或等于150 kDa），并且显著小于烟碱乙酰胆碱受体的还原形式（类似或等于300 kDa）。洗涤剂溶解的ClC-O通道，它总是显示两个Cl-扩散孔的活性复合物，因此最有可能建立作为一个同源二聚体的90 kDa的蛋白质纯化在这里。
The voltage-gated Cl- channel from Torpedo electroplax was purified in functional form by an immunoaffinity procedure. Channel activity was assayed by Cl-36(-) uptake into reconstituted liposomes and by direct recording after insertion into planar lipid bilayers. The purified channel displays the same ''double-barreled'' gating kinetics observed with native membranes, as well as the correct single-channel permeation characteristics. Preparations of active channels consist of a 90-kDa polypeptide, as expected from the known cDNA sequence. No associated subunits are present in the purified material. Direct protein sequencing confirms the absence of a cleavable signal sequence and demonstrates an N-terminus at Ser-2 of the cDNA-derived, sequence. This ''ClC-O'' protein is lightly glycosylated, losing only approximate to 2 kDa of sugar upon treatment with endoglycosidase H or N-glycanase. Most if not all of this glycosylation is found on Asn-365. This result necessitates revision of current transmembrane topology proposals, which have placed this residue on the cytoplasmic side of the membrane. Sedimentation in sucrose density gradients under activity-preserving conditions suggests the ClC-O channel is slightly larger than the Na/K-ATPase alpha/beta-protomer (similar or equal to 150 kDa) and substantially smaller than the reduced form bf the nicotinic acetylcholine receptor (similar or equal to 300 kDa). The detergent-solubilized ClC-O channel, which invariably displays two Cl- diffusion pores in the active complex, is therefore built most likely as a homodimer of the 90-kDa protein purified here.