Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.
Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.
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DOI：
10.1085/jgp.98.1.77
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发表时间：
1991-07
期刊：
影响因子：
--
通讯作者：
Slatin SL
中科院分区：
文献类型：
--
作者：
Abrams CK;Jakes KS;Finkelstein A;Slatin SL
离子传导通道的初级序列的可用性允许电压门控机制的可测试模型的发展。以前的工作平面磷脂双层和脂质囊泡表明，电压门控大肠杆菌素E1通道涉及易位的分子的肽段进入和穿过膜。在这里，我们确定组氨酸残基440作为门控电荷与此易位。使用定点诱变转换带正电荷的His 440的中性半胱氨酸，我们发现，关闭由该突变体在位置440处形成的通道的电压依赖性是不太陡峭的比野生型通道;电压依赖性的变化的幅度是一致的与残基440移动从反式到顺式侧的膜与通道关闭。反式pH变化对由半胱氨酸440突变体的羧甲基化衍生物形成的通道的离子选择性的影响独立地确定了在开放通道状态下，残基440位于膜的反式侧。在这些结果的基础上，我们提出，大肠杆菌素E1通道的电压门控开放伴随着插入到从残基420到残基459延伸的螺旋发夹环的双层中，并且电压门控关闭与该环从双层的内部回到顺式侧的挤出相关。
The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.