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首页> 外文期刊>The Journal of Neuroscience: The Official Journal of the Society for Neuroscience >Single-channel analysis of KCNQ K+ channels reveals the mechanism of augmentation by a cysteine-modifying reagent.
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Single-channel analysis of KCNQ K+ channels reveals the mechanism of augmentation by a cysteine-modifying reagent.

机译：KCNQ K +通道的单通道分析揭示了半胱氨酸修饰试剂增强的机制。

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摘要

The cysteine-modifying reagent N-ethylmaleimide (NEM) is known to augment currents from native M-channels in sympathetic neurons and cloned KCNQ2 channels. As a probe for channel function, we investigated the mechanism of NEM action and subunit specificity of cloned KCNQ2-5 channels expressed in Chinese hamster ovary cells at the whole-cell and single-channel levels. Biotinylation assays and total internal reflection fluorescence microscopy indicated that NEM action is not caused by increased trafficking of channels to the membrane. At saturating voltages, whole-cell currents of KCNQ2, KCNQ4, and KCNQ5 but not KCNQ3 were augmented threefold to fourfold by 50 microm NEM, and their voltage dependencies were negatively shifted by 10-20 mV. Unitary conductances of KCNQ2 and KCNQ3 (6.2 and 8.5 pS, respectively) were much higher that those of KCNQ4 and KCNQ5 (2.1 and 2.2 pS, respectively). Surprisingly, the maximal open probability (P(o)) of KCNQ3 was near unity, much higher than that of KCNQ2, KCNQ4, and KCNQ5. NEM increased the P(o) of KCNQ2, KCNQ4, and KCNQ5 by threefold to fourfold but had no effect on their unitary conductances, suggesting that the increase in macroscopic currents can be accounted for by increases in P(o). Analysis of KCNQ3/4 chimeras determined the C terminus to be responsible for the differential maximal P(o), channel expression, and NEM action between the two channels. To further localize the site of NEM action, we mutated three cysteine residues in the C terminus of KCNQ4. The C519A mutation alone ablated most of the augmentation by NEM, suggesting that NEM acts via alkylation of this residue.
机译：已知半胱氨酸修饰剂N-乙基马来酰亚胺（NEM）会增加交感神经元和克隆的KCNQ2通道中天然M通道的电流。作为通道功能的探针，我们在全细胞和单通道水平研究了中国仓鼠卵巢细胞中表达的克隆的KCNQ2-5通道的NEM作用机制和亚基特异性。生物素化测定法和全内反射荧光显微镜检查表明，NEM作用不是由向膜的通道运输增加引起的。在饱和电压下，KCNQ2，KCNQ4和KCNQ5而不是KCNQ3的全电池电流通过50 microm NEM增大了三倍至四倍，并且它们的电压依存关系负移了10-20 mV。 KCNQ2和KCNQ3的单位电导（分别为6.2和8.5 pS）比KCNQ4和KCNQ5的单位电导（分别为2.1和2.2 pS）高得多。令人惊讶的是，KCNQ3的最大开放概率（P（o））接近于1，远高于KCNQ2，KCNQ4和KCNQ5。 NEM使KCNQ2，KCNQ4和KCNQ5的P（o）增加了三倍至四倍，但对它们的单位电导率没有影响，这表明宏观电流的增加可以由P（o）的增加来解释。 KCNQ3 / 4嵌合体的分析确定C端负责两个通道之间最大差异P（o），通道表达和NEM作用。为了进一步定位NEM作用的位点，我们在KCNQ4的C末端突变了三个半胱氨酸残基。单独的C519A突变消除了NEM的大部分扩增，这表明NEM通过该残基的烷基化起作用。

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