Li M, Unwin N, Stauffer K A, Jan Y N, Jan L Y
Howard Hughes Medical Institute, Department of Physiology, University of California, San Francisco 94143-0724.
Curr Biol. 1994 Feb 1;4(2):110-5. doi: 10.1016/s0960-9822(94)00026-6.
Voltage-gated K+ channels play an important role in the control of neuronal excitability and synaptic plasticity. Their low abundance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-gated K(+)-channel gene, Shaker, from Drosophila. The Shaker K(+)-channel polypeptide resembles one of the four internal repeats of a Na(+)- or Ca(2+)-channel alpha subunit, suggesting that this example of a K+ channel contains four identical or homologous subunits. Similar K(+)-channel polypeptides have been characterized from mammals, other vertebrate and invertebrate species, and from plants. Electrophysiological studies of K+ channels expressed in Xenopus oocytes suggest that they are indeed tetramers, and heteromultimeric K+ channels have been found in the mammalian brain. Until now, however, no K+ channel, nor any other member of the superfamily of voltage-gated ion channels, has been characterized by electron microscopy or other structural analysis.
We have purified Shaker K+ channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physical dimensions of these molecules, as well as their biochemical characteristics, are consistent with a tetrameric subunit composition. Moreover, the Shaker channel revealed by negative staining has the appearance of a four-fold symmetric tetramer, with a large, central vestibule that presumably constitutes part of the pathway for ions.
These first clear images of a voltage-gated ion channel reveal a marked four-fold symmetry. The integrity of the purified tetrameric complex indicates that the purification scheme used in this study may be further developed for future structural analysis of voltage-gated K+ channels.
电压门控钾离子通道在控制神经元兴奋性和突触可塑性方面发挥着重要作用。它们的低丰度和极高的异质性使得从天然来源纯化它们变得困难。我们之前从果蝇中克隆了一个电压门控钾离子通道基因，即Shaker。Shaker钾离子通道多肽类似于钠通道或钙通道α亚基的四个内部重复序列之一，这表明这个钾离子通道的例子包含四个相同或同源的亚基。类似的钾离子通道多肽已在哺乳动物、其他脊椎动物和无脊椎动物物种以及植物中得到表征。对非洲爪蟾卵母细胞中表达的钾离子通道的电生理研究表明它们确实是四聚体，并且在哺乳动物大脑中发现了异源多聚体钾离子通道。然而，到目前为止，还没有通过电子显微镜或其他结构分析对任何钾离子通道或电压门控离子通道超家族的其他成员进行表征。
我们已经将在昆虫Sf9细胞中表达的Shaker钾离子通道纯化至表观均一性，并使用电子显微镜对其进行成像。这些分子的物理尺寸以及它们的生化特性与四聚体亚基组成一致。此外，负染显示的Shaker通道呈现出四重对称的四聚体外观，有一个大的中央前庭，推测它构成了离子通道的一部分。
这些电压门控离子通道的首张清晰图像显示出明显的四重对称性。纯化的四聚体复合物的完整性表明，本研究中使用的纯化方案可能会进一步发展，用于未来对电压门控钾离子通道的结构分析。
