Tetrameric stoichiometry of a prokaryotic K+ channel
Tetrameric stoichiometry of a prokaryotic K+ channel
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DOI：
10.1021/bi970988i
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发表时间：
1997-08-19
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：
Heginbotham, L;Odessey, E;Miller, C
最近在原核生物中发现了与神经元K+通道序列相似的基因。这些假设的K+通道似乎是完整的膜蛋白，通过疏水性分析鉴定出多个跨膜序列，并且序列与所有已知真核K+通道中发现的孔衬里“p区”基序惊人地相似。本研究考察了来自lividans链霉菌的K+通道同源物SliK在洗涤剂胶束中的低聚状态和稳定性。合成的SliK基因在大肠杆菌中高水平表达，并纯化了该蛋白。在SDS-PAGE凝胶中，这种蛋白质的主要形式是19 kda多肽的低聚物，但高温或高pH等严酷的处理会将这种缓慢迁移的物质转化为单体形式。一种用于检测亚基化学计量学的“质量标记”策略表明，SliK是SDS和十二烷基麦芽糖苷胶束的同四聚体。四聚体结构可以被已知的阻止神经元K+通道功能表达的p区突变破坏。四聚体非常稳定，在室温下14天后没有转化为单体形式。虽然没有观察到SliK介导的阳离子通量活性，但该蛋白的四聚体行为表明，SliK可能提供了一个直接攻击K+通道p区序列结构的系统。
Genes with sequences reminiscent of neuronal K+ channels have recently been identified in prokaryotes. These putative K+ channels appear to be integral membrane proteins, with multiple transmembrane sequences identified by hydrophobicity analysis and a sequence strikingly similar to the pore-lining ''P-region'' motif found in all known eukaryotic K+ channels. This study examines the oligomeric stale and stability in detergent micelles of SliK, a K+ channel homologue from Streptomyces lividans. A synthetic gene for SliK was expressed at high levels in Escherichia coli, and the protein was purified. The predominant form of the protein runs in SDS-PAGE gels as an oligomer of the 19-kDa polypeptide, but harsh treatments such as heat or high pH convert this slowly-migrating material into monomeric form. A ''mass-tagging'' strategy developed to examine subunit stoichiometry shows that SliK is a homotetramer in SDS and dodecyl maltoside micelles. The tetrameric structure can be disrupted by P-region mutations known to prevent the functional expression of neuronal K+ channels. The tetramer is remarkably stable, showing no conversion to the monomeric form after 14 days at room temperature. Although SliK-mediated cation flux activity was not observed, the tetrameric behavior of the protein argues that SliK may provide a system for a direct attack on the structure of a K+ channel P-region sequence.