PHYSIOLOGY OF ION CHANNEL FORMATION BY TYPE E1 COLICINS

E1 型大肠杆菌素离子通道形成的生理学

• 批准号: 3292691
• 负责人: JAMES O BULLOCK
• 金额: $ 7.93万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-03-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292691
• 关键词: Escherichia coli; acidity /alkalinity; bacterial proteins; binding proteins; calcium channel; cations; colicines; conformation; electric field; ion transport; membrane channels; membrane model; membrane permeability; membrane proteins; membrane reconstitution /synthesis; membrane structure; peptidases; phospholipids; physiology; plasmids; protein biosynthesis; protein structure; proteins; sodium channel

The central objective of this study is the elucidation of molecular processes which underlie the functional properties of proteins which form channels in biological membranes. Two channel forming colicins, E1 and B, will be incorporated into planar lipid bilayers to investigate two such problems of general interest: the mechanism of ion transport and selectivity of membrane channels, and the nature of the conformational changes which accompany insertion of proteins into membranes and the gating of channels. Colicin proteins are encoded on extrachromosomal plasmids in host bacterial cells and are produced in large quantities in a water-soluble form. Thus, the primary structures of these proteins are accessible for chemical and genetic manipulation. The channels formed by colicin E1 and colicin B have many functional features by which they may be distinguished. These features argue for their selection for this study. Colicin channels conduct both positively and negatively charged ions. While even the smallest ions are conducted at fairly slow rates, no large ion has yet been identified which behaves as if it were excluded from the channel. We propose to use a series of ionic substances as probes of the channel lumen. In addition to studies of the permeation and block of colicin channels by these ions, the competition between blocking and permeant ions will be examined. These studies will provide information about the channel's steric and electrostatic topography and the types of ionic interactions which are allowed within the channel. Colicin channels can exist in a membrane either as open channels or closed channels. We propose to determine whether any closed channels have structures which span the entire membrane. To do this, we will test the ability of chemical and biological agents to attack the colicin protein from the opposite side of a membrane. The ion selectivities of the colicins depend upon pH. Recent results have suggested that when colicin E1 is initially exposed to a neutrally charged planar bilayer at pH 5, the membrane insertion process traps the protein in a state whose selectivity is characteristic of pH 8. To test this hypothesis, colicin channels will be subjected to cyclical changes in pH while their selectivity is monitored. The demonstration of a time independent hysteresis loop in the selectivity will be sufficient grounds to rule out insertion as the critical event in this phenomenon and have significant implications for the nature of the conformational changes controlling selectivity.

本研究的中心目标是阐明分子 构成蛋白质功能特性的过程 在生物膜中形成通道。 两通道 形成大肠菌素 E1 和 B，将掺入平面脂质中 双层来研究两个普遍感兴趣的问题： 离子传输机制和膜通道的选择性， 以及伴随的构象变化的性质 将蛋白质插入膜和通道门控。 大肠菌素蛋白在染色体外质粒上编码 宿主细菌细胞并在一个环境中大量生产 水溶性形式。 因此，这些的主要结构 蛋白质可用于化学和遗传操作。 大肠杆菌素E1和大肠杆菌素B形成的通道有很多 可以用来区分它们的功能特征。 这些 特征为他们选择本研究提供了依据。 大肠杆菌素通道 传导带正电和负电的离子。 虽然即使 最小的离子以相当慢的速率传导，没有大离子 尚未确定其行为就像被排除在外 频道。 我们建议使用一系列离子物质作为 通道管腔的探头。 除了研究 这些离子渗透和阻断大肠菌素通道， 阻挡离子和渗透离子之间的竞争将是 检查了。 这些研究将提供有关 通道的空间和静电拓扑以及类型 通道内允许的离子相互作用。 大肠菌素 通道可以作为开放通道或开放通道存在于膜中 封闭渠道。 我们建议确定是否有任何关闭 通道具有跨越整个膜的结构。 要做的事 为此，我们将测试化学和生物制剂的能力 从膜的另一侧攻击大肠杆菌素蛋白。 大肠杆菌素的离子选择性取决于 pH 值。 最近的 结果表明，当大肠杆菌素 E1 最初暴露于 pH 5 时带中性电荷的平面双层，膜 插入过程将蛋白质捕获在一种状态，其选择性为 pH 8 的特征。为了检验这一假设，大肠杆菌素通道 会受到pH值的周期性变化，而它们的选择性 被监控。 与时间无关的磁滞现象的演示 选择性循环将有足够的理由排除 插入作为这种现象中的关键事件并且有 对构象性质具有重要意义 改变控制选择性。