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: 3292688
• 负责人: JAMES O BULLOCK
• 金额: $ 6.97万
• 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 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292688
• 关键词: Escherichia coli; colicines; electric field; ion transport; membrane permeability; membrane reconstitution /synthesis; peptidases

We propose an investigation of the physiology and kinetic behavior of proteins that form channels in biological membranes. Type E1 colicins will be incorporated into planar lipid bilayers to investigate two as yet unsolved problems: the mechanism of assembly of membrane-spanning proteins, and the mechanism of ion transport and selectivity of membrane channels. Colicin proteins are encoded on extrachromosomal plasmids in host bacterial cells. Colicin E1 (56,000 Daltons) is produced in large quantities in a water soluble form by induced cells and its plasmid has been sequenced. Thus, it will be accessible to chemical and genetic manipulation. These features argue for its selection for this study. In preliminary studies we have established that the aqueous form binds tightly to membranes in a voltage independent step. Functional ion channels are formed subsequently only in response to applied voltage. We propose to determine at which step the protein inserts into the membrane, thereby exposing part of its structure in the aqueous phase on the opposite side. To do this, we will examine the ability of proteolytic enzymes and group specific reagents to attack the colicin protein from the opposite side of a planar bilayer. New methods for the formation of planar bilayers will be used to determine whether the bound form of the proteins is associated exclusively with the planar membrane, or with surface monolayers or other hydrophobic surfaces as well. Properties of the ion transport pathway will be investigated using a series of test ions of differing size, charge, shape, and chemical constituent groups. Determining which of these ions can permeate the pore and which can block it will provide information regarding the topography of the aqueous pore of the channel. Competition between blocking ions and permeant ions or other blockers will reveal which types of ionic interactions are allowed in this channel. These studies will provide the basis for understanding the general conformation and assembly of colicin channels and the basis for understanding the consequences of specific amino acid substitutions and deletions on channel properties.

我们建议对生理学和动力学行为进行研究 在生物膜中形成通道的蛋白质。 E1 型大肠杆菌素将 被纳入平面脂质双层以研究两个 未解决问题：跨膜组装机理 蛋白质、离子传输和膜选择性的机制 渠道。 大肠菌素蛋白在染色体外质粒上编码 宿主细菌细胞。 大肠杆菌素 E1（56,000 道尔顿）大量生产 诱导细胞及其质粒以水溶性形式产生的量 已被测序。 因此，它将可以通过化学和遗传手段获得 操纵。 这些特征为本研究选择它提供了依据。 在 初步研究我们已经确定水性形式紧密结合 以与电压无关的步骤到达膜。 功能性离子通道是 随后仅响应于施加的电压而形成。 我们建议 确定蛋白质在哪一步插入膜，从而 将其部分结构暴露在另一侧的水相中。 为此，我们将检查蛋白水解酶和组的能力 从反面攻击大肠菌素蛋白的特定试剂 平面双层。 形成平面双层的新方法将是 用于确定蛋白质的结合形式是否相关 仅与平面膜，或与表面单层或其他 疏水表面也是如此。 离子传输途径的特性将 使用一系列不同尺寸、电荷的测试离子进行研究， 形状和化学组成基团。 确定这些离子中的哪一个 可以渗透毛孔，可以堵塞毛孔，提供信息 关于通道的水孔的地形。 竞赛 阻断离子和渗透离子或其他阻断剂之间的关系将揭示哪一个 该通道中允许发生各种类型的离子相互作用。 这些研究 将为理解一般构象提供基础 大肠杆菌素通道的组装和理解的基础 特定氨基酸取代和缺失对通道的影响 特性。