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.

我们提出了对生理学和动力学行为的研究 在生物膜中形成通道的蛋白质。 E1类colicins类型将 被纳入平面脂质双层，以调查两个 未解决的问题：跨膜跨度的机制 蛋白质，以及离子转运和膜选择性的机制 频道。 结肠蛋白蛋白编码在外染色体外质粒上 宿主细菌细胞。 菌Colicin E1（56,000 daltons）大量生产 诱导细胞及其质粒具有水溶性形式的数量 被测序。 因此，化学和遗传可以使用 操纵。 这些功能主张其选择这项研究。 在 初步研究我们已经确定水性形式结合紧密 在电压独立步骤中进入膜。 功能离子通道是 随后仅对施加电压形成。 我们建议 确定蛋白质在哪个步骤中插入膜，从而确定 在另一侧的水相中暴露其结构的一部分。 为此，我们将检查蛋白水解酶和组的能力 从A的另一侧攻击结肠蛋白的特定试剂 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 专门用平面膜或表面单层或其他 疏水表面也是如此。 离子传输途径的特性将 可以使用一系列大小，电荷的测试离子进行研究 形状和化学成分组。 确定这些离子中的哪一个 可以渗透孔，哪些可以阻止其提供信息 关于通道水孔的地形。 竞赛 在阻塞离子和渗透离子或其他阻滞剂之间将揭示哪个 在此通道中允许使用离子相互作用的类型。 这些研究 将为理解一般构象和 结肠毒素渠道的组装和理解的基础 特定氨基酸取代和通道上缺失的后果 特性。