MOLECULAR GENETIC DISSECTION OF THE VDAC ION CHANNEL

VDAC 离子通道的分子遗传学剖析

• 批准号: 2178063
• 负责人: MICHAEL A FORTE
• 金额: $ 28.12万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-11-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178063
• 关键词: MOLECULAR; GENETIC; DISSECTION; VDAC; ION

Voltage-dependent ion channels form aqueous pores in membranes that open and close in response to changes in transmembrane potential. Although many channels of this kind have been the subject of intense molecular and biophysical investigation, a true molecular picture of how voltage sensor movement is coupled to channel opening and the precise nature of the underlying protein conformational changes remain poorly understood despite much speculation. The long term goal of this proposal is to use the outer mitochondrial membrane channel VDAC as a model of how a voltage "sensor" couples to other regions of a protein to produce the molecular rearrangements generating channel gating. Over the past period of funding, we have examined the functional consequences of a large number of charge changes introduced by site-directed mutagenesis throughout the yeast VDAC (YVDAC) molecule. These functional studies have led to a model of the transmembrane topology of the yeast VDAC protein in the open state, defined regions of the protein that are removed from the pore during channel closure and identified specific residues forming the voltage sensor. In addition, human VDAC (HVDAC) genes have been identified and characterized. Over the course of the next funding period, we will build on these studies by first testing the hypothesis that a similar overall transmembrane topology of VDAC proteins generates VDAC's highly conserved physiology. To test this hypothesis, we will examine the folding pattern of HVDAC 1 by functional studies of mutant HVDAC 1 proteins. HVDAC 1 has a very different primary sequence yet identical physiological properties. These studies should allow us to test the generality of models developed in our studies of YVDAC. Second, we will test our models of the topology of the open VDAC channel and the conformational changes accompanying voltage-gating at the protein level by site-specific biotinylation of single cysteine residues (cys). Single cys residues will be engineered into the yeast protein and modified with thiol specific reagents that attach a biotin at this site. The accessibility of the biotin-modified cys to streptavidin (strp) and the location of the strp if bound will be used to localize this residue with respect to the membrane in a variety of protein conformations both in the outer membranes and in bilayers. Third, we will begin to test the idea that VDAC's channel properties in the cell are regulated by association with cytoplasmic and intermitochondrial membrane space proteins co-ordinated into a complex with VDAC. By use of the yeast two hybrid system, we have identified three novel molecules which interact strongly with yeast VDAC. Our goals are to characterize the proteins encoded by these and other genes identified in our screens, convincingly demonstrate their interaction with VDAC either in vitro or in vivo, and determine their potential effect in modulating VDAC's channel characteristics in planar phospholipid bilayers.

电压依赖性离子通道在膜中形成水孔 并响应跨膜电位的变化而接近。虽然很多 这种通道一直是强烈的分子和 生物物理研究，电压传感器的真实分子图 移动与渠道开放和开放的确切性质结合起来 尽管 很多猜测。该提议的长期目标是使用外部 线粒体膜通道VDAC作为电压“传感器”的模型 夫妻到蛋白质的其他区域产生分子 重排生成通道门控。在过去的资金期间， 我们检查了大量电荷的功能后果 在整个酵母VDAC中，由定向诱变引入的变化 （YVDAC）分子。这些功能研究导致了 酵母VDAC蛋白的跨膜拓扑结构处于开放状态， 定义的蛋白质区域，这些区域在 通道闭合并鉴定出形成电压的特定残基 传感器。此外，已经确定了人VDAC（HVDAC）基因 特征。在下一个资金期间，我们将建造 在这些研究中，首先检验了一个相似总体的假设 VDAC蛋白的跨膜拓扑产生VDAC的高度保守 生理。为了检验该假设，我们将检查折叠模式 HVDAC 1通过突变HVDAC 1蛋白的功能研究。 HVDAC 1具有 一个非常不同的主要序列但相同的生理特性。 这些研究应该使我们能够测试开发模型的一般性 在我们对YVDAC的研究中。其次，我们将测试拓扑模型 开放的VDAC频道和随附的构象变化 通过位点特异性生物素化在蛋白质水平上的电压门控 单半胱氨酸残基（CYS）。单一CYS残留将设计 进入酵母蛋白，并用特异性试剂修饰 在此站点附加生物素。生物素修饰的CYS的可访问性 到链霉亲和素（strp）和strp的位置，如果将使用绑定 在各种膜上定位该残基 外膜和双层中的蛋白质构象。第三， 我们将开始测试单元格中VDAC通道属性的想法 通过与细胞质和蒙上微粒的关联来调节 膜空间蛋白与VDAC协调成络合物。通过使用 酵母两个混合系统，我们已经确定了三个新的分子 与酵母VDAC强烈相互作用。我们的目标是表征 这些蛋白质由我们筛选中鉴定出的这些基因编码的蛋白质， 令人信服地展示了它们与VDAC的相互作用，无论是在体外还是在 体内，并确定它们在调节VDAC渠道中的潜在效果 平面磷脂双层的特征。