Exploration of Channel Specialization in Transport of Metabolites

跨膜通道参与了许多细胞过程，如代谢产物、蛋白质和转录因子通过生物膜的运输。显然，大通道渗透小离子的能力并不一定反映它们允许大离子或大分子穿过膜的能力。然而，最初为研究离子选择性通道而开发的相同的基本电生理学技术，被常规地应用于所有通道组。一种新的方法被开发出来，它为监测几乎任何分子的通道渗透性打开了可能性。该方法允许在单通道水平上测量在小离子存在的情况下诸如代谢物和大分子之类的相对高分子分子的传输。这可能是一种非常普遍的方法，应该适用于测量各种不同通道中广泛的生理重要溶质的渗透率。通过使用线粒体通道，称为VDAC，它旨在表明，虽然具有大孔的通道对小离子的选择性较差，但它们可能对它们进化来运输的较大分子表现出高选择性。新的方法将用于测量代谢物在通道中的分配及其在通道内的流动性。这是通过分析当这些代谢物现在通过单一通道从而阻碍较小离子通过时产生的平均电流和电流噪声的变化来实现的。初步证据表明，VDAC根据代谢物的三维结构，而不仅仅是大小和电荷来选择代谢物。三磷酸腺苷可以穿透毛孔，但具有相似大小和电荷的谷氨酸四肽不会。这种选择性的性质将通过使用其他物理性质与三磷酸腺苷相似的分子来检验。此外，孔内有利于ATP渗透的电荷分布的重要性将得到验证。将使用一组定点突变体。为了探索这种选择性是一种保守性质的可能性，我们将分析从非常不同的物种分离出来的VDAC通道。将检查同一生物体内不同的VDAC亚型，以确定它们是否表现出反映其特殊功能的不同选择性模式。此外，将研究细胞色素c的渗透，以确定小蛋白是否能够通过VDAC。如果成功，这项工作将获得对大通道运输特性的必要洞察，以开发关于其代谢物选择性的想法。活着的有机体和单个细胞的分隔能力对生命至关重要。这种划分允许每个地区保持一个特别适合其所服务功能的环境。被称为通道的蛋白质通过允许选定的分子和离子穿过隔室和膜之间的屏障来帮助这种隔间。这个项目探索了一种假设，即允许大分子通过的通道是以高度选择性的方式实现的。这不同于目前的假设，即这些较大的途径是非选择性的。由于开发了一种新的方法来检测和量化较大分子通过通道形成的路径，这项研究可以继续下去。测量了较大分子对小离子流动的干扰。如果成功，这项研究将改变我们对大通道在细胞生活中的作用的看法。

Transmembrane channels are involved in many cellular processes, such as transport of metabolites, proteins and transcription factors through biological membranes. It is evident that the ability of large channels to permeate small ions does not necessarily reflect their ability to allow larger ions or macromolecules to cross the membrane. However, the same basic electrophysiological techniques, initially developed for studies of ion-selective channels, are routinely applied to all groups of channels. A new approach was developed that opens the possibility to monitor channel permeability for virtually any molecule. The method allows measurement, at the single channel level, of the transport of relatively high molecular weight molecules such as metabolites and macromolecules in the presence of small permeant ions. This is potentially a very general method and should be applicable to measuring the permeation of a wide-range of physiologically important solutes through a variety of different channels. By using the mitochondrial channel, called VDAC, it is intended to show that while the channels with large pores are poorly selective for small ions, they may show high selectivity for the larger molecules they evolved to transport. The new approach will be used to measure the partitioning of metabolites into the channel and their mobility within the channel. This is achieved by analyzing the change in average current and current noise produced when these metabolites now through a single channel and thus obstruct the passage of the smaller ions. Preliminary evidence demonstrates that VDAC selects among metabolites based on their 3-dimensional structure, not merely size and charge. ATP permeates the pore, but a glutamate tetrapeptide with similar size and charge does not. The nature of this selectivity will be examined by using other molecules whose physical properties resemble those of ATP. In addition, the importance of charge distribution within the pore that favors ATP permeation over other molecules will be verified. A panel of site-directed mutants will be used. To explore the possibility that this selectivity is a conserved property, VDAC channels isolated from very different species will be analyzed. Different VDAC isoforms within the same organism will be examined to determine whether they exhibit different selectivity patterns reflecting their specialized functions. Furthermore, apocytochrome c permeation will be studied in order to determine whether small proteins might be able to go through VDAC. If successful, this work will gain needed insight into the transport characteristics of large channels to develop ideas about their metabolite selectivity.The ability of living organisms and individual cells to compartmentalize is critical to life. This compartmentation allows each region to maintain an environment especially tuned to the function it serves. Proteins called channels aid in this compartmentation by allowing selected molecules and ions to cross the barriers between the compartments, the membranes. This project explores the hypothesis that channels that allow the passage of large molecules do so in a highly selective way. This differs from the current presumption that these larger pathways are nonselective. The research can be pursued thanks to the development of a new method to both detect and quantitate the passage of larger molecules through the pathways formed by channels. The interference of the flow of small ions by the larger molecules is measured. If successful, this research will change the way we think about the role of large channels in the lives of cells.