Permeabilities and Pore Structures of Gap Junctions

描述(由申请人提供)：越来越多的证据表明，缝隙连接通道对其渗透体具有惊人的选择性，这是由通道的连接蛋白组成决定的，上一个资金周期的工作对此做出了重大贡献。这种选择性的基础是复杂的，但理解它对于确定这些细胞间通道在正常和致病细胞功能中所起的不同作用至关重要。这些通道的选择性渗透性的控制原理很可能不同于研究得更好的离子通道，并将需要新一代工具。目前的提案寻求开发这些工具，并将它们应用于绘制通过这些通道的渗透性的分子决定因素图。对由不同连接蛋白组成的缝隙连接通道进行比较分析，这些连接蛋白表现出非常不同的渗透特性，应该确定这些孔内可能构成其不同特性的结构多样性(目标1)。然后，将在不同的连接蛋白中使用扫描和定向诱变和标记策略，以绘制渗透剂的大小和电荷的选择性决定因素，以及天然永久物质的亲和力位置(目标2)。我们还首次对细胞间隙连接通道及其在细胞表面的相应半通道的渗透性特征和孔结构进行了全面的比较(目标#3)。初步证据表明，这两种通道亚型之间可能存在显著差异，而越来越多的证据表明，半通道在几个过程中具有生理意义，包括细胞凋亡等，这使得理解这些差异变得至关重要。这里提出的比较研究也应该解决文献中关于孔隙衬里的实际性质的争议。虽然缝隙连接家族的不同成员被认为是多种人类疾病的遗传原因，从白内障、皮肤病和周围神经瘫痪到最常见的遗传性感音神经性耳聋，但我们仍然几乎不了解这些基因的破坏如何导致所观察到的表型。目前的工作将通过定义这些细胞间毛孔的分子结构以及这如何调节穿过它们的东西，在解决这个问题方面取得重大进展。

DESCRIPTION (provided by applicant): A growing body of evidence, contributed to significantly by work from the previous funding cycle, has demonstrated that gap junction channels display a surprising level of selectivity for their permeants, dictated by the connexin composition of the channels. The basis of this selectivity is complex, but understanding it will be critical to defining the diverse role these intercellular channels play in normal and pathogenic cell function. It is likely that the principles governing selective permeabilities of these channels will be distinct from the much better studied ion channels, and will require a new generation of tools. The current proposal seeks to develop these tools and to apply them to a mapping of the molecular determinants of permeabilty through these channels. A comparative analysis of gap junction channels comprised of different connexins that display very different permeability properties should define the structural diversity within these pores that may underlie their different properties (Aim #1). Scanning and targetted mutagenic and labeling strategies will then be used in the different connexins to map selectivity determinants for size and charge of permeants, as well as mapping sites of affinity for natural permants (Aim #2). We also propose the first comprehensive comparison of peremability characteristics and pore structure of intercellular gap junction channels and their corresponding hemichannels on the cell surface (Aim #3). Initial evidence suggests that there may be significant differences between these two channel isoforms, and growing evidence for the physiological significance of hemichannels in several processes, including cell death via apoptosis, etc., makes understanding such differences critical. The comparative studies proposed here should also resolve a controversy in the literature over the actual nature of the pore lining. While different members of the gap junction family have been mapped as the genetic causes of a surprising variety of human diseases, from catarracts, skin disease and peripheral nerve paralysis to the most common form of hereditary sensorineural deafness, we still have almost no understanding of how damage to these genes can cause the phenotypes observed. The current work will make a significant stride in resolving this issue by defining the molecular structure of these intercellular pores and how this can regulate what goes through them.