Permeabilities and Pore Structures of Gap Junctions

描述（由申请人提供）：越来越多的证据（由上一个资助周期的工作显著贡献）表明，间隙连接通道对其渗透物显示出惊人的选择性水平，这取决于通道的连接蛋白组成。这种选择性的基础是复杂的，但是理解它对于定义这些细胞间通道在正常和致病细胞功能中发挥的不同作用至关重要。很可能这些通道的选择性渗透性的原则将不同于更好地研究离子通道，并将需要新一代的工具。目前的建议旨在开发这些工具，并将其应用于通过这些渠道的渗透性的分子决定因素的映射。由显示非常不同的渗透性性质的不同连接蛋白组成的间隙连接通道的比较分析应该定义这些孔内的结构多样性，这可能是它们不同性质的基础（目标#1）。然后，将在不同的连接蛋白中使用扫描和靶向突变和标记策略，以绘制渗透物大小和电荷的选择性决定因素，以及绘制对天然永久物的亲和力位点（目标#2）。我们还提出了细胞间间隙连接通道及其在细胞表面上的相应半通道的渗透性特征和孔结构的第一次全面比较（目标#3）。最初的证据表明，这两种通道亚型之间可能存在显著差异，并且越来越多的证据表明半通道在几个过程中的生理意义，包括通过凋亡等的细胞死亡，理解这些差异至关重要。这里提出的比较研究也应该解决在文献中的争议的实际性质的孔衬里。虽然差距连接家族的不同成员已被定位为各种令人惊讶的人类疾病的遗传原因，从白内障，皮肤病和周围神经麻痹到最常见的遗传性感觉神经性耳聋，但我们仍然几乎不了解这些基因的损伤如何导致观察到的表型。目前的工作将通过定义这些细胞间孔的分子结构以及如何调节通过它们的物质来解决这个问题。

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.