PERMEABILITIES AND PORE STRUCTURE OF GAP JUNCTIONS

在过去的几年里，缝隙连接通讯与人类健康的广泛相关性已经被至少5种非常不同的疾病与连接蛋白基因缺陷的直接关联所图示，这表明缝隙连接的连接蛋白表型可能赋予特殊的特性，导致不同的功能。这一论点得到了更多表型的强烈支持，这些表型是由小鼠连接蛋白的遗传消融引起的。尽管细胞质区连接蛋白序列的差异性引起了人们对其差异调控的浓厚兴趣，但缝隙连接的主要功能是低分子代谢物在细胞之间的扩散。通过不同的连接蛋白过滤这些信号的专一性肯定会给细胞间通信的主题带来全新的复杂性。这项建议试图通过定义孔隙的结构、其门控元件和选择性过滤器来建立我们之前证明的连接蛋白对离子、较大的痕量和自然代谢物之间的选择性，并利用这一点来开发模型来解释选择性的机制。这些信息是理解缝隙连接的生理作用的核心，以及它们结构中相当细微的缺陷如何导致人类各种疾病状态。建议的具体步骤是：(1)使用SCAM，一种半胱氨酸扫描突变的形式，在其中引入巯基试剂并测试其阻断通道的能力，以定位形成孔的连接蛋白的残基；(2)测试SCAM通道阻断剂在不同门控状态下对通道中不同位置的可达性，以确定通道门的物理位置(S)(3)通过对不同特性的较大探针家族的定量分析，进一步表征连接蛋白的渗透选择性，并通过对连接蛋白(S)的诱变，定义赋予人工和天然渗透剂选择性特性的位置(4)利用泊松-能斯特-普朗克理论，结合在AIMS 1和2收集的孔的结构信息，建立一个通过缝隙连接孔扩散的三维模型。

The broad relevance of gap junctional communication to human health has been graphically illustrated over the last few years by the direct association of no less than 5 very distinct diseases with defects in connexin genes, indicating that the connexin phenotype of a gap junction may impart specialized properties that lead to diverse functions. This contention is strongly supported by the even greater variety of phenotypes that result from genetic ablation of connexins in mice. Although the variability in connexin sequences in the cytoplasmic domains has led to significant interest in their differential regulation, the major function that the gap junctions subserve is the diffusion of low MW metabolites between cells. Specificity in the filtering of these signals by different connexins would certainly impose a whole new layer of complexity to the topic of intercellular communication. This proposal seeks to build on our previous demonstration of selectivity among connexins for ions, larger traces, and natural metabolites by defining the structure of the pore, its gating elements and selectivity filters, and to use this to develop models to explain the mechanism of selectivity. This information is central to understanding the physiological role of gap junctions, and how quite subtle defects in their structure can lead to a diverse array of disease states in man. Specific steps proposed are:(1) Use SCAM, a form of cysteine scanning mutagenesis in which sulfhydryl reagents are introduced and tested for ability to block the channel, to map the residues of connexins that form the pore; (2) Test the accessability of the SCAM channel blocking reagents to different sites in the channel during different gating states to define the physical locations of the channel gate(s) (3) Further characterize the permselectivity properties of connexins with quantitative assays of families of larger probes that vary in specific properties, and, through mutagenesis of the connexin(s), define the sites that confer selecytivity properties for both artificial and natural permeants (4) Develop a 3-dimensional model of diffusion through the gap junction pore using Poisson- Nernst-Planck theory, incorporating structural information on the pore gleaned in Aims 1 and 2.