PERMEABILITIES AND PORE STRUCTURE OF GAP JUNCTIONS

在过去的几年里，间隙连接通讯与人类健康的广泛相关性已经通过不少于5种非常不同的疾病与连接蛋白基因缺陷的直接关联而得到了生动的说明，这表明间隙连接的连接蛋白表型可能赋予导致不同功能的特殊性质。 这一论点得到了小鼠中连接蛋白基因消除所导致的更大种类的表型的有力支持。 虽然细胞质结构域中连接蛋白序列的可变性导致了对其差异调节的显著兴趣，但差距连接的主要功能是低分子量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特异性肯定会给细胞间通讯的主题带来全新的复杂性。 该提案旨在建立在我们以前的示范连接蛋白之间的选择性离子，较大的痕迹，和天然代谢物通过定义孔的结构，它的门控元件和选择性过滤器，并使用它来开发模型来解释选择性的机制。 这些信息对于理解缝隙连接的生理作用，以及它们结构上的细微缺陷如何导致人类多种疾病状态是至关重要的。建议的具体步骤是：（1）使用SCAM，一种半胱氨酸扫描诱变的形式，其中引入巯基试剂并测试其阻断通道的能力，以绘制形成孔的连接蛋白残基;（2）测试SCAM通道阻断剂在不同门控状态期间对通道中不同位点的可接近性，以定义通道门控的物理位置（3）进一步表征连接蛋白的选择透过性性质，通过定量测定在特定性质上不同的较大探针家族，并通过连接蛋白的诱变，确定对人工和天然渗透物都赋予选择性性质的位点（4）开发3-使用Poisson-Nernst-Planck理论，结合在目标1和2中收集的孔的结构信息，通过差距连接孔的扩散的三维模型。

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.