PERMEABILITIES AND PORE STRUCTURE OF GAP JUNCTIONS

在过去的几年中，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，已经图解地说明了间隙连接通讯与人类健康的广泛相关性，这表明间隙连接的连接蛋白表型可能赋予导致多种功能的特殊特性。这一论点得到了小鼠连接蛋白基因消融导致的更大种类表型的有力支持。尽管细胞质结构域中连接蛋白序列的可变性引起了对其差异调控的极大兴趣，但间隙连接的主要功能是细胞间低分子量代谢物的扩散。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增加一个全新的复杂性。本提案旨在通过定义孔的结构、其门控元件和选择性过滤器，建立我们之前对离子、较大痕量和天然代谢物的连接蛋白之间的选择性的论证，并利用这一点来开发模型来解释选择性的机制。这一信息对于理解间隙连接的生理作用，以及其结构中相当细微的缺陷如何导致人类多种疾病状态至关重要。提出的具体步骤是：(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的残基；(2)测试在不同门控状态下，SCAM通道阻断试剂对通道中不同位点的可达性，以确定通道门的物理位置。(3)通过对具有不同特定性质的较大探针家族的定量分析，进一步表征连接蛋白的超选择性特性，并通过连接蛋白的诱变，(4)利用泊松-能思-普朗克理论，结合目标1和目标2中收集到的孔结构信息，建立一个通过间隙连接孔的三维扩散模型。

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.