Architecture of the transmembrane pore formed by connexin 43

描述（由申请人提供）：连接蛋白是形成间隙连接通道的整合膜蛋白，间隙连接通道介导Mr <1，000的离子和亲水分子的细胞间渗透，因此是相邻细胞之间的电和化学耦合的基础。连接蛋白对胚胎发育和细胞及组织的正常功能至关重要，并且它们还参与遗传和获得性的病理过程。六个连接蛋白单体，每个包含四个跨膜α螺旋（M1至M4），形成间隙连接半通道。缝隙连接通道通过缝隙连接半通道的端对端对接形成，两个相邻细胞各一个缝隙连接半通道。现有的结构信息不足以识别单个跨膜螺旋，因此我们不知道哪些螺旋形成孔以及螺旋如何在间隙连接通道和半通道中折叠。本提案旨在填补这些知识空白。我们的总体目标是了解间隙连接通道和半通道孔的渗透性的结构基础，我们的中心假设是跨膜螺旋M1和M3排列在孔中，但跨膜螺旋的折叠与当前模型中提出的不同。我们的具体目标是：1）确定Cx43跨膜螺旋残基对孔的水性环境的可接近性，2）通过测量螺旋间距离鉴定间隙连接半通道中的单个Cx43螺旋，和3）确定半通道内跨膜螺旋的折叠是否形成显示分子大小显著差异的连接蛋白，氨基酸序列和结构域结构（Cx43和Cx26）相同。为了实现这些目标，我们将采用生物化学、细胞生物学和生物物理学技术，包括使用新开发的实验系统，在该系统中，我们可以在选定的位置处使用包含单个供体和已知数量的受体的间隙连接半通道中的发光共振能量转移来测量螺旋间距离。我们的建议将导致最好的可用间隙连接半通道模型。重要性：连接蛋白对包括心脏在内的许多器官的正常发育至关重要，连接蛋白的突变会导致包括耳聋在内的许多遗传疾病。阐明间隙连接通道和半通道孔的结构对于理解连接蛋白突变引起疾病的机制是必要的。

DESCRIPTION (provided by applicant): Connexins are integral membrane proteins that form the gap-junctional channels that mediate cell-to- cell permeation of ions and hydrophilic molecules of Mr < 1,000, hence underlying electrical and chemical coupling between neighboring cells. Connexins are essential for embryonic development and normal function of cells and tissues, and they also participate in pathological processes, both genetic and acquired. Six connexin monomers, containing four transmembrane a helices each (M1 to M4), form a gap-junctional hemichannel. Gap-junctional channels are formed by end-to-end docking of gap- junctional hemichannels, one from each of two adjacent cells. The available structural information is insufficient to identify individual transmembrane helices, and therefore we do not know which helices form the pore and how the helices fold in the gap-junctional channels and hemichannels. This proposal aims to address these gaps in knowledge. Our general goal is to understand the structural bases for the permeability properties of the gap-junctional channel and hemichannel pore, and our central hypothesis is that transmembrane helices M1 and M3 line the pore, but the folding of the transmembrane helices is different from that proposed in the current models. Our specific aims are: 1) to determine the accessibility of Cx43 transmembrane-helix residues to the aqueous environment of the pore, 2) to identify the individual Cx43 helices in gap-junctional hemichannels by measuring inter-helix distances, and 3) to determine whether the folding of transmembrane helices within hemichannels formed connexins that display significant differences in molecular size, amino-acid sequence and domain structure (Cx43 and Cx26) are the same. To accomplish these aims, we will employ biochemical, cell- biological and biophysical techniques, including the use of a newly-developed experimental system where we can measure inter-helical distances using luminescence resonance energy transfer in gap- junctional hemichannels containing a single donor and a known number of acceptors, at selected positions. Our proposal will result in the best available gap-junctional hemichannel model. Significance: connexins are essential for the normal development of many organs, including the heart, and mutations of connexins cause a number of genetic diseases, including deafness. Elucidation of the architecture of gap-junctional channel and hemichannel pores is necessary to understand the mechanisms of disease due to connexin mutations.