Architecture of the transmembrane pore formed by connexin 43

描述(申请人提供)：连接蛋白是一种完整的膜蛋白，它形成缝隙连接通道，调节MR&lt；1000的离子和亲水性分子在细胞之间的渗透，从而在相邻细胞之间进行潜在的电和化学耦合。连接蛋白对胚胎发育和细胞和组织的正常功能是必不可少的，也参与了遗传和后天的病理过程。6个连接蛋白单体，每个含有4个跨膜α螺旋(M1到M4)，形成一个缝隙连接半通道。缝隙连接通道是由缝隙连接半通道的端到端对接形成的，来自两个相邻单元中的每一个。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了孔，以及螺旋如何在缝隙连接通道和半通道中折叠。这项提议旨在解决知识方面的这些差距。我们的总体目标是了解缝隙连接通道和半通道孔渗透特性的结构基础，我们的中心假设是跨膜螺旋M1和M3排列在孔内，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环境的可及性，2)通过测量缝隙连接半管的螺旋间距来识别单个Cx43螺旋，以及3)确定半管内跨膜螺旋的折叠是否形成连接蛋白，这些连接蛋白在分子大小、氨基酸序列和结构域结构(Cx43和Cx26)上表现出显著的差异。为了实现这些目标，我们将使用生化、细胞生物学和生物物理技术，包括使用一种新开发的实验系统，在选定的位置，我们可以使用缝隙连接半通道中的发光共振能量转移来测量螺旋间距离，该缝隙连接半通道包含一个单一供体和已知数量的受体。我们的建议将产生最好的可用的缝隙连接半通道模型。意义：连接蛋白对包括心脏在内的许多器官的正常发育是必不可少的，连接蛋白的突变会导致许多遗传性疾病，包括耳聋。阐明缝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的疾病机制是必要的。

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.