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Architecture of the transmembrane pore formed by connexin 43

连接蛋白 43 形成的跨膜孔的结构

基本信息

批准号：
8080841
负责人：
GUILLERMO A ALTENBERG
金额：
$ 26.85万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8080841
关键词：
6-carboxyfluorescein Abbreviations Address Amino Acid Sequence Amino Acids Architecture Biochemical Biological Brain C-terminal Capillary Endothelial Cell Cell physiology Cells Chemicals Connexin 43 Connexins Connexon Coupling Cryoelectron Microscopy Cysteine Development Disease Docking Embryonic Development Environment Extracellular Space Face Genetic Goals Heart Hereditary Disease Individual Integral Membrane Protein Iodoacetates Ions Kidney Knowledge Length Liver Measures Mediating Methods Modeling Mutation Oocytes Organ Pathologic Processes Permeability Polyethylene Glycols Positioning Attribute Property Protein Isoforms Proteins Rana Research Research Personnel Series Side Structure Sulfhydryl Reagents System Techniques Time Tissues Transmembrane Domain Uncertainty Uterus aqueous base deafness extracellular improved luminescence resonance energy transfer methanethiosulfonate molecular size monomer mutant novel strategies programs reconstitution stoichiometry

项目摘要

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.

描述（由申请人提供）：连接蛋白是完整的膜蛋白，形成间隙连接通道，介导Mr<1,000的离子和亲水分子的细胞间渗透，因此在相邻细胞之间形成电和化学耦合。连接蛋白对于胚胎发育以及细胞和组织的正常功能至关重要，它们还参与遗传和后天的病理过程。六个连接蛋白单体，每个包含四个跨膜螺旋（M1 至 M4），形成间隙连接半通道。间隙连接通道是通过间隙连接半通道的端到端对接形成的，两个相邻细胞各有一个。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孔以及螺旋如何在间隙连接通道和半通道中折叠。该提案旨在解决这些知识差距。我们的总体目标是了解间隙连接通道和半通道孔隙渗透特性的结构基础，我们的中心假设是跨膜螺旋 M1 和 M3 排列在孔隙中，但跨膜螺旋的折叠与当前模型中提出的折叠不同。我们的具体目标是：1) 确定 Cx43 跨膜螺旋残基对孔水环境的可及性，2) 通过测量螺旋间距离来识别间隙连接半通道中的单个 Cx43 螺旋，3) 确定半通道内跨膜螺旋的折叠是否形成在分子大小、氨基酸序列方面表现出显着差异的连接蛋白域结构（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，我们可以在选定位置使用包含单个供体和已知数量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。我们的建议将产生最佳的间隙连接半通道模型。意义：连接蛋白对于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许多遗传疾病，包括耳聋。阐明间隙连接通道和半通道孔的结构对于了解连接蛋白突变引起的疾病机制是必要的。