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：
7475614
负责人：
GUILLERMO A ALTENBERG
金额：
$ 27.4万
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 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7475614
关键词：
6-carboxyfluorescein 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 Helix (Snails)Hereditary Disease Individual Integral Membrane Protein Iodoacetates Ions Kidney Knowledge Length Liver Measures Mediating Methods Modeling Mutation Numbers 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 ear helix 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）：连接蛋白是组成膜蛋白，形成了间隙 - 连接通道，可介导MR <1,000的离子和亲水性分子的细胞对细胞渗透，从而在相邻细胞之间进行电和化学偶联。连接素对于细胞和组织的胚胎发育和正常功能至关重要，它们也参与遗传和获得的病理过程。六个连接蛋白单体包含四个跨膜A螺旋（M1至M4），形成一个间隙 - 连接的半通道。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，这是两个相邻细胞中的每个。可用的结构信息不足以识别单个跨膜螺旋，因此我们不知道哪种螺旋形成孔，以及螺旋螺旋如何在间隙 - 连接通道和半频道中折叠。该建议旨在解决知识中的这些差距。我们的一般目标是了解间隙 - 连接通道和半通道孔的渗透率特性的结构碱基，我们的中心假设是孔孔M1和M3线孔，但是孔的折叠与当前模型中提出的跨膜螺旋的折叠不同。 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和域结构（CX43和CX26）相同。为了实现这些目标，我们将采用生化，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，我们可以在包含单个捐赠者和所选位置的含量为已知捐赠者的间隙 - 连接半渠道中使用发光谐振能量转移来测量螺旋之间的距离。我们的建议将导致最佳的间隙缝合半通道模型。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，连接蛋白的突变会引起许多遗传疾病，包括耳聋。需要阐明间隙通道和半通道孔的结构，对于了解康纳蛋白突变引起的疾病机制是必要的。