Structure/Function of Gap Junctions

间隙连接的结构/功能

基本信息

批准号：
7216386
负责人：
Thaddeus Andrew Bargiello
金额：
$ 46.68万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-02-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7216386
关键词：
Accounting Adopted Amino Acids Binding Biochemical Biological Assay Charcot-Marie-Tooth Disease Chemical Agents Closure Connexin 43 Connexins Cysteine Data Dependence Depth Disease Disulfides Elements Etiology Freezing Future Gap Junctions Gene Family Helix (Snails)Hydrophobic Interactions Individual Ion Channel Knowledge Lead Length Maps Membrane Metals Methods Modeling Molecular Molecular Conformation Mutation N-terminal Nature Pattern Peptides Pliability Positioning Attribute Proline Property Reagent Recombinants Reporting Research Personnel Resolution Rotation Solutions Specific qualifier value Structural Models Structure Testing Xenopus oocyte aqueous base connexin 32 connexin pore disulfide bond ear helix electron density extracellular gap junction channel image processing insight interest loss of function mutant oleylamide peptide structure research study sensor voltage

项目摘要

DESCRIPTION (provided by applicant): There is considerable controversy surrounding current models of the structure of channels formed by the connexin gene family. A recent model of the Cx32 channel (Fleishman et al. 2004) that is based on the structure of Cx43, obtained by image processing of frozen hydrate 2D crystals (Linger et al. 1999) uses the third transmembrane segment, M3, to form the majority of the aqueous channel pore. This view is supported by results of SCAM (substituted cysteine accessibility method) studies of Cx32 intercellular channels (Skerrett et al., 2002) but not by the SCAM studies reported by Zhou et al. (1997) and Kronengold et al. (2003). These authors indicate that M1 and a portion of the first extracellular loop E1 form the pore of Cx32*43E1 and Cx46 functional hemichannels. We propose to use disulphide-trapping methods to test the helical contact points predicted by these disparate models. Our preliminary studies strongly support the view that the pore of connexin channels is formed primarily by M1 and demonstrate that the Cx32*43E1 hemichannel can be locked in a state dependent conformation by the formation of Cd2+bridges between substituted cysteine residues in adjacent M1/E1 helices. Our results suggest, that the closure of connexin channels by loop-gating results from a rotation of the M1/E1 segment. We propose to continue studies of disulphide bond formation between substituted cysteines in the M1/E1 region to determine the proximity relations of residues located deeper in the hemichannel pore and to establish their functional correlates. The ability to lock channel channels in open and closed conformations provides a means to explore the nature of conformational changes that underlie voltage gating. We propose to use state-dependent lock to establish the relation between Vj and loop-gating, two forms of voltage gating that are common to all connexins. We will continue to use NMR to solve the structure of wild type and mutant N-termini. Our past studies have suggested that the structure of N-terminus is determined largely by hydrophobic interactions among conserved non-polar residues and by the presence of highly flexible turn in the vicinity of the 12th residue. We propose solve the structure of mutant peptides to test these hypotheses.

描述（由申请人提供）：围绕着连接蛋白基因家族形成的通道结构的当前模型存在很大的争议。 CX32通道的最新模型（Fleishman等，2004）基于CX43的结构，该结构是通过冷冻水合物2D晶体的图像处理获得的（Linger等，1999）使用的第三个跨膜段M3，以形成大多数水通道孔。这种观点得到了CX32间细胞间通道的骗局（Skerrett等，2002）的骗局（替代性半胱氨酸可及性法）的结果的支持，但不是由Zhou等报道的骗局研究。（1997）和Kronengold等。（2003）。这些作者表明，第一个细胞外环E1的M1和一部分形成了CX32*43E1和CX46功能半通道的孔。我们建议使用二硫键捕获方法测试这些不同模型预测的螺旋接触点。我们的初步研究强烈支持以下观点：连接蛋白通道的孔主要由M1形成，并证明CX32*43E1 Hemichannel可以通过形成CD2+桥的形成在邻近的M1/E1/E1螺旋壳中的替代囊肿残基之间的CD2+桥。我们的结果表明，通过循环门控通过M1/E1段旋转导致连接蛋白通道的闭合。我们建议继续研究M1/E1区域中取代的半胱氨酸之间的二硫化物键形成，以确定位于半通道孔中更深层的残基的接近性关系并建立其功能相关性。锁定通道通道以开放和封闭构象的能力为探索电压门控构象变化的性质提供了一种手段。我们建议使用与状态依赖性锁一起建立VJ和环门之间的关系，即所有连接素共有的两种形式的电压门控。我们将继续使用NMR来解决野生型和突变的N末端的结构。我们过去的研究表明，N末端的结构在很大程度上取决于保守的非极性残基之间的疏水相互作用以及在第12个残基附近存在高度柔性的转弯。我们建议求解突变肽的结构以检验这些假设。