Structure/Function of Gap Junctions

间隙连接的结构/功能

基本信息

批准号：
7585665
负责人：
Thaddeus Andrew Bargiello
金额：
$ 48.07万
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 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7585665
关键词：
Accounting Adopted Amino Acids Binding Biochemical Biological Assay Charcot-Marie-Tooth Disease Chemical Agents Connexin 43 Connexins Cysteine Data Dependence Disease Disulfides Elements Etiology Freezing Future Gap Junctions Gene Family Hydrophobic Interactions Individual Ion Channel Knowledge Lead Length Maps Membrane Metals Methods Modeling Molecular Molecular Conformation Mutation N-terminal Nature Pattern Peptides 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 electron density extracellular flexibility gap junction channel image processing insight interest loss of function mutant oleylamide peptide structure research study sensor voltage

项目摘要

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.

围绕当前模型的通道结构的当前模型存在很大争议连接蛋白基因家族。 CX32频道的最新模型（Fleishman等，2004），该模型基于 CX43的结构，通过冷冻水合物2D晶体的图像处理获得（Linger等，1999）使用第三跨膜段M3，形成大多数水通道孔。支持此观点通过骗局（替代的半胱氨酸可及性法）的结果CX32研究（Skerrett等，2002），但不是Zhou等报道的骗局研究。（1997）和Kronengold等。（2003）。这些作者指出，M1和第一个细胞外环E1的一部分形成了孔的孔 CX32*43E1和CX46功能性半通道。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这种观点连接蛋白通道的孔主要由M1形成，并证明CX32*43E1 半通道可以通过CD2+桥的形成在状态依赖构象中在相邻M1/E1螺旋中取代半胱氨酸残基。我们的结果表明，连接蛋白的关闭通过循环门控的通道是由M1/E1段的旋转导致的。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键键形成，以确定接近度位于半通道孔深处的残基的关系并建立其功能相关性。这能够以开放和封闭构型将通道通道锁定的能力为探索性质提供了一种手段电压门控的构象变化。我们建议使用与国家依赖的锁定建立 VJ和环路门控之间的关系，两种形式的电压门控为所有连接素的共同点。我们将继续使用NMR解决野生型和突变型N末端的结构。我们过去的研究有建议N末端的结构主要取决于保守的非极性残基，并通过在第12个残基附近的高度柔性转弯。我们建议求解突变肽的结构以检验这些假设。