Structure/Function of Gap Junctions

间隙连接的结构/功能

• 批准号: 7391588
• 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
• 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/7391588
• 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

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.

目前，围绕由水形成的河道结构模型存在相当大的争议。 连接蛋白基因家族Cx 32通道的最新模型（Fleishman et al. 2004）是基于 通过冷冻水合物2D晶体的图像处理获得的Cx43的结构（Linger等人，1999）使用 第三跨膜区段M3，以形成大部分的水通道孔。这一观点得到支持 通过Cx 32细胞间通道的SCAM（取代半胱氨酸可及性方法）研究结果 （Skerrett等人，2002），但不是由周等人（1997）和Kronengold等人（1998）报道的SCAM研究。 （2003年）的报告。这些作者指出，M1和第一胞外环E1的一部分形成了 Cx 32 * 43 E1和Cx46功能性半通道。我们建议使用二硫化物捕获方法来测试 这些不同的模型预测的螺旋接触点。我们的初步研究强烈支持这一观点 连接蛋白通道的孔主要由M1形成，并证明Cx 32 * 43 E1 半通道可以通过在半通道之间形成Cd 2+桥而锁定在状态依赖性构象中。 取代的半胱氨酸残基在相邻的M1/E1螺旋。我们的研究结果表明，连接蛋白的关闭 通过循环门控的通道是M1/E1段旋转的结果。我们建议继续研究 M1/E1区中取代的半胱氨酸之间的二硫键形成，以确定 位于半通道孔更深处的残基的关系，并建立其功能相关性。的 在开放和闭合构象中锁定通道通道的能力提供了一种探索 构成电压门控的基础的构象变化。我们建议使用状态依赖锁来建立 Vj和环路选通之间的关系，这是所有连接点共有的两种电压选通形式。我们 将继续使用NMR来解决野生型和突变体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是由疏水相互作用决定的， 保守的非极性残基和在第12个残基附近存在高度柔性的转角。 我们建议解决突变肽的结构来验证这些假设。