Structure/Function of Gap Junctions

围绕着目前由 连接蛋白基因家族。Cx32通道的最新模型(Fleishman等人2004)，这是基于 Cx43的结构，通过冷冻水合物2D晶体的图像处理获得(Linger et al.1999)使用 第三跨膜段，M3，形成水通道的大部分孔。这种观点是受支持的 用取代半胱氨酸可及性方法研究Cx32细胞间通道的结果 (Skerrett等人，2002)，但不是通过周等人报告的骗局研究。(1997)和Kronengold等人。 (2003)。这些作者指出，M1和第一细胞外环E1的一部分形成了 Cx32*43E1和Cx46功能半通道。我们建议使用二硫化物捕捉法来测试 由这些不同的模型预测的螺旋接触点。我们的初步研究有力地支持了这一观点 连接蛋白通道的孔道主要由M1形成，证明了Cx32*43E1 半通道可以通过形成Cd~(2+)-桥而被锁定在依赖于状态的构象中 相邻M1/E1螺旋中取代的半胱氨酸残基。我们的结果表明，缝隙连接蛋白的关闭 通过环路选通的通道由M1/E1段的旋转产生。我们建议继续研究 M1/E1区取代半胱氨酸之间的二硫键形成以确定其近似性 研究位于半通道孔深处的残留物之间的相互关系，并建立它们之间的功能关系。这个 在开放和封闭的构象中锁定水道的能力提供了一种探索 电压门控背后的构象变化。我们建议使用状态依赖锁来建立 VJ和环路门控之间的关系，这是所有连接蛋白共同的两种电压门控形式。我们 将继续使用核磁共振技术解决野生型和突变型N-末端的结构问题。我们过去的研究已经 认为N末端的结构很大程度上是由相互间的疏水作用决定的 保守的非极性残基和第12残基附近高度灵活的转折。 我们建议通过解决突变多肽的结构来验证这些假说。

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.