Structure/Function of Gap Junctions

描述(由申请人提供)：围绕连接蛋白基因家族形成的通道结构的当前模型存在相当大的争议。Cx32通道的最新模型(Fleishman等人2004)，其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)使用第三跨膜段M3来形成水通道孔的大部分。这一观点得到了对Cx32细胞间通道的SCAM(取代半胱氨酸可及性方法)研究结果的支持(Skerrett等人，2002年)，但没有得到周等人报告的SCAM研究的支持。(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功能半通道的孔。我们建议使用二硫化物捕捉法来测试这些不同模型预测的螺旋接触点。我们的初步研究有力地支持了连接蛋白通道的孔主要由M1形成的观点，并证明了Cx32*43E1半通道可以通过在相邻M1/E1螺旋上取代的半胱氨酸残基之间形成CD2+桥而被锁定在一种状态依赖的构象中。我们的结果表明，通过环门控关闭连接蛋白通道是M1/E1节段旋转的结果。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，以确定位于半通道孔深处的残基的邻近关系，并建立它们的功能关联。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电压门控背后的构象变化的本质的方法。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来解决野生型和突变型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保守的非极性残基之间的疏水相互作用和第12残基附近高度柔性转弯的存在决定的。我们建议通过解决突变多肽的结构来验证这些假说。

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.