STRUCTURE-FUNCTION OF CONNEXIN PORES

描述(申请人提供)：这项建议通过使用孔道阻滞剂来探索连接蛋白通道的结构和功能，集中在两种缺陷导致神经病理的连接蛋白上。连接蛋白形成缝隙连接通道，这是离子细胞和细胞质分子之间直接运动的途径，包括最已知的第二信使。严重的病理由连接蛋白缺陷引起，包括脱髓鞘、耳聋、皮肤疾病和白内障，这取决于受影响的连接蛋白亚型。尽管有强烈的生物学和医学兴趣，但连接蛋白通道的决定性特性--孔道调节细胞间选择性分子通透性的能力--的机制尚未阐明。由于缺乏进入和结合在孔中的分子试剂，对连接蛋白孔结构和功能的研究一直受到阻碍。这类试剂(“孔道阻滞剂”)在阐明其他通道渗透的结构和功能方面具有不可估量的价值。该项目应用新发现的缝隙连接蛋白孔道阻滞剂来研究缝隙连接蛋白孔道，从而获得人们期待已久的关键信息。初步研究已经确定了两类基于碳水化合物的缝隙连接蛋白孔阻滞剂，并确定了它们作为缝隙连接蛋白通道研究工具的可行性。对于第一类，设计了新的氨基苯甲酸甘氨酰胺(ABGs)的甘氨酰胺衍生物，并将其与一组大小索引的麦芽糖偶联。所得到的ABG-糖结合物作为可逆的、高亲和力的阻滞剂，以大小和连接蛋白特定的方式通过连接蛋白孔进行分子渗透，而麦芽糖或ABG本身不能阻止分子渗透。第二类阻滞剂是环糊精(CDS)，它是环化的葡萄糖。它们还以一种可逆的、特定大小的方式阻止连接蛋白毛孔。对于这两类阻滞剂，阻断所需的分子大小和孔的相对宽度之间的相关性(使用一系列大小索引的可渗透糖确定)表明它们的作用部位在孔内。拟议的研究建立在这项工作的基础上。目的1研究ABG-糖结合物孔内结合的化学决定因素和机理。目的2开创ABG-糖在连接蛋白通道研究中的应用。目的3利用自然产生和修饰的CDS来探测连接蛋白的孔。这些项目主要利用一个特征良好的重建系统来研究异源表达的连接蛋白通道，以获得其他方法无法获得的信息。预计这些气孔阻滞剂的开发和应用将使生物物理和细胞研究成为可能，并为确定发育和疾病中细胞间通讯的分子机制提供信息。在本提案中，这一分析将应用于分别导致X连锁Charcot-Marie-Tooth病神经病和感觉神经性耳聋的Cx32和Cx26缺陷。

DESCRIPTION (provided by applicant): This proposal explores the structure-function of the connexin channel by the use of pore blockers, focusing on two connexins in which defects cause neurological pathologies. Connexin protein forms gap junction channels, which are pathways for direct movement between cells of ions and cytoplasmic molecules, including most known second messengers. Serious pathologies arise from connexin defects, including demyelination, deafness, skin disorders and cataracts, depending on the connexin isoform affected. Despite acute biological and medical interest, the mechanism of the defining property of connexin channels - the ability of the pore to mediate selective molecular permeability between cells - has not been elucidated. Investigation of the structure and function of connexin pores has been hampered by the absence of molecular reagents that enter and bind in the pore. This class of reagents ("pore blockers") has been of inestimable value in elucidation of the structure-function of permeation of other channels. This project applies newly identified connexin pore blockers to investigate the connexin pore, and to thus obtain key information that has been long desired. Preliminary studies have identified two classes of carbohydrate-based connexin pore blockers, and established their feasibility as investigational tools of connexin channels. For the first class, novel glycinamide derivatives of aminobenzoic acid glycinamides (ABGs) were designed and conjugated to a size-indexed set of maltosaccharides. The resulting ABG-glycoconjugates act as reversible, high affinity blockers of molecular permeation through connexin pores in a size- and connexin-specific manner, whereas the maltosaccharides or the ABGs alone do not block. The second class of blockers are cyclodextrins (CDs), which are cyclized glucosaccharides. They also block connexin pores in a reversible, size-specific manner. For both classes of blockers, the correlation between the size of the molecule required for block and the relative width of the pore (determined using a size-indexed series of permeable sugars) indicate that their site of action is within the pore. The proposed studies build on this work. Aim 1 investigates the chemical determinants and mechanism of the intra-pore binding of the ABG- glycoconjugates. Aim 2 initiates application of the ABG-sugars to the study of connexin channels. Aim 3 utilizes naturally-occurring and modified CDs to probe the connexin pore. The projects primarily utilize a well- characterized reconstitution system to study heterologously-expressed connexin channels to obtain information unavailable by other means. It is anticipated that the development and application of these pore blockers will enable and inform the biophysical and cellular studies required to define the molecular mechanisms of intercellular communication in development and disease. In the present proposal, this analysis will be applied to Cx32 and Cx26, defects in which cause X-linked Charcot-Marie-Tooth disease neuropathy and sensorineural deafness, respectively.