STRUCTURE-FUNCTION OF CONNEXIN PORES

连接蛋白孔的结构-功能

• 批准号: 7675278
• 负责人: Andrew L Harris
• 金额: $ 33.97万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7675278
• 关键词: 4-Aminobenzoic Acid; Acute; Address; Affect; Affinity; Aminobenzoic Acids; Binding; Biochemical; Biological; Carbohydrates; Cataract; Cell physiology; Cells; Charcot-Marie-Tooth Disease; Chemicals; Congenital Heart Defects; Connexins; Cyclodextrins; Data; Defect; Demyelinations; Development; Disease; Glycoconjugates; Goals; Health; Hereditary Disease; Human Genetics; Investigation; Ion Channel; Ions; Link; Mediating; Medical; Molecular; Molecular Structure; Movement; Neurons; Neuropathy; Pathology; Pathway interactions; Peripheral; Permeability; Pharmacology; Play; Point Mutation; Positioning Attribute; Property; Protein Isoforms; Proteins; Reagent; Relative (related person); Role; Second Messenger Systems; Sensorineural Hearing Loss; Series; Signal Transduction; Signaling Molecule; Site; Structure; System; Trauma; Variant; Width; Work; base; connexin pore; deafness; design; gap junction channel; glycine amide; indexing; intercellular communication; interest; mutant; neurological pathology; novel; reconstitution; research study; response; second messenger; skin disorder; sugar; tool

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.

描述（由申请人提供）：该提案通过使用孔阻断剂探索连接蛋白通道的结构-功能，重点关注两种连接蛋白，其中缺陷导致神经病理学。连接蛋白蛋白形成间隙连接通道，其是离子和细胞质分子（包括大多数已知的第二信使）在细胞之间直接移动的途径。严重的病理由连接蛋白缺陷引起，包括脱髓鞘、耳聋、皮肤病和白内障，这取决于受影响的连接蛋白同种型。尽管急性生物学和医学的兴趣，连接蛋白通道的定义属性的机制-孔介导细胞之间的选择性分子渗透性的能力-尚未阐明。连接蛋白孔的结构和功能的研究一直受到阻碍的情况下，进入和结合在孔的分子试剂。这类试剂（“孔阻断剂”）在阐明其他通道渗透的结构-功能方面具有不可估量的价值。本计画应用新近发现的连接蛋白孔阻断剂来研究连接蛋白孔，并借此获得长久以来所期待的关键资讯。初步研究已经确定了两类基于碳水化合物的连接蛋白孔阻断剂，并确定了它们作为连接蛋白通道研究工具的可行性。对于第一类，新的氨基苯甲酸甘氨酰胺衍生物（ABG）的设计和共轭的麦芽糖醇的大小索引集。所得的ABG-糖缀合物作为可逆的，高亲和力阻断剂的分子渗透通过连接蛋白孔的大小和连接蛋白特异性的方式，而麦芽糖醇或ABG单独不阻止。第二类阻断剂是环糊精（CD），它是环化的葡萄糖。它们还以可逆的、大小特异性的方式阻断连接蛋白孔。对于这两类阻断剂，阻断所需的分子大小与孔的相对宽度之间的相关性（使用可渗透糖的大小索引系列确定）表明它们的作用位点在孔内。拟议的研究建立在这项工作的基础上。目的1研究ABG-糖缀合物孔内结合的化学决定因素和机制。目的2：首次将ABG糖应用于连接蛋白通道的研究。目的3利用天然存在的和修饰的CD来探测连接蛋白孔。该项目主要利用一个良好表征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。预计这些孔阻断剂的开发和应用将使生物物理和细胞研究能够确定发育和疾病中细胞间通讯的分子机制并为其提供信息。在本提案中，该分析将应用于Cx 32和Cx 26，这两种缺陷分别导致X连锁腓骨肌萎缩症神经病变和感觉神经性耳聋。