Mutant cochlear connexins associated with deafness

与耳聋相关的突变耳蜗连接蛋白

• 批准号: 7147778
• 负责人: GUILLERMO A ALTENBERG
• 金额: $ 10.15万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147778
• 关键词: SDS polyacrylamide gel electrophoresis; Xenopus; cochlea; deafness; gap junctions; membrane channels; mutant; protein purification; protein structure function; protein transport; technology /technique development

DESCRIPTION (provided by applicant): Gap junctions are aqueous channels permeable to ions and hydrophilic molecules of Mr < 1,000. Each gapjunctional channel is formed by head-to-head association of two connexons (connexin hexamers), one from each of the adjacent cells. The cochlea (responsible for mechano-transduction of sound waves into electrical impulses) has a highly-developed gap-junctional network that is essential for hearing. In most cells of the normal cochlea gap-junctional channels are heteromeric assemblies formed by the connexins Cx26 and Cx30. Infant deafness due to mutations of Cx26 is very common and our long-term objective is to elucidate at the molecular level the mechanisms by which mutations of connexins cause deafness. The properties of wild-type Cx26, Cx30 and heteromeric Cx26/Cx30 connexons, and the effects of mutations that cause deafness on the properties of heteromeric connexons that include wild-type connexins, are poorly understood. This is in part due to the fact that the few groups that have carried out functional experiments on Cx26 and Cx30 mutants work with complex systems, performing measurements that depend on the gapjunctional communication between two neighboring cells. This kind of experiments is essential to understand the effects of the mutations, but cannot fully address the molecular mechanism of the alterations. The specific aims of this proposal are: 1) to develop an expression/purification/reconstitution system that yields large amounts of functional wild-type and mutant Cx26 and Cx30, and 2) to test the usefulness of the purified connexon system to determine the functional properties of connexons formed by wild-type and mutant connexins. We will adapt the methodology that we developed recently for Cx43, which should allow us to obtain large amounts of functional Cx26 and Cx30, and connexin mutants. We will study the permeability properties of wild-type and mutant homomeric Cx26 and Cx30 connexons and heteromeric Cx26/Cx30 connexons formed by wild-type connexins as well as connexons containing Cx26 or Cx30 mutants. An integrative approach comparing the properties of gap- junctional channels and connexons will allow us to determine whether specific mutations alter gap-junctional communication at the level of the gap-junctional channel (e.g., docking between GJH) or connexon (e.g., non-permeable connexons).

描述（由申请人提供）：间隙连接是可渗透Mr<1,000的离子和亲水分子的水通道。每个间隙连接通道由两个连接子（连接蛋白六聚体）头对头连接形成，每个连接子来自相邻细胞。耳蜗（负责将声波机械传导为电脉冲）具有高度发达的间隙连接网络，这对于听力至关重要。在正常耳蜗间隙连接通道的大多数细胞中，是由连接蛋白 Cx26 和 Cx30 形成的异聚体组装体。 Cx26 突变导致的婴儿耳聋非常常见，我们的长期目标是在分子水平上阐明连接蛋白突变导致耳聋的机制。野生型 Cx26、Cx30 和异聚 Cx26/Cx30 连接子的特性，以及导致耳聋的突变对包括野生型连接蛋白在内的异聚连接子特性的影响，人们知之甚少。部分原因是少数几个对 Cx26 和 Cx30 突变体进行功能实验的小组使用复杂的系统，进行依赖于两个相邻细胞之间间隙连接通讯的测量。此类实验对于了解突变的影响至关重要，但不能完全解决突变的分子机制。该提案的具体目标是：1) 开发一种表达/纯化/重构系统，产生大量功能性野生型和突变型 Cx26 和 Cx30，2) 测试纯化连接子系统的有用性，以确定功能性连接子系统的有效性。由野生型和突变型连接蛋白形成的连接子的特性。我们将采用最近为 Cx43 开发的方法，这将使我们能够获得大量功能性 Cx26 和 Cx30 以及连接蛋白突变体。我们将研究野生型和突变型同聚 Cx26 和 Cx30 连接子以及由野生型连接蛋白形成的异聚 Cx26/Cx30 连接子以及含有 Cx26 或 Cx30 突变体的连接子的渗透性特性。比较间隙连接通道和连接子特性的综合方法将使我们能够确定特定突变是否改变间隙连接通道（例如，GJH之间的对接）或连接子（例如，非渗透性）水平上的间隙连接通讯。连接子）。