Mutant cochlear connexins associated with deafness

描述(申请人提供)：缝隙连接是离子和亲水分子渗透的水溶液通道，MR&lt；1000。每个缝隙连接通道是由两个连接蛋白(连接蛋白六聚体)头对头结合形成的，每个相邻细胞都有一个。耳蜗(负责将声波机械转换为电脉冲)有一个高度发达的缝隙连接网络，这对听力是必不可少的。在大多数正常耳蜗细胞中，缝隙连接通道是由连接蛋白Cx26和Cx30形成的异构体集合体。Cx26基因突变引起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和异构体Cx26/Cx30连接蛋白的特性，以及导致耳聋的突变对包括野生型连接蛋白在内的异构体连接蛋白特性的影响，目前还知之甚少。这在一定程度上是由于这样一个事实，即对Cx26和Cx30突变体进行功能实验的少数几个小组使用复杂的系统，执行依赖于两个相邻细胞之间缝隙连接通信的测量。这类实验对于了解突变的影响是必不可少的，但不能完全解决突变的分子机制。该方案的具体目的是：1)建立一个表达/纯化/重组系统，以产生大量具有功能的野生型和突变型Cx26和Cx30；以及2)测试纯化的连接蛋白系统的有用性，以确定由野生型和突变型连接蛋白形成的连接蛋白的功能特性。我们将采用我们最近为Cx43开发的方法，这应该允许我们获得大量的功能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源连接蛋白Cx26和Cx30以及由野生型连接蛋白形成的异构体Cx26/Cx30连接蛋白以及含有Cx26或Cx30突变的连接蛋白的通透性。通过比较缝隙连接通道和连接蛋白的性质，我们可以确定特定的突变是否会改变缝隙连接通道(如GJH之间的对接)或连接蛋白(如非渗透性连接蛋白)水平上的缝隙连接通讯。

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).