Mutant cochlear connexins associated with deafness

描述（由申请人提供）：缝隙连接是Mr < 1000的离子和亲水分子可渗透的水通道。每个间隙连接通道是由两个连接子（连接蛋白六聚体）的头对头连接形成的，每个连接子来自相邻的细胞。耳蜗（负责声波转化为电脉冲的机械传导）具有高度发达的间隙连接网络，这对听力至关重要。在大多数正常耳蜗细胞中，间隙连接通道是由连接蛋白Cx26和Cx30组成的异质组合。由Cx26突变引起的婴儿耳聋是非常常见的，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和异质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子在内的异质连接子特性的影响，目前尚不清楚。这在一定程度上是由于少数几个对Cx26和Cx30突变体进行功能实验的小组在复杂的系统中工作，执行依赖于两个相邻细胞之间间隙通信的测量。这类实验对于了解突变的影响是必不可少的，但不能完全解决改变的分子机制。本提案的具体目的是：1)开发一种表达/纯化/重组系统，以产生大量功能性野生型和突变型Cx26和Cx30； 2)测试纯化的连接子系统对确定野生型和突变型连接子形成的连接子的功能特性的有用性。我们将采用我们最近为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源Cx26和Cx30连接子，以及由野生型连接子和含有Cx26或Cx30突变体的连接子形成的异质Cx26/Cx30连接子的通透性。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会在间隙连接通道（例如GJH之间的对接）或连接子（例如非渗透连接子）的水平上改变间隙连接通信。

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).