Analysis of cSlo interacting proteins in the inner ear

描述(摘自申请者摘要)：听力损失影响约35 百万美国人。它还影响到65和65人中约35%的人 年纪更大了。这一组中听力损失的主要原因是 毛细胞。我们感兴趣的是了解 确定毛细胞的功能，以及如何控制这些事件。毛细胞 是声音的主要换能器，用来转换机械能 声音传给了一个编码的神经元。这项提案试图进一步了解如何 毛细胞区分声音的频率。具体地说，它旨在 扩展了最近关于一级结构的实验证据 钙门控钾通道，Slo，到电共振；一种现象， 允许单个毛细胞区分不同频率的 声音。我们将寻找与之结合并改变其功能的蛋白质。 频道因此改变了其对特定声音频率的响应能力。 将使用一些策略来识别这些蛋白质，包括 基于与已知与Slo结合的蛋白质的同源性的简并RT PCR， 和酵母双杂交 利用Slo的羧基末端作为“诱饵”进行检测。一旦这些蛋白质 已被克隆和鉴定，标记融合蛋白的免疫沉淀 将被用来确认这些互动实际上是真实的。抗体会 在细菌中表达这些蛋白质后，针对它们进行了提升。这些 蛋白质将与Slo通道共表达，试图确定 他们如何改变其功能(S)。不同脑组织的电生理学特性 将研究在有和没有这些Slo结合的情况下Slo通道的异构体 蛋白质。此外，共聚焦免疫荧光显微镜将用于 确定这些蛋白质如何改变头发中Slo的亚细胞分布 细胞。将尝试确定这些蛋白质是否分布 不同的是在基底乳头内。这将使用RT-PCR和 乳头的个别碎片和免疫组织化学。常量 将继续进行监测，以确定这些病毒的基因组位置 蛋白质(由基因组计划揭示)映射到任何一种综合征形式 听力损失(鉴于其他钾通道缺陷导致的综合征 听力损失)。

DESCRIPTION (from applicant's abstract): Hearing loss affects approximately 35 million Americans. It also affects approximately 35 percent of those 65 and older. The primary cause of hearing loss in this group is a result of a loss of hair cells. Our interest is in understanding the molecular events that determine hair cell function, and how these events are controlled. Hair cells are the primary transducers of sound serving to convert the mechanical energy of sound to a coded a neuronal one. This proposal seeks to understand further how hair cells discriminate between frequencies of sound. Specifically, it aims to extend on the recent experimental evidence relating the primary structure of the calcium gated potassium channel, Slo, to electrical resonance; a phenomenon that allows individual hair cells to discriminate between different frequencies of sound. We will look for proteins that bind to and alter the function of this channel thus altering its ability to respond to particular frequencies of sound. A number of strategies will be used to identify these proteins including degenerate RT PCR based on homology to proteins that re known to bind to Slo, and yeast two hybrid assays utilizing the carboxy  terminus of Slo as "bait". Once these proteins have been cloned and identified, immunoprecipitation of tagged fusion proteins will be used to confirm that the interactions are in fact real. Antibodies will be raised against these proteins after expressing them in bacteria. These proteins will be co-expressed with the Slo channel in an attempt to determine how they alter its function(s). The electophysiological properties of different isoforms of the Slo channel will be studied with and without these Slo binding proteins. In addition confocal immuno-fluorescence microscopy will be used to determine how these proteins alter the subcellular distribution of Slo in hair cells. Attempts will be made to determine if these proteins are distributed differentially within the basilar papilla. This will employ both RT PCR of individual fragments of the papilla and immunohistochemistry. Constant surveillance will be maintained to determine if the genomic locus of these proteins (revealed by the genome project) maps to any of the syndromic forms of hearing loss (in view of other potassium channel defects causing syndromic hearing loss).