Analysis of cSlo interacting proteins in the inner ear

内耳中 cSlo 相互作用蛋白的分析

• 批准号: 6516327
• 负责人: DHASAKUMAR S NAVARATNAM
• 金额: $ 14.74万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6516327
• 关键词: auditory discrimination; cell component structure /function; cell line; chickens; confocal scanning microscopy; ear hair cell; electrophysiology; gene delivery system; gene expression; immunocytochemistry; immunoprecipitation; infant animal; labyrinth; neural information processing; nucleic acid sequence; polymerase chain reaction; potassium channel; protein binding; protein localization; protein protein interaction; protein structure function; sound; tissue /cell culture; transfection; western blottings; yeast two hybrid system

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).

描述（来自申请人的摘要）：听力损失影响大约35 数百万美国人 它还影响了65岁和65岁以下儿童中的35%， 老了 听力损失的主要原因是由于听力损失。 毛细胞 我们的兴趣在于了解 确定毛细胞的功能，以及这些事件是如何控制的。 毛细胞 是声音的主要转换器，用于将机械能转换为 一个编码的神经元信号。 本建议旨在进一步了解如何 毛细胞区分声音的频率。 具体而言，它旨在 扩展最近的实验证据有关的一级结构的 钙门控钾通道，Slo，电共振;一种现象， 允许单个毛细胞区分不同频率的 声音 我们将寻找能结合并改变这种功能的蛋白质 通道，从而改变其响应特定频率的声音的能力。 许多策略将用于识别这些蛋白质，包括 简并RT PCR基于与已知与Slo结合的蛋白质的同源性， 酵母双杂交 利用羧基的测定  SLO的终点站作为“诱饵”。 一旦这些蛋白质 已被克隆和鉴定，标记的融合蛋白的免疫沉淀 将被用来确认相互作用实际上是真实的。 抗体将 在细菌中表达这些蛋白质后， 这些 蛋白质将与Slo通道共表达，以试图确定 如何改变其功能。 不同的电生理特性 Slo通道的亚型将在有和没有这些Slo结合的情况下进行研究 proteins. 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Slo在头发中的亚细胞分布 细胞 将尝试确定这些蛋白质是否分布在 在基底乳头内有差异。 这将使用RT PCR和 乳头的单个片段和免疫组织化学。 恒定 将继续进行监测，以确定这些基因组位点是否 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综合征 听力损失）。