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: 6748577
• 负责人: DHASAKUMAR S NAVARATNAM
• 金额: $ 14.16万
• 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/6748577
• 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 百万美国人。 它还影响了这 65 人和 35% 的人 年纪大了。 该群体听力损失的主要原因是听力损失 毛细胞。 我们的兴趣是了解分子事件 确定毛细胞功能以及如何控制这些事件。 毛细胞 是声音的主要换能器，用于转换机械能 声音到编码的神经元。 该提案旨在进一步了解如何 毛细胞区分声音的频率。 具体来说，它的目的是 扩展了最近有关一级结构的实验证据 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。 我们将寻找结合并改变其功能的蛋白质 通道从而改变其响应特定声音频率的能力。 将使用许多策略来识别这些蛋白质，包括 基于与已知与 Slo 结合的蛋白质同源性的简并 RT PCR， 和酵母两种杂交 利用 Slo 的羧基末端作为“诱饵”进行测定。 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 将用于确认交互实际上是真实的。 抗体会 在细菌中表达这些蛋白质后，可以针对这些蛋白质进行培养。 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。 不同的电生理特性 将在有或没有这些 Slo 结合的情况下研究 Slo 通道的亚型 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 基底乳头内有差异。 这将同时采用 RT PCR 乳头的单个片段和免疫组织化学。 持续的 将维持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）映射到任何综合症形式 听力损失（鉴于其他钾通道缺陷导致综合征 听力损失）。