Auditory Neurogenetics

描述(申请人提供)：听觉转导在很大程度上取决于Atp2b2基因(编码PMCA2蛋白)。我们的实验室和其他实验室已经在老鼠和人类基因中发现了几个突变(2007年发表在Tempel和Shling上的综述)。我们已经开发了一系列带有Atp2b2改变的Deafwaddler小鼠的等位基因突变，使我们能够研究耳蜗中功能性PMCA2的逐步减少的影响。我们已经使用这个等位基因系列来证明杂合性部分功能丧失突变是单倍体不足的(McCullough和Tempel，2004)，并提供了一个年龄相关性听力损失(AHL)的模型(McCullough和Tempel，2005；Walker等人，2008)。在这项建议中，我们将通过检测耳聋患者等位基因中的噪声诱发听力损失(NIHL)来增加我们对PMCA2在耳蜗中的作用的了解。我们将开发一种过度表达PMCA2的新小鼠品系，以观察这些小鼠是否对AHL或NIHL具有抵抗力。我们还将研究PMCA2在听觉脑干神经元中的作用。我们假设PMCA2在调节这些快速放电神经元中的钙离子(Ca~(2+))中起关键作用；这一作用以前没有被探索过，因为研究主要集中在PMCA2在毛细胞中的功能。最后，我们将开发一个细胞表达系统来研究PMCA2的功能。我们将使用PMCA2的半胱氨酸扫描突变来确定硫醇试剂在细胞外应用时可以迅速阻止钙泵活性的位置。除了提供对药物靶向的潜在位置的洞察外，这还应该为研究PMCA2在毛细胞(HC)转导和神经元传递中的细胞作用提供一种方法。在一个日益嘈杂和寿命更长的社会中，了解与年龄相关的听力损失和噪音导致的听力损失的基因是重要的。我们对质膜钙泵的研究表明，它定位于听觉毛细胞的静纤毛细胞，当它的功能因小鼠或人类的突变而降低时，它们会有显著的听力损失。我们正在研究钙泵的工作原理，希望了解如何使其保护毛细胞免受损害，从而防止听力损失。

DESCRIPTION (provided by applicant): Auditory transduction depends critically on the Atp2b2 gene (encoding the PMCA2 protein). Our lab and others have identified several mutations in mouse and human genes (reviewed in Tempel and Shilling, 2007). We have developed an allelic series of deafwaddler mouse mutants with altered Atp2b2 that allow us to study the effects of stepwise reductions of functional PMCA2 in the cochlea. We have used this allelic series to show that heterozygous partial loss of function mutations are haplo-insufficient (McCullough and Tempel, 2004) and provide a model of age-related hearing loss (AHL) (McCullough and Tempel, 2005; Walker et al., 2008). In this proposal we will add to our knowledge of the role of PMCA2 in the cochlea by examining noise-induced hearing loss (NIHL) in alleles of deafwaddler. We will develop a new line of mice that over-express PMCA2 to see if these mice are resistant to AHL or NIHL. We will also study the role of PMCA2 in neurons of the auditory brainstem. We hypothesize that PMCA2 plays a critical role in regulating calcium (Ca2+) in these fast-firing neurons; a role previously unexplored because studies have focused on PMCA2 function in hair cells. Finally, we will develop a cellular expression system for studying PMCA2 function. We will use cysteine-scanning mutagenesis of PMCA2 to define sites where thiol reagents, when applied extracellularly can rapidly block Ca2+ pump activity. Besides providing insight into potential sites for drug targeting, this should provide a way to study the cellular role of PMCA2 in hair cell (HC) transduction and neuronal transmission. In an increasingly noisy and longer-lived society, knowledge of the genes contributing to age-related hearing loss and noise induced hearing loss is important. Our work on the plasma membrane calcium pump shows that it is localized to the stereocilia of the auditory hair cells and that when its function is reduced by mutations in mouse or human, they have significant hearing loss. We are studying how the calcium pump works, hoping to learn ways to make it protect hair cells from damage, thereby preventing hearing loss.