Regulation of alpha9/10-nAChR and SK2 function and localization at auditory synap

描述(申请人提供)：内耳中的橄榄耳蜗型尼古丁突触在正常听力中起着关键作用。橄榄耳蜗径纤维起源于大脑，并直接支配机械感觉毛细胞。在发育过程中，这些突触是正常听力敏感度、频率选择性和调位映射形成所必需的。然而，人们对橄榄耳蜗突的正确组装和功能所需的分子机制知之甚少。A9/10-烟碱型乙酰胆碱受体(NAChRs)介导毛细胞的突触传递。A9/10-nAChRs与小电导钙激活钾(SK2)通道的功能性偶联是正常功能所必需的。本研究的目的是确定A9/10-nAChRs和SK2通道突触定位和功能偶联所需的蛋白质。拟议的研究将：1)确定毛细胞中橄榄耳蜗突触后位置的分子组成；2)检验我们的假设，即特定的适配蛋白与A9/10-nAChRs和SK2通道结合并将它们拴在突触后支架上；3)测试适配蛋白和支架蛋白在调节A9/10-nAChRs和SK2突触定位中的体内作用。在目标1的研究中，将使用免疫荧光和共聚焦显微镜来鉴定鸟类毛细胞突触后复合体的蛋白质成分。体外和体内共沉淀实验将被用来确定A10和SK2的结合伙伴，这些结合伙伴将A9/10-nAChRs和SK2通道连接到突触后复杂成分。目的研究A9/10-nAChRs和SK2通道的接头蛋白和APC在体内指导突触定位和功能偶联的作用。我们将使用逆转录病毒介导的优势负肽在体内的表达，选择性地阻断靶向蛋白质的相互作用：我们将使用免疫荧光和定量共聚焦显微镜测试A9/10-nAChRs和SK2在表达优势负肽的毛细胞中突触定位的变化。功能偶联的变化将使用全细胞膜片钳记录来分析。 总体而言，这些研究将为指导感觉毛细胞中功能性橄榄耳蜗型突触组装的分子机制提供新的见解。在正常发育的毛细胞中阐明这些机制，对于开发促进再生毛细胞中突触形成的策略，并在感音性耳聋的动物模型中恢复听力至关重要。感觉神经性耳聋是一种影响数百万美国人的永久性疾病。

DESCRIPTION (provided by applicant): Olivocochlear nicotinic synapses in the inner ear play a critical role in normal hearing. Olivocochlear pathway fibers originate in the brain and directly innervate mechanosensory hair cells. These synapses are required for normal hearing sensitivity, frequency selectivity and tonotopic map formation during development. However, little is known about the molecular mechanisms required for the proper assembly and function of Olivocochlear synapses. a9/10-nicotinic acetylcholine receptors (nAChRs) mediate synaptic transmission in hair cells. Functional coupling of a9/10-nAChRs to small conductance Ca2+activated potassium (SK2) channels is essential for normal function. The objective of the proposed studies is to identify proteins required for synaptic localization and functional coupling of a9/10-nAChRs and SK2 channels. The proposed studies will: 1) define the molecular composition of Olivocochlear postsynaptic sites in hair cells, 2) test our hypothesis that specific adapter proteins bind to a9/10-nAChRs and SK2 channels and tether them to the postsynaptic scaffold, and 3) test the in vivo roles of the adapter proteins and the scaffold protein adenomatous polyposis coli (APC) in regulating the synaptic localization of a9/10-nAChRs and SK2. In Aim 1 studies, immunofluorescence and confocal microscopy will be used to identify protein components of the avian hair cell postsynaptic complex. In vitro and in vivo co-precipitation assays will be used to identify binding partners of a10 and SK2 that connect a9/10-nAChRs and SK2 channels to postsynaptic complex components. Aim 2 studies will test the roles of the adapter proteins and APC in directing the synaptic localization and functional coupling of a9/10-nAChRs and SK2 channels in vivo. We will use retroviral-mediated expression of dominant negative peptides in vivo that selectively block the targeted protein interactions: We will test for changes in the synaptic localization of a9/10-nAChRs and SK2 in hair cells expressing the dominant negative versus control peptides using immunofluorescence and quantitative confocal microscopy. Changes in functional coupling will be assayed using whole-cell patch-clamp recordings. Overall, these studies will provide novel insights into molecular mechanisms that direct functional Olivocochlear synapse assembly in sensory hair cells. Elucidating these mechanisms in normal developing hair cells will be essential for developing strategies to promote synapse formation in regenerating hair cells and restore hearing in animal models of sensorineural hearing loss, a permanent condition that affects millions of Americans.