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

描述（由申请人提供）：内耳中的橄榄耳蜗烟碱突触在正常听力中起关键作用。橄榄耳蜗通路纤维起源于大脑并直接支配机械感觉毛细胞。这些突触是发育过程中正常听觉灵敏度、频率选择性和音调定位图形成所必需的。然而，关于橄榄耳蜗突触的正确组装和功能所需的分子机制知之甚少。α 9/10-烟碱乙酰胆碱受体（nAChR）介导毛细胞中的突触传递。α 9/10-nAChR与小电导Ca 2+激活的钾（SK 2）通道的功能性偶联对于正常功能是必需的。所提出的研究的目的是鉴定α 9/10-nAChR和SK 2通道的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点的分子组成，2）测试我们的假设，即特异性衔接蛋白结合α 9/10-nAChR和SK 2通道并将它们拴系到突触后支架，以及3）测试衔接蛋白和支架蛋白腺瘤性结肠息肉病（APC）在调节α 9/10-nAChR和SK 2的突触定位中的体内作用。在目标1研究中，免疫荧光和共聚焦显微镜将被用来确定鸟类毛细胞突触后复合物的蛋白质成分。将使用体外和体内共沉淀测定来鉴定将α 9/10-nAChR和SK 2通道连接至突触后复合物组分的α 10和SK 2的结合配偶体。目的2研究将测试衔接蛋白和APC在体内指导α 9/10-nAChR和SK 2通道的突触定位和功能偶联中的作用。我们将使用逆转录病毒介导的显性负性肽在体内的表达，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检测表达显性负性肽的毛细胞中α 9/10-nAChR和SK 2的突触定位与对照肽的变化。将使用全细胞膜片钳记录分析功能偶联的变化。 总的来说，这些研究将提供新的见解的分子机制，直接功能橄榄耳蜗突触组装感觉毛细胞。阐明正常发育毛细胞中的这些机制对于制定促进再生毛细胞中突触形成的策略和恢复感音神经性听力损失动物模型的听力至关重要，感音神经性听力损失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.