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

描述（申请人提供）：内耳的耳蜗烟碱突触在正常听力中起着至关重要的作用。耳蜗通路纤维起源于大脑，直接支配机械感觉毛细胞。在发育过程中，这些突触对于正常的听觉灵敏度、频率选择性和张力图的形成是必需的。然而，人们对耳蜗突触正常组装和功能所需的分子机制知之甚少。9/10-烟碱乙酰胆碱受体（nAChRs）介导毛细胞突触传递。a9/10-nAChRs与小电导Ca2+活化钾（SK2）通道的功能偶联对于正常功能至关重要。本研究的目的是鉴定a9/10-nAChRs和SK2通道突触定位和功能偶联所需的蛋白质。拟进行的研究将：1)确定耳蜗毛细胞突触后位点的分子组成；2)验证我们的假设，即特异性的连接蛋白与a1 /10- nachrs和SK2通道结合并将其系在突触后支架上；3)测试连接蛋白和支架蛋白adenomatous polyposis coli （APC）在体内调节a1 /10- nachrs和SK2突触定位中的作用。在Aim 1研究中，免疫荧光和共聚焦显微镜将用于鉴定禽毛细胞突触后复合体的蛋白质成分。体外和体内共沉淀法将用于鉴定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合物组分。Aim 2研究将在体内测试适配器蛋白和APC在指导a9/10-nAChRs和SK2通道突触定位和功能偶联中的作用。我们将在体内使用逆转录病毒介导的显性阴性肽的表达，选择性地阻断目标蛋白的相互作用：我们将使用免疫荧光和定量共聚焦显微镜检测表达显性阴性肽和对照肽的毛细胞中a1 /10- nachrs和SK2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进行分析。

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.