Regulation of auditory calcium channels

描述（由申请人提供）：在耳蜗内毛细胞（IHC）中，Cav1.3 L型电压门控Ca 2+通道介导Ca 2+信号，触发神经递质从IHC胞吐到听觉神经传入。Cav1.3的这种功能对听力至关重要：Cav1.3的功能丧失改变会导致人类和动物模型的耳聋。Cav1.3通道与其他细胞类型相比，在IHC中表现出不同的特性，但对这些差异的基础或它们与IHC声音编码的相关性知之甚少。填补这一知识空白有望揭示Cav1.3通道在听觉通路中第一个突触的独特作用所需的基本过程。我们研究的长期目标是确定调节电压门控Cav Ca 2+通道的机制，以发现导致人类疾病的原因以及如何治愈人类疾病。为此，我们已经确定了新形式的Cav1.3调制的IHC。首先，我们发现IHC中Cav1.3通道的细胞表面密度是由与harmonin（一种与Usher综合征发病机制有关的蛋白质）的相互作用控制的。Harmonin通过泛素-蛋白体（UPS）系统增强Cav1.3的降解，并且该过程在Usher综合征小鼠模型中被破坏。第二，我们发现CaBP 2，一种与钙调蛋白（CaM）相关的Ca 2+结合，抑制Ca 2+依赖性Cav1.3失活;这种作用受到CaBP 2基因中导致常染色体隐性听力损失的人类突变的损害。第三，我们发现Cav1.3与RIBEYE相关，RIBEYE是IHC和其他感觉细胞类型中“带状”突触的主要成分。这种相互作用不仅可以调节Cav1.3在IHC中的定位，而且可以调节其功能 活动区基于我们的研究结果，我们假设Cav1.3与harmonin，CaBP 2和RIBEYE等蛋白质的大分子组装决定了IHC中Ca 2+信号的强度和定位，因此对听觉传递至关重要。本提案的目的是使用分子、遗传和电生理技术来验证这一假设。其基本原理是，拟议的研究将揭示塑造IHC突触功能的基本信号复合物，以及这些复合物的失调如何导致遗传性或获得性听力损失的病理生理学。

DESCRIPTION (provided by applicant): In cochlear inner hair cells (IHCs), Cav1.3 L-type voltage-gated Ca2+ channels mediate Ca2+ signals that trigger exocytosis of neurotransmitter from IHCs onto auditory nerve afferents. This function of Cav1.3 is crucial for hearing: loss-of-function alterations in Cav1.3 cause deafness in humans and animal models. Cav1.3 channels exhibit distinct properties in IHCs compared to other cell-types, but little is known about what underlies these differences or their relevance for sound encoding by the IHCs. Filling this gap in knowledge is expected to reveal fundamental processes that are required for the unique role of Cav1.3 channels at this first synapse in the auditory pathway. The long-term goal of our research is to define the mechanisms that regulate voltage- gated Cav Ca2+ channels in order to discover what causes, and how to cure, human disease. To this end, we have identified new forms of Cav1.3 modulation in IHCs. First, we found that the cell-surface density of Cav1.3 channels in IHCs is controlled by interactions with harmonin, a protein implicated in the pathogenesis of Usher syndrome. Harmonin enhances degradation of Cav1.3 by the ubiquitin-proteosome (UPS) system, and this process is disrupted in a mouse model of Usher syndrome. Second, we discovered that CaBP2, a Ca2+ binding related to calmodulin (CaM), inhibits Ca2+-dependent inactivation of Cav1.3; this effect is impaired by a human mutation in the CaBP2 gene that causes autosomal-recessive hearing loss. Third, we found that Cav1.3 associates with RIBEYE, the major component of "ribbon" synapses in IHCs and other sensory cell-types. This interaction may regulate not only the localization, but also the function of Cav1.3 at the IHC active zone. Based on our findings, we hypothesize that the macromolecular assembly of Cav1.3 with proteins such as harmonin, CaBP2, and RIBEYE, dictate the strength and localization of Ca2+ signals in IHCs, and is therefore crucial for auditory transmission. The objective of this proposal is to test this hypothesis using molecular, genetic, and electrophysiological techniques. The rationale is that the proposed research will reveal essential signaling complexes that shape the synaptic function of IHCs, and how dysregulation of such complexes may contribute to the pathophysiology of inherited or acquired forms of hearing loss.