Regulation of auditory calcium channels

描述(申请人提供)：在耳蜗内毛细胞中，Cav1.3 L类型的电压门控性钙通道介导钙信号，触发神经递质从耳蜗内毛细胞向听神经传入的胞吐。Cav1.3的这一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耳聋。与其他细胞类型相比，Cav1.3声道在IHC中表现出不同的特性，但人们对这些差异背后的原因或它们与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通道在听觉通路的第一个突触中发挥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钙通道的机制，以发现是什么导致以及如何治愈人类疾病。为此，我们确定了IHC中Cav1.3调制的新形式。首先，我们发现IHC中Cav1.3通道的细胞表面密度受与Harmonin的相互作用控制，Harmonin是一种与Usher综合征发病机制有关的蛋白质。Harmonin通过泛素-蛋白酶体(UPS)系统促进Cav1.3的降解，这一过程在Usher综合征小鼠模型中被破坏。其次，我们发现CaBP2，一种与钙调蛋白(CaM)相关的钙结合蛋白，抑制了CaV1.3的钙依赖失活；这种作用被导致常染色体隐性听力损失的人类CaBP2基因突变所削弱。第三，我们发现Cav1.3与Ribeye有关，Ribeye是IHC和其他类型感觉细胞中“带状”突触的主要成分。这种相互作用不仅可能调节Cav1.3在IHC的定位，还可能调节Cav1.3在IHC的功能 活动区。根据我们的发现，我们假设Cav1.3与Harmonin、CaBP2和Ribeye等蛋白质的大分子组装决定了IHC中钙信号的强度和定位，因此对听觉传递至关重要。这项提议的目的是使用分子、遗传和电生理技术来检验这一假说。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体，以及这些复合体的调节失调如何有助于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.