Mechanisms of the calcium-triggered neurotransmitter release machinery in hair cells

毛细胞中钙触发神经递质释放机制的机制

基本信息

批准号：
10424526
负责人：
ERDEM KARATEKIN
金额：
$ 55.11万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10424526
关键词：
Acute Anatomy Applications Grants Auditory Auditory Physiology Binding Binding Proteins Biochemical Biological Assay Biological Models Biophysical Process C2 Domain Calcium Calcium Binding Cell fusion Cell membrane Cells Communication Complex Couples DYSF gene Data Dependence Development Diffuse Docking Electric Capacitance Electrophysiology (science)Embryo Engineering Exocytosis Family Genetic study Glutamates Grant Hair Cells Head In Vitro Injections Instruction Laboratories Lead Learning Length Lipids Measurement Measures Mediating Membrane Membrane Fusion Mental Health Modeling Molecular Molecular Biology Mosaicism Movement Mus Mutation Neurons OTOF gene Phenotype Phospholipids Physiology Process Property Proteins Proteomics Reaction Recycling Role S-nitro-N-acetylpenicillamine SNAP receptor Signal Transduction Synapses Synaptic Transmission Synaptic Vesicles System Techniques Testing Time Total Internal Reflection Fluorescent Toxin Transgenic Organisms Transmembrane Domain Vesicle Work Zebrafish base deafness effectiveness testing experimental study genetic deafness human disease inhibitor knock-down lateral line member mutant nanodisk nervous system disorder neuromast neurotransmitter release novel plasmid DNA pressure presynaptic protein complex reconstitution recruit ribbon synapse sensor sensory stimulus sound synaptic function synaptotagmin syntaxin target SNARE proteins vesicle-associated membrane protein vesicular SNARE proteins

项目摘要

Hair cells of the auditory and vestibular systems signal sensory stimuli as graded changes in neurotransmitter release and employ unique anatomical and molecular components that differ from conventional synapses. Among the unique molecular features is the apparent lack of reliance on neuronal SNARE proteins and their various partners. Instead, hair cell exocytosis depends on a large protein called Otoferlin, by unknown mechanisms. In this proposal, we investigate the unique features of hair cell synaptic transmission using a combination of molecular biology, electrophysiological approaches in zebrafish, and novel in vitro membrane fusion assays. In Aim 1 we will determine whether Otoferlin can stimulate membrane fusion using well established cell-cell fusion assays with engineered cells and determine the requirements for SNAREs, lipids and calcium in otoferlin-dependent fusion. These experiments will also determine the functional domains required to mediate membrane fusion. Importantly, this strategy avoids difficulties with purification of Otoferlin that hindered advances in reconstituting Otoferlin-dependent fusion in the past. In Aim 2, we will measure the calcium-dependent membrane binding properties of Otoferlin and look for Otoferlin-interacting partners in native cells. In Aim 3, we use the zebrafish lateral line as a model system explore the role of SNAREs in hair cell exocytosis. In Aim 4, we test the effectiveness of truncation mutants to rescue synaptic function. Understanding hair cell synaptic function at the molecular level will ultimately aid in understanding how auditory information is processed and communicated. Moreover, mutations in Otoferlin lead to DFNB9 form of inherited deafness and thus study of its function has relevance for human disease. Otoferlin belongs to the ferlin class of proteins, which include myoferlin and dysferlin, which are also implicated in membrane fusion and human disease. It is reasonable to expect that what we learn in this project will be instructive for studies with other ferlins. Lastly, the fundamental understanding of presynaptic processes in these specialized cells will have broader implications for cellular communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders.

听觉和前庭系统的毛细胞将感觉刺激信号传递为神经递质的分级变化释放和利用与传统突触不同的独特的解剖和分子成分。独特的分子特征之一是明显缺乏对神经元 SNARE 蛋白及其各种合作伙伴。相反，毛细胞的胞吐作用依赖于一种名为 Otoferlin 的大蛋白质，其来源未知。机制。在本提案中，我们使用以下方法研究了毛细胞突触传递的独特特征：分子生物学、斑马鱼电生理学方法和新型体外膜的结合融合测定。在目标 1 中，我们将确定 Otoferlin 是否可以充分利用来刺激膜融合建立了工程细胞的细胞-细胞融合测定，并确定了 SNARE、脂质的要求和耳铁蛋白依赖性融合中的钙。这些实验还将确定功能域介导膜融合所需的。重要的是，该策略避免了 Otoferlin 纯化的困难这阻碍了过去重建 Otoferlin 依赖性融合的进展。在目标 2 中，我们将测量 Otoferlin 的钙依赖性膜结合特性并寻找 Otoferlin 的相互作用伙伴天然细胞。在目标 3 中，我们使用斑马鱼侧线作为模型系统探索 SNARE 在头发中的作用细胞胞吐作用。在目标 4 中，我们测试了截短突变体挽救突触功能的有效性。在分子水平上了解毛细胞突触功能将最终有助于了解听觉如何信息被处理和传达。此外，Otoferlin 的突变导致 DFNB9 形式的遗传性遗传耳聋及其功能的研究与人类疾病相关。 Otoferlin 属于 ferlin 类蛋白质，包括肌铁蛋白和 Dysferlin，它们也与膜融合和人类疾病。可以合理地预期，我们在这个项目中学到的东西将对其他项目的研究具有指导意义。费林斯。最后，对这些特殊细胞的突触前过程的基本了解将有助于一般而言，对细胞通信具有更广泛的影响，因此可能有助于我们理解心理健康和神经系统疾病的各个方面。