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

• 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
• 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

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作为神经递质的分级变化 释放并使用不同于常规突触的独特解剖和分子成分。 独特的分子特征之一是明显缺乏对神经元SNARE蛋白的依赖， 各种合作伙伴。相反，毛细胞的胞吐作用依赖于一种叫做Otoferlin的大蛋白质， 机制等在这个提议中，我们研究了毛细胞突触传递的独特功能， 分子生物学、斑马鱼电生理学方法和新型体外膜的组合 融合测定。在目标1中，我们将确定Otoferlin是否可以刺激膜融合， 用工程化细胞建立细胞-细胞融合试验，并确定对SNARE、脂质 和钙在耳铁蛋白依赖性融合中的作用。这些实验还将确定功能域 调节膜融合所必需的。重要的是，这种策略避免了纯化Otoferlin的困难 这阻碍了过去重建耳铁蛋白依赖性融合的进展。在目标2中，我们将测量 钙依赖性膜结合特性的Otoferlin，并寻找Otoferlin相互作用的合作伙伴， 原生细胞目的3：以斑马鱼侧线为模型系统，探讨SNARE在毛发中的作用 细胞胞吐作用在目标4中，我们测试截断突变体拯救突触功能的有效性。 在分子水平上理解毛细胞突触功能将最终有助于理解听觉是如何 信息被处理和传递。此外，Otoferlin的突变导致DFNB 9形式的遗传性 耳聋，因此对其功能的研究与人类疾病有关。Otoferlin属于Ferlin类， 蛋白质，包括肌钙蛋白和dysferlin，它们也涉及膜融合和人 疾病我们有理由期望，我们在这个项目中学到的东西将对其他研究具有指导意义。 费林。最后，对这些特化细胞中突触前过程的基本了解将使我们对突触前过程的研究更加深入。 更广泛的影响，细胞通信一般，因此，可能有助于我们的理解， 精神健康和神经系统疾病的各个方面。