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: 10197098
• 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/10197098
• 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/antagonist; 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向感觉刺激发出信号，作为神经递质的分级变化 释放并使用不同于传统突触的独特的解剖和分子组件。 在独特的分子特征中，明显缺乏对神经元SNAR蛋白和它们的 各种各样的合作伙伴。相反，毛细胞的胞吐依赖于一种名为Otoferlin的大蛋白质，目前尚不清楚 机制。在这项建议中，我们研究了毛细胞突触传递的独特特征 斑马鱼分子生物学、电生理方法与新型体外膜的结合 融合分析。在目标1中，我们将确定Otoferlin是否可以使用Well刺激膜融合 建立与工程细胞的细胞-细胞融合分析，并确定对SNARES、脂质的要求 和钙在耳铁蛋白依赖的融合中。这些实验还将确定功能域 介导膜融合所需的。重要的是，这种策略避免了奥托费林的纯化困难 这阻碍了过去在重建依赖奥托费林的融合方面的进展。在目标2中，我们将测量 Otoferlin的钙依赖膜结合特性及寻找Otoferlin相互作用伙伴 原生细胞。在目标3中，我们以斑马鱼的侧线为模型系统，探索圈套在毛发中的作用。 细胞胞吐作用。在目标4中，我们测试了截断突变体对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最终将有助于理解听觉 对信息进行处理和交流。此外，Otoferlin的突变导致DFNB9形式的遗传性 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myoferlin和dyferlin，它们也与膜融合和人类 疾病。有理由期待我们在这个项目中学到的东西将对其他人的研究具有指导意义 蕨类植物。最后，对这些特化细胞中的突触前过程的基本理解将具有 对蜂窝通信的更广泛的影响，因此，可能有助于我们理解 精神健康和神经紊乱的各个方面。