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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：
10636938
负责人：
ERDEM KARATEKIN
金额：
$ 51.11万
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/10636938
关键词：
Acceleration 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 Docking Electric Capacitance Electrophysiology (science)Embryo Engineering Exocytosis Family Genetic study Glutamates Grant Hair Cells Head In Vitro Injections Laboratories Lead Learning Length Lipids Measurement Measures Mediating Membrane Membrane Fusion Mental Health Modeling Molecular Molecular Biology Movement Mus Mutation Neurons OTOF gene Phenotype Phospholipids Physiology Process Property Proteins Proteomics Reaction Recycling Role SNAP receptor Signal Transduction Synapses Synaptic Transmission Synaptic Vesicles System Techniques Testing Time Total Internal Reflection Fluorescent Toxin Transgenic Organisms Transmembrane Domain Vesicle Visualization Whole-Cell Recordings Work Zebrafish deafness effectiveness testing experimental study genetic deafness human disease inhibitor knock-down lateral line member mosaic 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通过神经递质的分级变化来传递感觉刺激的信号