G-protein regulation of exocytotic transmitter release

G 蛋白调节胞吐递质释放

• 批准号: 7471353
• 负责人: KEVIN P CURRIE
• 金额: $ 33.54万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7471353
• 关键词: Address; Adenovirus Vector; Adrenal Glands; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; Catecholamines; Cells; Chromaffin Cells; Complex; Computer information processing; Condition; Coupled; Data; Disease; Distal; Electric Capacitance; Electrophysiology (science); Event; Exocytosis; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G-protein Beta gamma; GTP-Binding Proteins; Goals; Hormones; In Vitro; Investigation; Knowledge; Left; Maps; Mediating; Membrane; Modeling; Molecular; Neurons; Neurotransmitters; Peptide Mapping; Peptides; Physiological; Play; Point Mutation; Process; Protein Overexpression; Proteins; Recombinants; Regulation; Regulation of Exocytosis; Relative (related person); Reporting; Research Personnel; Role; SNAP receptor; Signal Pathway; Staging; Stimulus; Stress; Synaptic Transmission; Synaptic plasticity; Testing; Vesicle; Work; base; carbon fiber; chemical release; controlled release; flash photolysis; insight; intercellular communication; interest; millisecond; mutant; novel; patch clamp; receptor; response; sensor; synaptotagmin; synaptotagmin I; syntaxin; syntaxin 1; syntaxin 1A; tool; voltage

DESCRIPTION (provided by applicant): Release of chemical transmitters by regulated exocytosis underlies many forms of intercellular communication, including hormone release and synaptic transmission. G protein-coupled receptors (GPCRs) orchestrate complex regulation of exocytosis, and in particular inhibit transmitter release from neurosecretory cells. Inhibitory GPCRs can be auto- or hetero-receptors, are typically Gi/o coupled, and work by release of G protein beta-gamma subunits (Gbeta-gamma). The most intensively studied mechanism of inhibition involves modulation of voltage-gated Ca2+ channels (Ca-channels), but direct effects on the exocytotic apparatus have also been reported. Our preliminary data show that Gbeta-gamma binds to SNAP25 and syntaxin-1A, suggesting that Ca-channels and SNAREs might be targeted in parallel by Gbeta-gamma to inhibit exocytosis. The central goal of this proposal is to dissect the molecular basis by which Gbeta-gamma controls exocytotic transmitter release in neurosecretory cells. To enable the precise biophysical analyses required to address this goal, we will use adrenal chromaffin cells, a neurosecretory model that provides significant experimental advantages. Furthermore, catecholamines released from chromaffin cells play important physiological roles in the coordinated response to stress or danger. We will combine carbon fiber amperometry, patch clamp electrophysiology, and flash photolysis of caged compounds, along with novel molecular tools (mutant Gbeta-gamma subunits and inhibitory peptides) to dissect the roles of Ca-channels and SNAREs in Gbeta-gamma -mediated regulation of exocytosis. In aim #1 we will test the hypothesis that Gbeta-gamma acts in parallel at Ca-channels and other downstream targets to inhibit transmitter. In aim #2 we will characterize the interaction of Gbeta-gamma with the exocytotic machinery and test the hypothesis that Gbeta-gamma inhibits exocytosis by competing with synaptotagmin-l for binding to SNAP25. In aim #3 we will use mutational and peptide mapping to characterize the Gbeta-gamma - SNAP25 interaction and generate novel molecular tools to dissect its role in the regulation of chromaffin cell exocytosis. To summarize, our investigations will significantly advance knowledge of the molecular mechanisms that control neurotransmitter and hormone secretion, and provide insight into the pathological basis of diseases related to secretion and neuromodulation.

描述（由申请人提供）：通过调节的胞吐作用释放化学发射器是许多形式的细胞间通信，包括激素释放和突触传播。 G蛋白偶联受体（GPCR）调节胞吐作用的复杂调节，特别是抑制了从神经分泌细胞中释放的发射机。抑制性GPCR可以是自动或异核受体，通常是GI/O耦合，并且通过释放G蛋白β-Gamma亚基（GBETA-GAMMA）来起作用。最深入研究的抑制作用机制涉及对电压门控Ca2+通道（CA通道）的调节，但也报道了对胞吐仪的直接影响。我们的初步数据表明，GBETA-GAMMA与SNAP25和Syntaxin-1a结合，这表明GBETA-GAMMA可能会并行抑制胞吐作用。该建议的核心目的是剖析GBETA-GAMMA控制神经分泌细胞中胞吐发射器的分子基础。为了实现解决此目标所需的精确生物物理分析，我们将使用肾上腺染色体细胞，肾上腺染色蛋白细胞是一种神经分泌模型，具有显着的实验优势。此外，从铬蛋白细胞释放的儿茶酚胺在对压力或危险的协调反应中起着重要的生理作用。我们将结合碳纤维安培计量学，贴片夹电生理学以及笼中化合物的闪光分解，以及新型的分子工具（突变的GBETA-GBETA-GAMMA亚基和抑制性肽），以剖析GBETA-GAMMA介导的exocytosis中的Ca-chnannels和SENRES在GBETA-GAMMA介导的调节中的作用。在AIM＃1中，我们将检验以下假设：GBETA-GAMMA在CA通道和其他下游靶标并抑制发射机的情况下并行作用。在AIM＃2中，我们将表征GBETA-GAMMA与胞吐机制的相互作用，并检验以下假设：GBETA-GAMMA通过与SynaptoTagmin-L竞争与SNAP25结合，从而抑制了胞吐作用。在AIM＃3中，我们将使用突变和肽映射来表征GBETA -GAMMA -SNAP25相互作用，并生成新型的分子工具，以剖析其在调节铬脂细胞外胞菌病的调节中的作用。总而言之，我们的研究将大大提高控制神经递质和激素分泌的分子机制的了解，并洞悉与分泌和神经调节有关的疾病的病理基础。