Probing the Synapse for pH-Microdomains

探测突触的 pH 微域

• 批准号: 8719822
• 负责人: GREGORY TALISKER MACLEOD
• 金额: $ 18.14万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8719822
• 关键词: ATP phosphohydrolase; Acids; Action Potentials; Binding Proteins; Biochemical Process; Biochemical Reaction; Buffers; Ca(2+)-Transporting ATPase; Cell membrane; Characteristics; Chemicals; Cleaved cell; Cytosol; Disease; Drosophila genus; Environment; Enzymes; Epilepsy; Event; Exploratory/Developmental Grant; Extracellular Space; Face; Failure; Fluorescence; Genome; Glutamate Receptor; Glutamates; Goals; Imaging Techniques; Investigation; Ion Channel; Laboratories; Lead; Left; Life; Location; Measures; Membrane; Membrane Proteins; Modification; Monitor; Motor; Motor Neurons; Nerve; Nerve Endings; Nervous system structure; Neuromuscular Junction; Neurons; Neurophysiology - biologic function; Neuropilins; Pathology; Platelet-Derived Growth Factor; Process; Property; Proteins; Protons; Recovery; Reporting; Research Personnel; Speed; Stroke; Synapses; Synaptic Cleft; Synaptic Vesicles; Synaptic plasticity; Techniques; Testing; Transgenic Organisms; Vascular Endothelial Growth Factor Receptor; density; extracellular; farnesylation; fluorescence imaging; fly; insight; interstitial; neurotransmission; new technology; novel; operation; postsynaptic; presynaptic; prevent; protein complex; public health relevance; ratiometric; receptor; tool; voltage

Project Summary / Abstract All forms of life rely on biochemical processes and these processes are either accelerated or inhibited according to the concentration of protons (pH) in their immediate vicinity. In the nervous system, pH buffering mechanisms provide a stable pH environment for biochemical reactions. Volume-averaged estimates of pH reveal only modest fluctuations in cytosolic and interstitial pH. Yet changes in pH, much like changes in Ca2+, are likely to be spatially non- uniform, and pH microdomains of substantial magnitude may develop close to the membranes across which acid equivalents flow. As many membrane-associated receptors, transporters, ion channels and enzymes are pH sensitive, pH-microdomains could have a significant impact on the fundamental neuronal properties underpinning normal operations of the nervous system. Our long range goal is to understand the influence of pH-microdomains on neuronal processes such as membrane excitability, neurotransmission and short term synaptic plasticity, and the extent to which near-membrane pH can influence the recovery of neural function after ischemic events. Our central hypothesis is that, as Ca2+ is ejected across the plasma-membrane, substantially acidic pH-microdomains develop at the cytosolic face of plasma-membrane Ca2+- ATPases (PMCAs) as a result of H+ exchange for Ca2+. The synaptic cleft will also alkalinize as a result of PMCA activity. Technological limitations have prevented investigations into the magnitude of pH microdomains, and their temporal and spatial characteristics. In an investigation of pH microdomains at the synapse, we will overcome current limitations by targeting pH Indicators to the plasma-membrane of pre- and post-synaptic compartments of the Drosophila neuromuscular junction (NMJ), and to the synaptic cleft. This approach requires the creation of a number of transgenic flies with ratiometric Genetically Encoded pH Indicators (GEpHIs) fused to proteins with well characterized distributions at the NMJ. We also introduce a technique to trap chemical pH indicators in the synaptic cleft through the introduction of a tetracysteine motif to an extracellular loop of the endogenous presynaptic voltage-gated Ca2+- channel. High speed fluorescence imaging techniques will be used to measure changes in fluorescence during the action potentials which initiate neurotransmission. Changes in fluorescence will be calibrated to quantify the underlying changes in pH.

项目摘要 /摘要 各种形式的生活都依赖生化过程，这些过程要么加速或 根据质子（pH）在附近的浓度（pH）的浓度。在 神经系统，pH缓冲机制为生化提供了稳定的pH环境 反应。 pH值的体积平均估计值仅显示胞质和 间质ph。然而，pH的变化（就像Ca2+的变化一样）在空间上可能是非 - 均匀的均匀和pH微域可能会在接近膜的附近发展 酸等效物的流动。与膜相关的受体，转运蛋白，离子一样 通道和酶是pH敏感的，pH-微生素可能会对 神经系统正常手术的基本神经元特性。 我们的远距离目标是了解pH-微生素对神经元过程的影响 例如膜兴奋性，神经传递和短期突触可塑性，以及 近膜pH可以影响缺血后神经功能的恢复的程度 事件。我们的中心假设是，随着Ca2+在等离子体膜上弹出， 在血浆膜的胞质面部CA2+ - 的胞质面上形成了基本酸性的pH-微生物域 ATPases（PMCAS）是H+交换Ca2+的结果。突触裂缝也将碱化为 PMCA活性的结果。技术局限性阻止了调查 pH微区的大小及其时间和空间特征。在 调查突触时pH微区域的研究，我们将通过 将pH指标靶向到pH前和突触后室的等离子体膜 果蝇神经肌肉连接（NMJ）和突触裂缝。这种方法需要 用比例遗传编码的pH指示器创建许多转基因苍蝇 （Gephis）与NMJ的分布良好的蛋白质融合在一起。我们还介绍了 通过引入的 四环素半胱氨酸基序至内源性突触前电压门控Ca2+ - 的细胞外环 渠道。高速荧光成像技术将用于测量 启动神经传递的动作电位期间的荧光。变更 荧光将进行校准以量化pH的潜在变化。