Proton as co-transmitter of neuronal signaling

质子作为神经元信号传导的共同递质

• 批准号: 6984401
• 负责人: MARTIN MORAD
• 金额: $ 18.62万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6984401
• 关键词: PC12 cells; acetylcholine; acidity /alkalinity; cholinergic receptors; chromaffin cells; confocal scanning microscopy; fluorescence microscopy; laboratory rat; membrane transport proteins; neural transmission; neurons; nicotinic receptors; protonation; synapses; synaptic vesicles; tissue /cell culture; voltage /patch clamp

DESCRIPTION (provided by applicant): The overall goal of this proposal is to examine whether protons can serve as co-transmitters in cholinergic neuronal synaptic signaling. The rationale for this possibility carne from our recent findings that acidification uniquely modulated the recombinant neuronal nicotinic acetylcholine receptors (nAChRs) in part based on their desensitization kinetics and their beta subunit composition. In sharp contrast to reports that acidification generally inhibits ligand- and voltage-gated cation channels, we found that rapid (approximately 20 ms) acidification enhanced the alpha3/beta4 neuronal nAChR current and accelerated its activation and decay kinetics. Since it is well known that: a) repetitive firing in the brain causes brief acidic shifts, b) ACh is stored in acidic vesicles, and c) release of vesicular protons inhibits presynaptic Ca2+ channels, we hypothesize that brief intermittent acidification of synaptic cleft modulates the postsynaptic nAChRs providing focal plasticity to synaptic signaling. The experiments proposed here are aimed at: 1) examining, on a time scale approximating synaptic transmission, how acidification modulates the native neuronal nAChRs in primary culture of chromaffin cells, cortical, and cerebellar neurons; 2) imaging the microdomains of acidification and probing for presence of "proton sparks" with transmitter release in PC12 and chromaffin cell cultures; 3) evaluating directly the roles of released protons in modulation of synaptic signaling in cortical and cerebellar neurons. To test this hypothesis under feasible experimental conditions, we will use two models of neurosecretion: 1) PC12 and chromaffin cells and 2) cultures of cortical and cerebellar neurons, where agonist and protons may be applied rapidly (approximately 2 ms) and briefly, and membrane currents measured simultaneously. In chromaffin cells and cortical neurons, we shall quantify the effect of protons in modulation of nAChRs, and image the acidification profiles resulting from the release of secretory vesicles into paracellular space, using custom-made pH-sensitive dyes, in combination with rapid (240-480 f/s) confocal microscopy and a novel high-resolution optical technique (Total Internal Reflection Fluorescence Microscopy, TIRF), developed in our laboratory. Our finding, we believe, will provide novel insights into synaptic signaling that may be critical in the pathogenesis of neurodegenerative diseases such as Alzheimer's, where proton content of synaptic vesicle may be altered leading to impaired cholinergic signaling.

描述（由申请人提供）：该提案的总体目标是检查质子是否可以充当胆碱能神经元突触信号传导中的共递质。这种可能性的基本原理来自我们最近的发现，即酸化独特地调节重组神经元烟碱乙酰胆碱受体（nAChR），部分基于其脱敏动力学和β亚基组成。与酸化通常抑制配体门控和电压门控阳离子通道的报道形成鲜明对比，我们发现快速（约 20 ms）酸化增强了 α3/β4 神经元 nAChR 电流并加速其激活和衰减动力学。由于众所周知：a) 大脑中的重复放电会导致短暂的酸移，b) 乙酰胆碱储存在酸性囊泡中，c) 囊泡质子的释放会抑制突触前 Ca2+ 通道，因此我们假设突触间隙的短暂间歇性酸化会调节突触后 nAChR 为突触信号传递提供局部可塑性。这里提出的实验旨在：1）在接近突触传递的时间尺度上检查酸化如何调节嗜铬细胞、皮质和小脑神经元原代培养物中的天然神经元 nAChR； 2) 对酸化微区进行成像，并探测 PC12 和嗜铬细胞培养物中递质释放的“质子火花”的存在； 3）直接评估释放的质子在皮质和小脑神经元突触信号调节中的作用。为了在可行的实验条件下检验这一假设，我们将使用两种神经分泌模型：1) PC12 和嗜铬细胞，2) 皮质和小脑神经元培养物，其中可以快速（约 2 毫秒）且短暂地应用激动剂和质子，并且同时测量膜电流。在嗜铬细胞和皮质神经元中，我们将量化质子在 nAChR 调节中的作用，并使用定制的 pH 敏感染料结合快速 (240 -480 f/s）共焦显微镜和我们实验室开发的新型高分辨率光学技术（全内反射荧光显微镜，TIRF）。我们相信，我们的发现将为突触信号传导提供新的见解，这对于阿尔茨海默氏症等神经退行性疾病的发病机制可能至关重要，其中突触小泡的质子含量可能发生改变，导致胆碱能信号传导受损。