Probing the Synapse for pH-Microdomains

项目概要/摘要 所有形式的生命都依赖于生物化学过程，这些过程要么加速， 根据其紧邻的质子浓度（pH）抑制。在 pH缓冲机制为生物化学提供了稳定的pH环境， 反应. pH的体积平均估计值仅显示胞质和胞浆中的适度波动。 然而，pH的变化，很像Ca 2+的变化，可能是空间非线性的。 均匀的，和pH值的微域的实质性幅度可能发展接近膜 酸等价物流过的通道。由于许多膜相关受体、转运蛋白、离子 通道和酶是pH敏感的，pH微区可能对 支持神经系统正常运作的基本神经元特性。 我们的长期目标是了解pH微区对神经元过程的影响 如膜兴奋性、神经传递和短期突触可塑性， 近膜pH对缺血后神经功能恢复的影响程度 事件我们的中心假设是，当Ca 2+被喷射穿过质膜时， 在质膜Ca 2 +-Ca 2+的胞质表面形成基本上酸性的pH微区。 ATP酶（PMCAs）作为H+交换Ca 2+的结果。突触间隙也会碱化， 这是PMCA活动的结果。技术限制阻碍了对 pH微区的大小及其时空特征。中 在突触的pH微域的调查，我们将克服目前的限制， 将pH指示剂靶向到突触前和突触后区室的质膜， 果蝇的神经肌肉接头（NMJ）和突触间隙。这种方法需要 用比率遗传编码pH指示剂创建许多转基因果蝇 （GEpHI）融合至在NMJ处具有良好表征的分布的蛋白质。我们还引入了 一种技术，通过引入一种 四半胱氨酸基序的内源性突触前电压门控Ca 2 +- 频道高速荧光成像技术将用于测量 在启动神经传递的动作电位过程中产生荧光。变化 将校准荧光以量化pH的潜在变化。

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.