Probing the Synapse for pH-Microdomains

项目摘要/摘要 所有形式的生命都依赖于生化过程，这些过程要么加速，要么 根据其附近的质子浓度(pH值)而被抑制。在 神经系统、pH缓冲机制为生化提供稳定的pH环境 反应。PH的体积平均估计显示，胞浆和 间质pH。然而，酸碱度的变化，就像钙离子的变化一样，很可能是空间上的 均匀的、相当大的pH微区可以在靠近膜的地方形成 酸当量流过的地方。正如许多膜相关受体、转运体、离子 通道和酶是pH敏感的，pH微域可能会对 神经系统正常运作所需的基本神经特性。 我们的长期目标是了解pH微域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，以及 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，当钙离子被喷射到质膜上时， 强酸性pH微区在质膜Ca~(2+)的胞液面上形成。 ATP酶(PMCA)是氢交换钙的结果。突触裂隙也会碱化为 是PMCA活动的结果。技术上的限制阻碍了对 PH微域的大小及其时空特征。在一个 研究突触的pH微域，我们将克服目前的限制，通过 将pH指示剂定位于突触前和突触后隔区的质膜 果蝇神经肌肉接头(NMJ)和突触间隙。此方法需要 用比例基因编码的pH指示器创造若干转基因果蝇 (GEPHIs)与蛋白融合，在NMJ具有良好的分布特征。我们还介绍了一个 通过引入一种在突触间隙中捕获化学pH指示剂的技术 四半胱氨酸基序与内源性突触前电压门控钙离子的胞外环路 频道。将使用高速荧光成像技术来测量 启动神经传递的动作电位中的荧光。中的更改 将对荧光进行校准，以量化pH值的潜在变化。

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.