Characterization of D3cpv cameleon sensitivity to zinc

描述（由申请人提供）：Ca2+稳态的不稳定在中风期间发生的缺血性神经元死亡中起关键作用。为了确定改善卒中预后的药理学靶点，需要测量细胞内Ca2+浓度（[Ca2+]i）。目前最常用的测量[Ca2+]i的方法是监测小分子Ca2+指示剂如fura-2或fura-2FF的信号。不幸的是，这种方法缺乏选择性。也就是说，这些指标不仅结合Ca2+，还结合Zn2+，并且在缺血神经元中[Ca2+]i和[Zn2+]i都增加。因此，这种方法产生矛盾的结果。需要一种更有选择性的方法来监测[Ca2+]i。基因工程Ca2+指标，cameleon，构成了一个新的有前途的研究工具来监测[Ca2+]i。然而，目前尚不清楚该工具对[Ca2+]i和[Zn2+]i的特异性如何。本研究的目的是澄清这一问题。我们的目的是表征D3cpv cameleon对锌的敏感性，并确定当[Zn2+]i升高同时发生时，该工具是否可用于监测[Ca2+]i。为此，将在转染D3cpv的小鼠海马原代神经元中监测D3cpv荧光信号，同时将神经元暴露于不同浓度的Zn2+中，并通过质膜运输Zn2+。D3cpv作为监测[Ca2+]i的工具的可靠性将在实验中进行测试，其中D3cpv和fura-2FF信号将在谷氨酸处理的神经元中同时监测。这些实验将确定是否使用D3cpv，可以解决fura-2FF信号的歧义。

DESCRIPTION (provided by applicant): Destabilization of Ca2+ homeostasis plays a critical role in the ischemic neuronal death that takes place during a stroke. To identify pharmacological targets to improve the outcome of stroke, one needs to measure the intracellular Ca2+ concentration ([Ca2+]i). The most common currently used method to measure [Ca2+]i is to monitor the signal of small molecule Ca2+ indicators such as fura-2 or fura-2FF. Unfortunately, this method suffers from a lack of selectivity. Namely, these indicators bind not only Ca2+ but also Zn2+ and both [Ca2+]i and [Zn2+]i increase in ischemic neurons. Consequently, this method yields ambivalent results. A more selective method to monitor [Ca2+]i is needed. Genetically engineered Ca2+ indicators, cameleons, constitute a novel promising research tool to monitor [Ca2+]i. However, it is not yet known how specific for [Ca2+]i versus [Zn2+]i this tool is. The goal of the proposed research is to clarify this issue. We aim to characterize the sensitivity of the D3cpv cameleon to zinc and determine whether this tool can be used to monitor the [Ca2+]i when [Zn2+]i elevations take place concurrently. To this end, the D3cpv fluorescent signal will be monitored in primary mouse hippocampal neurons transfected with D3cpv while the neurons will be exposed to various Zn2+ concentrations in the presence of an agent that transports Zn2+ across the plasma membrane. The reliability of D3cpv as a tool to monitor [Ca2+]i will be tested in the experiments in which D3cpv and fura-2FF signals will be monitored simultaneously in glutamate-treated neurons. These experiments will determine whether using D3cpv, one can resolve fura-2FF signal ambiguities. PUBLIC HEALTH RELEVANCE: The proposed research will examine whether a novel genetically engineered Ca2+ indicator, the D3cpv cameleon, reliably reports intracellular Ca2+ concentrations when intracellular Zn2+ elevations take place concurrently. The research is relevant to public health because intracellular Ca2+ and Zn2+ increases play a role in the mechanisms of neuronal death during stroke.