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着关键作用。为了确定改善中风结局的药理学靶标，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。即，这些指示剂不仅与Ca2+结合，还与Zn2+结合，并且在缺血神经元中[Ca2]i和[Zn2]i均增加。因此，这种方法会产生矛盾的结果。需要一种更具选择性的方法来监测[Ca2]i。基因工程 Ca2 指标 Cameleons 构成了一种新的、有前途的监测 [Ca2 ]i 的研究工具。然而，尚不清楚该工具对于 [Ca2 ]i 与 [Zn2 ]i 的特异性如何。拟议研究的目的是澄清这个问题。我们的目的是表征 D3cpv 驼峰对锌的敏感性，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，将在用 D3cpv 转染的原代小鼠海马神经元中监测 D3cpv 荧光信号，同时将神经元在存在跨质膜转运 Zn2 的试剂的情况下暴露于各种 Zn2 浓度。 D3cpv 作为监测 [Ca2]i 工具的可靠性将在实验中得到测试，其中 D3cpv 和 fura-2FF 信号将在谷氨酸处理的神经元中同时监测。这些实验将确定使用 D3cpv 是否可以解决 fura-2FF 信号模糊性。 公共健康相关性：拟议的研究将检验一种新型基因工程 Ca2 指示剂 D3cpv Cameleon 是否能够在细胞内 Zn2 升高同时发生时可靠地报告细胞内 Ca2 浓度。这项研究与公共健康相关，因为细胞内 Ca2 和 Zn2 的增加在中风期间神经元死亡的机制中发挥着作用。

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.