Characterization of D3cpv cameleon sensitivity to zinc

描述（由申请人提供）：Ca 2+稳态的不稳定在卒中期间发生的缺血性神经元死亡中起关键作用。为了确定改善卒中结局的药理学靶点，需要测量细胞内Ca 2+浓度（[Ca 2 +]i）。目前最常用的测量[Ca 2 +]i的方法是监测小分子Ca 2+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，这些指示剂不仅结合Ca 2+，而且还结合Zn 2+，并且在缺血神经元中[Ca 2 +]i和[Zn 2 +]i均增加。因此，这种方法产生矛盾的结果。需要一种更具选择性的方法来监测[Ca 2 +]i。转基因的钙指示剂，骆驼，构成了一种新的有前途的研究工具，以监测[Ca 2 +]i。然而，尚不清楚该工具对[Ca 2 +]i相对于[Zn 2 +]i的特异性如何。这项研究的目的是澄清这个问题。我们的目的是表征D3 cpv骆驼对锌的敏感性，并确定该工具是否可用于监测[Zn 2 +]i升高同时发生时的[Ca 2 +]i。为此，将在用D3 cpv转染的原代小鼠海马神经元中监测D3 cpv荧光信号，同时将神经元暴露于各种Zn 2+浓度，存在将Zn 2+转运穿过质膜的试剂。将在实验中测试D3 cpv作为监测[Ca 2 +]i的工具的可靠性，其中将在谷氨酸处理的神经元中同时监测D3 cpv和fura-2FF信号。这些实验将确定使用D3 cpv是否可以解决fura-2FF信号模糊性。 公共卫生相关性：拟议的研究将检查一种新的基因工程Ca 2+指示剂，D3 cpv cameleon，是否可靠地报告细胞内Ca 2+浓度时，细胞内Zn 2+升高同时发生。该研究与公共卫生相关，因为细胞内Ca 2+和Zn 2+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.