Characterization of D3cpv cameleon sensitivity to zinc

描述(由申请人提供)：钙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。为了确定改善卒中预后的药物靶点，需要测量细胞内钙浓度([Ca~(2+)]_i)。目前最常用的测定[Ca~(2+)]i的方法是监测Fura-2或Fura-2FF等小分子钙指示剂的信号。不幸的是，这种方法缺乏选择性。也就是说，这些指标不仅与钙结合，而且还与锌结合，在缺血神经元中[钙]i和[锌]i均增加。因此，这种方法产生了矛盾的结果。需要一种更具选择性的方法来监测[钙]i。基因工程的钙指示剂，骆驼，构成了一种新的有希望的研究工具来监测[钙]i。然而，目前还不知道这个工具对[钙]i和[锌]i的特异性有多高。拟议研究的目的就是澄清这一问题。我们的目标是表征D3cpv Cameleon对锌的敏感性，并确定当[锌]i同时升高时，该工具是否可以用于监测[Ca~(2+)]_i。为此，将监测D3cpv转基因小鼠海马神经元中的D3cpv荧光信号，同时将这些神经元暴露在不同浓度的锌离子中，并在一种通过质膜运输锌离子的试剂的存在下进行处理。在谷氨酸处理的神经元中同时监测D3cpv和Fura-2Ff信号的实验中，将测试D3cpv作为监测[钙]i的工具的可靠性。这些实验将确定使用D3cpv是否可以解决Fura-2FF信号的模糊性。 与公共卫生相关：拟议的研究将检查一种新的基因工程钙指示剂D3cpv Cameleon在细胞内锌浓度同时升高时是否可靠地报告细胞内钙浓度。这项研究与公众健康相关，因为细胞内钙和锌的增加在中风时神经元死亡的机制中发挥了作用。

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.