Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.
Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.
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DOI：
10.1371/journal.pone.0113873
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Pieribone VA
中科院分区：
文献类型：
--
作者：
Han Z;Jin L;Chen F;Loturco JJ;Cohen LB;Bondar A;Lazar J;Pieribone VA
ArcLight是一种具有大ΔF/ΔV的基因编码荧光蛋白电压探针，是玻璃海鞘电压敏感磷酸酶的电压传感结构域与携带单一突变（荧光蛋白中的A227 D）的超黄道荧光蛋白之间的融合体。如果没有这种突变，探针在响应电压偏转时仅产生非常小的荧光变化（± 1%）。这种突变提供的大信号允许在体外和体内的单次试验中光学检测动作电位和亚阈值电事件。然而，目前尚不清楚这种单一突变如何产生具有随膜电位变化对其荧光输出的如此大的调节的探针。在这项研究中，我们确定了超黄道pHluorin（相对于eGFP）中的哪些残基对ArcLight响应至关重要，因为基于eGFP的类似构建的探针如果携带这些关键残基，也会表现出较大的响应幅度。我们发现，D147负责确定这些探针中使用的荧光蛋白的pH敏感性，但其本身不会导致具有大信号的电压探针。我们还提供了证据表明，电压依赖的信号的弧光不简单地感知环境pH值的变化。一项双光子偏振显微镜研究表明，ArcLight对膜电位变化的反应包括超黄道pHluorin的重新定向。我们还探索了不同的变化，包括修改接头长度，删除非必需氨基酸的超黄道pHluorin，添加法尼基化位点，使用串联荧光蛋白和其他pH敏感的荧光蛋白。
ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.
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影响因子：
16.6
作者：
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通讯作者：
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Pieribone VA
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作者：
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