A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites
A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites
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DOI：
10.1038/srep34447
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发表时间：
2016-09-28
影响因子：
4.6
通讯作者：
Enikolopov, Grigori N.
中科院分区：
文献类型：
--
作者：
Barykina, Natalia V.;Subach, Oksana M.;Enikolopov, Grigori N.
遗传编码钙指示剂（GECIs）主要由两个或一个荧光团为基础的传感器。一种类型的基于双荧光团的传感器，携带Opsanus肌钙蛋白C（TnC）作为Ca 2+结合部分，具有两个钙离子结合位点，提供对钙离子的线性响应。基于单荧光团的传感器具有四个Ca 2+结合位点，但更适合于体内实验。在此，我们描述了一种新的设计为一个荧光团为基础的GECI与两个Ca（2+-）结合位点。工程传感器称为NTnC，使用TnC作为Ca 2+结合部分，插入mNeonGreen荧光蛋白中。与标准GECI GCaMP 6s相比，单体NTnC在体外具有更高的亮度和pH稳定性。此外，NTnC显示出对Ca 2+的反向荧光响应。使用NTnC，我们已经可视化的Ca 2+动力学的神经元培养物的自发活动过程中确认的控制NTnC和它的突变体，其中的亲和力Ca 2+被消除。使用全细胞膜片钳，我们已经证明，神经元中的NTnC动力学类似于GCaMP 6，并允许单个动作电位的稳健检测。最后，我们已经使用NTnC可视化Ca 2+神经元活动在清醒和自由移动的小鼠使用双光子显微镜或nVista微型显微镜在V1皮质区在体内。
Genetically encoded calcium indicators (GECIs) are mainly represented by two-or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca(2+-)binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope.