BACKGROUND Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

中文翻译：

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基于循环排列海葵红色荧光蛋白 eqFP578 的基因编码 Ca2+ 指示剂。


背景基因编码钙离子（Ca2+）指示剂（GECI）是测量体外和体内Ca2+动态和神经元活动不可或缺的工具。由于用于激发的光波长较长，基于红色荧光蛋白 (RFP) 的 GECI 相对于基于绿色荧光蛋白的 GECI 具有固有的优势。较长波长的光与降低光毒性和更深地穿透组织有关。红色 GECI 还可以使用蓝色或青色可激发荧光团实现多色可视化。结果在此，我们报告了基于 RFP 的新 GECI K-GECO1 的开发、结构和验证，该 GECI 基于源自海葵 Entacmaea 四色的循环排列 RFP。我们表征了 K-GECO1 在培养的 HeLa 细胞、分离的神经元、干细胞衍生的心肌细胞、器官型脑切片、体内斑马鱼脊髓和体内小鼠大脑中的性能。结论 K-GECO1 是基于 RFP eqFP578 支架的新 GECI 谱系的原型。它具有高灵敏度和快速动力学，与当前最先进的指示剂相似或更好，并且溶酶体积累减少，蓝光光活化最小化。 K-GECO1 谱系的进一步改进可能会导致进一步改进的变体，其整体性能超过最高度优化的红色 GECI。