Substitution with a Single Cysteine in the Green Fluorescent Protein-Based Calcium Indicator GCaMP3 Enhances Calcium...

Genetically encoded calcium indicators (GECI) such as GCaMP3 are attracting significant attention as a good option for measuring intracellular calcium levels. Recently, a modified GCaMP3 called dCys-GCaMP3 was developed by replacing two threonine residues with cysteines. dCys-GCaMP3 proved to be a better calcium indicator, but it was not clear how and why the two cysteine residues were able to enhance the protein's calcium sensitivity. The aim of the present study was to investigate the possible roles of these cysteine residues in dCys-GCaMP3. dCys-GCaMP3 (Thr330Cys;Thr364Cys) exhibited enhanced fluorescence intensity compared to the canonical GCaMP3 in calcium imaging experiments. However, substitution of a single residue at position 330 with cysteine (Thr330Cys) also afforded comparable sensitivity to GCaMP3. In contrast, the other single residue substitution at position 364 with cysteine (Thr364Cys) failed to enhance calcium sensitivity, showing that cysteine at position 330 is essential to improve calcium sensitivity. Thr330Cys substitution in the GCaMP3 or "Cys³³⁰-GCaMP3" showed significantly reduced background fluorescence, and the fluorescence intensity was proportional to the amount of DNA used to transfect the cells used in the study. The substitute had to be cysteine, because replacement with other amino acids such as alanine, valine, and aspartate did not improve GCaMP3's calcium sensitivity. Cys³³⁰-GCaMP3 outperformed a synthetic calcium-specific indicator, Fluo-3, in various calcium imaging experiments. Thus, the present study asserts that substituting the threonine at position 330 in GCaMP3 with cysteine is essential to enhance calcium sensitivity, and suggests that Cys³³⁰-GCaMP3 can be used as a potent fluorescent calcium indicator to measure intracellular calcium levels.