Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase

Abstract

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K⁺), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K⁺-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K⁺-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K⁺. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems.

Figure 1

Mechanistic comparison of this work with other common bioluminescent indicators of in vivo imaging importance. (a) Reaction-based bioluminescent indicators: a caged luciferin is utilized, where a specific activity can remove the caging group and activate the luciferin, allowing for the specific detection of bioactivity. (b) Sensory luciferase-based bioluminescent indicators: a luciferase is engineered to be responsive to a specific analyte by strategically inserting and fusing a sensory domain to the luciferase. (c) Sensory luciferin-based bioluminescent indicators (this work): an analyte-binding moiety (e.g., a K⁺-binding crown ether) is strategically introduced to the luciferin, leading to the analyte-responsive modulating of the bioluminescence reaction. CG, caging group; BL, bioluminescence.

Figure 2

Design of potassiorin and initial evaluation with BREP luciferase. (a) Illustration of the DTZ structure and the installation of a K⁺-binding crown ether ring to derive potassiorin. The C2, C6, and C8 derivatizations on DTZ are highlighted. (b) Schematic illustration of the domain arrangements of BREP, a fusion of mScarlet-I and teLuc through a three amino acid linker. (c) Illustration of a modeled structure of NanoLuc (cyan ribbon) in complex with CTZ (magenta sticks). The C2, C6, and C8 derivatizations on CTZ are highlighted. (d, e) Bioluminescence emission spectra of BREP in the presence of potassiorin (d) or DTZ (e) with or without 150 mM KCl. Presented are the averages from three technical replicates. (f) Bioluminescence intensities of BREP and potassiorin at 590 nm in the presence of the indicated concentrations of K⁺ or Na⁺. n = 3 technical replicates. A one-site binding model was used to fit the data and derive the apparent dissociation constants (K_d). BL, bioluminescence.

Figure 3

In vitro characterization of BRIPO. (a) Schematic illustration of BRIPO with mutations from BREP highlighted. (b, c) Bioluminescence emission spectra of BRIPO in the presence of potassiorin (b) or DTZ (c) with or without 150 mM KCl. Presented are the averages from three technical replicates. (d) Bioluminescence spectra of BRIPO and potassiorin with the indicated concentrations of KCl. Presented are the averages from three technical replicates. (e) Bioluminescence intensities of BRIPO and potassiorin at 590 nm in the presence of the indicated concentrations of K⁺ or Na⁺. n = 3 technical replicates. A one-site binding model was used to fit the data and derive the apparent dissociation constants (K_d). (f) Normalized bioluminescence intensity of BRIPO and potassiorin in the presence of different metal ions: Na⁺ (15 mM), Zn²⁺ (10 μM), Ca²⁺ (2 mM), Fe²⁺ (10 μM), Cu²⁺ (100 nM), Mn²⁺ (10 μM), K⁺ (150 mM), Mg²⁺ (2 mM). n = 3 technical replicates. BL, bioluminescence.

Figure 4

Imaging K⁺ efflux in cultured cell lines. (a) Schematic illustration of nigericin-mediated K⁺ efflux in HEK 293T cells in a low K⁺ buffer. (b) Representative pseudocolored bioluminescence images of BRIPO-expressing HEK 293T cells in the presence of potassiorin (top) or DTZ (bottom) before (left) and after (right) treatment with a combination of nigericin, ouabain, and bumetanide. Scale bar: 50 μm. (c) Quantification of bioluminescence intensity changes of individual cells from experiments in (b). Data are presented as mean ± s.d. (n = 39 cells for the potassiorin group, n = 57 cells for the DTZ group). (d) Schematic illustration of a stable HEK 293T cell line treated with arachidonic acid to open the mTrek channel and induce K⁺ efflux. (e) Representative pseudocolored bioluminescence images of BRIPO-expressing HEK 293T cells stably expressing mTrek and other ion channels in the presence of potassiorin (top) or DTZ (bottom) before (left) and after (right) treatment with arachidonic acid. Scale bar: 50 μm. (f) Quantification of bioluminescence intensity changes of individual cells from experiments in (e). Data are presented as mean ± s.d. (n = 33 cells for the potassiorin group, n = 42 cells for the DTZ group). In (c) and (f), the baselines were corrected using a monoexponential decay model, and the P value was derived from unpaired two-tailed t-tests. The GraphPad Prism software does not provide extract P values below 10^–15. This figure is created with BioRender.com. BL, bioluminescence. Arb. units, arbitrary units.

Figure 5

Imaging K⁺ efflux in primary mouse neurons and the brains of live mice. (a) Representative fluorescence and pseudocolored bioluminescence images of BRIPO-expressing primary mouse neurons. Glutamate was used to induce potassium efflux. Scale bar, 50 μm. (b) Quantification of bioluminescence intensity changes of individual responsive neurons upon glutamate treatment. Data are presented as mean ± s.d. (n = 25 cells for the potassiorin group, n = 31 cells for the DTZ group). (c) Schematic illustration of stereotactic intracranial administration of AAVs containing the BRIPO gene and other general experiment procedures. (d) Quantification of bioluminescence intensity changes of individual animals. Data are presented as mean ± s.e.m. (n = 5 mice for the potassiorin group, n = 6 mice for the DTZ group). In (b) and (d), the baselines were corrected using a monoexponential decay model. and the P value was derived from unpaired two-tailed t-tests. The GraphPad Prism software does not provide extract P values below 10^–15. This figure is created with BioRender.com.