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29 March 2024 Engineering luminopsins with improved coupling efficiencies
Ashley N. Slaviero, Nipun Gorantla, Jacob Simkins, Emmanuel L. Crespo, Ebenezer C. Ikefuama, Maya O. Tree, Mansi Prakash, Andreas Björefeldt, Lauren M. Barnett, Gerard G. Lambert, Diane Lipscombe, Christopher I. Moore, Nathan C. Shaner, Ute Hochgeschwender
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Neurophotonics, Vol. 11, Issue 2, 024208 (March 2024). https://doi.org/10.1117/1.NPh.11.2.024208
Abstract

Significance

Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research.

Aim

We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs.

Approach

We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons.

Results

Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical.

Conclusions

Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.
CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
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Ashley N. Slaviero, Nipun Gorantla, Jacob Simkins, Emmanuel L. Crespo, Ebenezer C. Ikefuama, Maya O. Tree, Mansi Prakash, Andreas Björefeldt, Lauren M. Barnett, Gerard G. Lambert, Diane Lipscombe, Christopher I. Moore, Nathan C. Shaner, and Ute Hochgeschwender "Engineering luminopsins with improved coupling efficiencies," Neurophotonics 11(2), 024208 (29 March 2024). https://doi.org/10.1117/1.NPh.11.2.024208
Received: 22 November 2023; Accepted: 14 March 2024; Published: 29 March 2024
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KEYWORDS
Neurons
Engineering
Photocurrent
Bioluminescence
Light emitting diodes
Optogenetics
Light sources
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