Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca sensing.

Yang J, Cumberbatch D, Centanni S, Shi SQ, Winder D, Webb D, Johnson CH
Nat Commun. 2016 7: 13268

PMID: 27786307 · PMCID: PMC5476805 · DOI:10.1038/ncomms13268

Optogenetic techniques allow intracellular manipulation of Ca by illumination of light-absorbing probe molecules such as channelrhodopsins and melanopsins. The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring Ca levels are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. Using a new bright luciferase, we here develop a genetically encoded Ca sensor that is ratiometric by virtue of bioluminescence resonance energy transfer (BRET). This sensor has a large dynamic range and partners optimally with optogenetic probes. Ca fluxes that are elicited by brief pulses of light to cultured cells expressing melanopsin and to neurons-expressing channelrhodopsin are quantified and imaged with the BRET Ca sensor in darkness, thereby avoiding undesirable consequences of fluorescence irradiation.

MeSH Terms (12)

Animals Batrachoidiformes Calcium Fluorescence Resonance Energy Transfer HEK293 Cells HeLa Cells Humans Luciferases Luminescence Luminescent Measurements Microscopy, Fluorescence Optogenetics

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