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

Jie Yang, Derrick Cumberbatch, Samuel Centanni, Shu-qun Shi, Danny Winder, Donna Webb and Carl Hirschie Johnson ()
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Jie Yang: Vanderbilt University
Derrick Cumberbatch: Vanderbilt University
Samuel Centanni: Vanderbilt University
Shu-qun Shi: Vanderbilt University
Danny Winder: Vanderbilt University
Donna Webb: Vanderbilt University
Carl Hirschie Johnson: Vanderbilt University

Nature Communications, 2016, vol. 7, issue 1, 1-10

Abstract: Abstract 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.

Date: 2016
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DOI: 10.1038/ncomms13268

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