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

Area of Science:

• Neuroscience
• Biotechnology
• Cell Biology

Background:

• Optogenetics enables precise control of intracellular calcium (Ca++) using light-sensitive proteins like channelrhodopsins and melanopsins.
• Current optical methods for monitoring Ca++ rely on fluorescence, which can interfere with optogenetic probes and cause artifacts like autofluorescence.
• Luminescence offers an alternative optical technology that circumvents fluorescence-related issues.

Purpose of the Study:

• To develop a genetically encoded, ratiometric Ca++ sensor utilizing bioluminescence resonance energy transfer (BRET).
• To enable non-invasive optical monitoring of Ca++ dynamics elicited by optogenetic stimulation.
• To overcome limitations of fluorescence-based Ca++ sensors in optogenetics research.

Main Methods:

• Development of a novel, bright luciferase-based BRET sensor for ratiometric Ca++ detection.
• Co-expression of the BRET Ca++ sensor with optogenetic probes (melanopsin and channelrhodopsin) in cultured cells and neurons.
• Quantification and imaging of light-induced Ca++ fluxes using the BRET sensor under dark-field conditions.

Main Results:

• Successful development of a genetically encoded BRET Ca++ sensor with a large dynamic range.
• The BRET sensor effectively quantified and imaged light-elicited Ca++ fluxes in cells expressing melanopsin and neurons expressing channelrhodopsin.
• Monitoring was achieved in darkness, avoiding interference with optogenetic probes and autofluorescence.

Conclusions:

• The developed BRET Ca++ sensor provides a robust, non-invasive optical method for monitoring optogenetically controlled Ca++ signaling.
• This approach overcomes the limitations of fluorescence-based methods, offering improved compatibility with optogenetics.
• The sensor is well-suited for precise quantification and imaging of Ca++ dynamics in response to light stimulation.