title: Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca++sensing.
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.
authors: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
authors: Jie
authors: J
authors: Yang
authors: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
authors: Derrick
authors: D
authors: Cumberbatch
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
authors: Samuel
authors: S
authors: Centanni
authors: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
authors: Shu-Qun
authors: SQ
authors: Shi
authors: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
authors: Danny
authors: D
authors: Winder
authors: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
authors: Donna
authors: D
authors: Webb
authors: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.|Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
authors: Carl Hirschie
authors: CH
authors: Johnson
mesh_terms: D000818:Animals; D024541:Batrachoidiformes; D002118:Calcium / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D062308:Optogenetics / Q000379:methods*
chemical_list: D008156:Luciferases; D002118:Calcium