Optochemokine Tandem for Light-Control of Intracellular Ca2

An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-g...

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Bibliographic Details
Published in:PloS one 2016-10, Vol.11 (10), p.e0165344-e0165344
Main Authors: Feldbauer, Katrin, Schlegel, Jan, Weissbecker, Juliane, Sauer, Frank, Wood, Phillip G, Bamberg, Ernst, Terpitz, Ulrich
Format: Article
Language:English
Subjects:
Animals
Apoptosis
Biology and Life Sciences
Biophysics
Biotechnology
Calcium
Calcium (intracellular)
Calcium - metabolism
Calcium ions
Calcium permeability
Cell adhesion & migration
Cell cycle
Cell Line, Tumor
Chemistry
Chemokines
Chemokines - metabolism
Clathrin - metabolism
Confocal microscopy
CXCR4 protein
Cytosol
Cytosol - metabolism
Electrophysiology
Endocytosis
Endosomes
Engineering and Technology
G protein-coupled receptors
HEK293 Cells
Humans
Internalization
Intracellular
Kinases
Kinetics
Ligands
Light
Mice
Microscopy
Patch-Clamp Techniques
Physical Sciences
Physiology
Proteins
Rats
Receptors
Receptors, CXCR4 - metabolism
Research and Analysis Methods
Rhodopsin
Scanning microscopy
SDF-1 protein
Signal transduction
Signaling
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Summary:An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0165344