title: Optochemokine Tandem for Light-Control of Intracellular Ca2.
abstract: 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.
authors: Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany.
authors: Katrin
authors: K
authors: Feldbauer
authors: Department of Biotechnology and Biophysics, Biocenter, Julius Maximilian University, Würzburg, Germany.
authors: Jan
authors: J
authors: Schlegel
authors: Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany.
authors: Juliane
authors: J
authors: Weissbecker
authors: Department of Biotechnology and Biophysics, Biocenter, Julius Maximilian University, Würzburg, Germany.
authors: Frank
authors: F
authors: Sauer
authors: Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany.
authors: Phillip G
authors: PG
authors: Wood
authors: Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany.|Chemical and Pharmaceutical Sciences Department, Johann Wolfgang Goethe University, Frankfurt am Main, Germany.
authors: Ernst
authors: E
authors: Bamberg
authors: Department of Biotechnology and Biophysics, Biocenter, Julius Maximilian University, Würzburg, Germany.
authors: Ulrich
authors: http://orcid.org/0000-0003-3031-3422
authors: U
authors: Terpitz
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D018925:Chemokines / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D057809:HEK293 Cells; D006801:Humans; D008027:Light; D051379:Mice; D018408:Patch-Clamp Techniques; D051381:Rats; D019718:Receptors, CXCR4 / Q000378:metabolism
chemical_list: D018925:Chemokines; D002966:Clathrin; D019718:Receptors, CXCR4; D002118:Calcium