Open AccessOptochemokine Tandem for Light-Control of Intracellular Ca2+
Author(s) -
Katrin Feldbauer,
Jan Schlegel,
Juliane Weissbecker,
Frank Sauer,
Paul Wood,
Ernst Bamberg,
Ulrich Terpitz
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0165344
Subject(s) - g protein coupled receptor , endosome , microbiology and biotechnology , internalization , biophysics , intracellular , cytosol , endocytic cycle , rhodopsin , chemistry , transient receptor potential channel , biology , signal transduction , biochemistry , receptor , endocytosis , retinal , enzyme
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 Ca 2+ -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 Ca 2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca 2+ -sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca 2+ in response to light.