Optochemokine Tandem for Light-Control of Intracellular Ca2

PLoS One. 2016 Oct 21;11(10):e0165344. doi: 10.1371/journal.pone.0165344. eCollection 2016.

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.

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Cell Line, Tumor
  • Chemokines / metabolism*
  • Clathrin / metabolism
  • Cytosol / metabolism
  • Endocytosis
  • HEK293 Cells
  • Humans
  • Light*
  • Mice
  • Patch-Clamp Techniques
  • Rats
  • Receptors, CXCR4 / metabolism

Substances

  • Chemokines
  • Clathrin
  • Receptors, CXCR4
  • Calcium

Grants and funding

Our work was supported by the German Research Foundation (http://www.dfg.de) (SFB807 to E.B.) and by the Max-Planck-Society (http://www.mpg.de) (E.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.