TagsBooks chloride ChRs Cryptochrome Dimerizers Electroporation Epilepsy Expression fiberoptics FRET GECI GEVI Highlighters Holography hVoS Indicators LED Lines LOV Opsins Optics Optrodes Phototropin prosthetics protons Pumps Recordings Reporter Retina Reviews Scattering Therapeutics Thoughts Toxicity Viruses Voltage waveguides
Tag Archives: FRET
The simultaneous optogenetic sensing of the intracellular concentrations of Cl− and H+ is a challenging task, which requires probes with high sensitivity allowing reliable quantitative measurements without perturbation of cell functioning. The recent paper in Frontiers of Molecular Neurosciences by Mukhtarov et al. (2013) describes the intracellular calibration and functional characterization of three genetically encoded probes developed for these purposes. The first is recently proposed combined Cl−/pH sensor (ClopHensor), which was obtained by fusion of a red fluorescent protein (RFP) with a GFP variant, E2GFP, which contains a specific Cl−-binding site (Arosio et al., 2010). The second is PalmPalm-ClopHensor, a variant that is preferentially expressed at the plasma membrane thanks to the addition of two palmitoylation sites at the N-terminus. The third ClopHensor variant contains a two additional mutations (H148G/V224L) in the GFP moiety conferring improved Cl− affinity and reduced pH dependence.
For functional analysis, constructs were expressed in CHO cells and neurons. In CHO cells ClopHensor and the H148G/V224L mutant exhibits cytoplasmic intracellular distribution while the PalmPalm-ClopHensor construct, as expected, is preferentially localized in the vicinity of membranes (FIG.1).
In order to evaluate the dynamic range and sensitivity of constructs to ions, CHO cells were co-transfected with Cl−-selective glycine … Continue reading