Gq proteins are universally important for signal transduction in mammalian cells. The underlying kinetics and transformation from extracellular stimuli into intracellular signaling, however could not be investigated in detail so far. Here we present the human Neuropsin (hOPN5) for specific and repetitive manipulation of Gq signaling in vitro and in vivo with high spatio-temporal resolution. Properties and G protein specificity of hOPN5 are characterized by UV light induced IP3 generation, Ca2+ transients and inhibition of GIRK channel activity in HEK cells. In adult hearts from a transgenic animal model, light increases the spontaneous beating rate. In addition, we demonstrate light induced contractions in the small intestine, which are not detectable after pharmacological Gq protein block. All-optical high-throughput screening for TRPC6 inhibitors is more specific and sensitive than conventional pharmacological screening. Thus, we demonstrate specific Gq signaling of hOPN5 and unveil its potential for optogenetic applications.

No abstract

No abstract

Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam. For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps, we measured FRET from cyan (CFP) to yellow fluorescent protein (YFP) by spectral analysis and fluorescence lifetime imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response to pharmaceutical agents, e.g., Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples.

No abstract