FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons

摘要

Development of PKG FRET biosensors for cGMP. Schematic of the PKG and the Yellow and Red PKG biosensors. The Red PKG biosensor with the indicated conformational change upon cGMP binding. The D-domain of PKG ( PKG) is from S403 to E542 (PDB:4OFF and 4OFG; absence and presence of cGMP, respectively), T-sapphire (GFP) is from PDB:1GFL, Dimer2 (DsRed) is from PDB:1G7K. Similar structures would be expected from the Yellow PKG. , Homogenates of HEK293 cells transfected with Yellow PKG biosensor or Red PKG biosensor (where indicated) were incubated with increasing concentrations of cGMP and FRET, displayed as / (Yellow PKG) and / (Red PKG) ratios, was measured as described in Materials and Methods and normalized to the minimum (absence of cGMP) and maximum (highest cGMP concentration). Shown are data from one representative of four independent experiments. , Emission spectra of the indicated biosensors from HEK293 homogenates incubated with (solid line) and without (dashed line) 100 µM cGMP and excited at 405nm. The emission spectra were normalized to maximum fluorescence in the absence of cGMP. Maximal FRET response at the highest cGMP concentration. Data are mean ± SEM of four independent experiments. *  = 0.0002 vs. Red PKG (Two-tailed Student’s test)