Fig. 5

Characterization and application of GEPIIs in cells. a Representative pseudocolor ratio images of permeabilized HeLa cells expressing GEPII 1.0 in the presence of different [K⁺]. Scale bar represents 20 µm. b CFP (cyan dashed line), FRET (yellow dashed line) fluorescence intensities, and the respective FRET ratio signal (black solid line) of GEPII 1.0 expressed in HeLa cells. Permeabilized (5 µM digitonin) HeLa cells were treated with different [K⁺] as indicated using a semi-automatic perfusion system. c Normalized EC₅₀ curves for K⁺ of different GEPIIs expressed in HeLa cells (n = 6 for each ± SD). Cells were permeabilized using 5 µM digitonin and respective K⁺ concentrations were added. Respective ∆FRET ratio values, normalized to the maximal response, i.e., 100%, were plotted against [K⁺]. d Representative ratio images of HeLa cells expressing either cytosolic (upper panels), nuclear (middle panels) or mitochondrially targeted (lower panels) lc-LysM GEPII 1.0 under resting conditions (intact cells, left panels) or after cell permeabilization (10 µM digitonin) in the absence of extracellular K⁺ (right panels). Scale bar represents 20 µm. e Basal [K⁺] ± SEM calculated using lc-LysM GEPII 1.0 expressed in INS-1 (white bars), HeLa (black bars), HEK293a (red bars) and EA.hy926 cells (blue bars) within the cytosol (n = 7 independent experiments for each/n = 52 cells for INS-1/ n = 74 cells for HeLa/n = 62 cells for HEK293a/n = 29 cells for EA.hy926), nucleus (n = 7 independent experiments for each/n = 133 cells for INS-1/n = 35 cells for HeLa/n = 184 cells for HEK293a/n = 49 cells for EA.hy.926), or mitochondria (n = 7 independent experiments for each/n = 142 cells for INS-1/n = 62 cells for HeLa/n = 102 cells for HEK293a/n = 45 cells for EA.hy926)