(a) Alignment of the S3-S4 loop region of voltage-sensing domains (VSDs) from voltage sensitive phosphatases of four different species. Low, intermediate and high levels of homology are represented by white, grey and black background color, respectively. The locations of S3 and S4 were predicted by the Transmembrane Prediction Tool of Geneious v.7 (Biomatters). (b) G. gallus VSD with the R153Q mutation and an insertion of circularly permuted green fluorescent protein (cpGFP) from GCaMP3 at 4, 5, and 6 amino acids downstream of S3 exhibited bright membrane-localized fluorescence in HEK293A cells. Insertion at other positions caused poor membrane localization or dimness. Scale bar, 10 μm. (c) Fluorescence response in HEK293A cells to voltage steps from –150 mV to +50 mV relative to the holding potential of –70 mV for three sensor variants with different insertion points of cpGFP into G. gallus VSD (GgVSD). All three variants produced similar fluorescence responses, with an ~3% decrease in intensity from –70 mV to +30 mV. Samples sizes (n) correspond to individual HEK293A cells. Error bars are mean ± standard error of the mean (SEM). (d) We constructed sensor variants in which the fluorescent protein, originally cpGFP from GCaMP3, was replaced with other circularly-permuted GFPs. All fluorescent proteins were inserted between Ala-147 and Thr-148 of GgVSD. To normalize for variability in transfection efficiency or expression in HEK293A cells, each sensor was coexpressed with mCherry-CAAX using an internal ribosome entry site (see Supplementary Fig. 5a). For each sensor, the green and red fluorescence images were identically scaled and merged. The relative ratio of green to red fluorescence, an indication of sensor brightness, was larger for the sensor containing the OPT variant of superfolder GFP (cpsfGFP-OPT). Substitution with a circularly permuted version of Clover (cpClover) or of the superecliptic pHluorin A227D from ArcLight (cpsepHluorin A227D) largely abolished membrane-localized GFP fluorescence. Findings were reproducible across multiple (n > 5) fields of view, with representative images shown here. Scale bar, 10 μm.