Confocal images of HEK 293 cells. (A) SPARC, VSFP-1, Flare, Kv1.4-N-GFP, or NKCC1-YFP were expressed in HEK 293 cells and imaged via confocal microscopy. The images on the left show HEK 293 cells expressing the fluorescent construct. The images on the right are the same cells after the addition of di8-ANEPPS to the bathing medium. Di8-ANEPPS functions as a fluorescent plasma membrane marker. (B) The profiles show the green (FP voltage sensor) and red (di8-ANEPPS) fluorescence along the arrow in the images. The arrows indicated the location of the external membrane. Taken from Baker et al. (2007).

Properties of VSFP2.1. Response–voltage relationship and kinetics of VSFP2.1 at 22°C (A1–A3) and at 35°C (B1–B3). (A1, B1) Ratio of yellow/cyan fluorescence during a family of 500 ms voltage steps from a holding potential of −70 mV to test potentials of −140 to +40 mV (20 mV increments). Traces are grand averages over average responses from 4 cells (A1) and 6 cells (B1). (A2, B2) Ratio of yellow/cyan fluorescence versus test membrane voltage. Connected symbols are data from individual cells. Red lines are Boltzmann fits with V_1/2 values as indicated. (A3, B3) Activation and deactivation time constants. Taken from Dimitrov et al. (2007).

VSFP2.1 can monitor physiological neuronal membrane voltage dynamics. PC12 cells expressing VSFP2.1 were voltage-clamped with a voltage trace obtained from a current-clamped mouse olfactory bulb mitral cell. The mitral cell was stimulated to generate a series of action potentials by intracellular injection of a current pulse (A) or by electrical stimulation of the olfactory nerve (B). Traces in (A) are averages of 50 sweeps, upper traces (in B) are the average of 90 sweeps, and the lower four traces in (B) are single sweeps. Traces show membrane potential (V), yellow fluorescence (Fy), cyan fluorescence (Fc), and the ratio of yellow and cyan fluorescence (Fy/Fc). Fluorescence signals were digitally low pass filtered (0.2 kHz) and were not corrected for dye bleaching. Recordings were done at 35°C. Taken from Dimitrov et al. (2007).

Fist generation FP voltage sensors. FlaSh (left panel) was generated by fusing wtGFP to the C-terminus of Drosophila Shaker potassium channel. Simultaneous two-electrode voltage-clamp recording and photometry in Xenopus oocytes show current and fluorescence changes in response to voltage steps (V) between −60 and 10 mV, in 10 mV increments. Holding potential was −80 mV. FlaSh exhibits on and off gating currents (Ig) but no ionic current. Integrating the gating current gives the total gating charge (Q) moved during the pulse. FlaSh fluorescence (F) decreases reversibly in response to membrane depolarizations. Traces are the average of 20 sweeps. Modified from Siegel and Isacoff (1997). VSFP1 (middle panel) consist of an isolated voltage sensor domain coupled to a pair of cyan and yellow fluorescent proteins. Recordings were done in HEK 293 cells. Modified from Sakai et al. (2001). SPARC (right panel) was generated by inserting an FP between domains I and II of the rat skeletal muscle Na⁺ channel. Recordings were done in Xenopus oocytes. Modified from Ataka and Pieribone (2002).

Efficient plasma membrane targeting and voltage report of VSFP2s. (A) Confocal fluorescence and transmission images of PC12 cells transfected with VSFP2A, VSFP2B, VSFP2C, and VSFP2D. Scale bar is 30 μM. (B) Sample traces of cyan fluorescence, yellow fluorescence, and the ratio of yellow/cyan fluorescence (average of 27 traces). Lower traces indicate times of shutter opening and membrane depolarization from −70 to 150 mV. (C) Average changes in cyan fluorescence and yellow fluorescence induced by depolarization to 150 mV in VSFP2A (11 cells), VSFP2B (7 cells), VSFP2C (7 cells), and VSFP2D (7 cells). (D) Average changes in the ratio of yellow and cyan fluorescence induced by depolarization to 150 mV. (E) Ratio of yellow/cyan fluorescence versus test membrane voltage. Lines are Boltzmann fits. Modified from Dimitrov et al. (2007).

Neuronal expression and functionality of VSFP2.1. Primary mouse cortical cultures were transfected with a codon optimized version of VSFP2.1 at 10 DIV. Left panel shows a pyramidal-shaped neuron expressing this FP voltage sensor. Right panel shows response of membrane potential, yellow fluorescence, and cyan fluorescence to a hyperpolarizing current injected via a patch pipette. Fluorescence values are expressed as percentage of total measured light intensity, without correction for background fluorescence (Mutoh et al., unpublished observations).