Engineering and characterization of an enhanced fluorescent protein voltage sensor - PubMed (original) (raw)

Engineering and characterization of an enhanced fluorescent protein voltage sensor

Dimitar Dimitrov et al. PLoS One. 2007.

Abstract

Background: Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells.

Results: Here we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells.

Conclusions and significance: This biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Design and plasma membrane expression of VSFP2s.

A: A pair of CFP (donor) and YFP (acceptor) is attached to the 4-transmembrane-voltage-sensing domain (VSD) of Ci-VSP. B, C: Confocal fluorescence (B) and transmission images (C) of PC12 cells transfected with VSFP2D. Note the targeting of the fluorescent protein to the plasma membrane. Scale bar is 30 μm.

Figure 2

Figure 2. Fluorescence signals induced by membrane depolarization in PC12 cells.

A: 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 mV to 150 mV. B: Average changes in cyan fluorescence and yellow fluorescence induced by depolarization to 150 mV. Labels A–D indicate VSFP2A (11 cells), VSFP2B (7cells), VSFP2C (7cells), VSFP2D (7 cells). C: Average changes in the ratio of yellow and cyan fluorescence induced by depolarization to 150 mV. (D) Ratio of yellow/cyan fluorescence versus test membrane voltage. Lines are Boltzmann fits. Bars in B–D are SEM.

Figure 3

Figure 3. Characteristics of VSFP2.1. Response-voltage relationship and kinetics of VSFP2.1 at 35°C.

(A) 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 mV to +40 mV (20 mV increments). Traces are grand averages over average responses from 6 cells. (B) Ratio of yellow/cyan fluorescence versus test membrane voltage. Connected symbols are data from individual cells. Red line is Boltzmann fit with V1/2 value of −68.4 mV. (C) Voltage dependence of on and off time constants.

Figure 4

Figure 4. VSFP2.1 responds to physiological neuronal membrane signals.

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 sweep 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.

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