Design and characterization of a DNA‐encoded, voltage‐sensitive fluorescent protein

Abstract

Optical imaging of electrical activity has been suggested as a promising approach to investigate the multineuronal representation of information processing in brain tissue. While considerable progress has been made in the development of instrumentation suitable for high-speed imaging, intrinsic or extrinsic dye-mediated optical signals are often of limited use due to their slow response dynamics, low effective sensitivity, toxicity or undefined cellular origin. Protein-based and DNA-encoded voltage sensors could overcome these limitations. Here we report the design and generation of a voltage-sensitive fluorescent protein (VSFP) consisting of a voltage sensing domain of a potassium channel and a pair of cyan and yellow emitting mutants of green fluorescent protein (GFP). In response to a change in transmembrane voltage, the voltage sensor alters the amount of fluorescence resonance energy transfer (FRET) between the pair of GFP mutants. The optical signals respond in the millisecond time-scale of fast electrical signalling and are large enough to allow monitoring of voltage changes at the single cell level.

Rational Optimization and ImagingIn Vivoof a Genetically Encoded Optical Voltage Reporter

“…It is difficult to make precise predictions for GFP fusions with voltage-gated cation channels, because the mechanisms of fluorescence modulation are generally unclear. However, performance is likely to be poor given that the ⌬F/F of all existing channel-based probes is at least one order of magnitude lower than the predicted ⌬Q/Q of the tethered acceptor configuration (Siegel and Isacoff, 1997;Sakai et al, 2001;Ataka and Pieribone, 2002;Guerrero et al, 2002). This problem is compounded by the large number of gating charges associated with each fluorophore (Aggarwal and MacKinnon, 1996), which compromises performance even in the ideal case (Fig.…”

“…The simplest of these is the tethered model, which represents the general class of reporters in which the fluorophore is physically associated with mobile probe charges. This class includes both the tethered acceptor configuration and GFP fusions with voltage-gated cation channels (Siegel and Isacoff, 1997;Sakai et al, 2001;Ataka and Pieribone, 2002;Guerrero et al, 2002). The tethered-DPA/ EGFP-⌬CAAX pair ( y ϭ 10 Å; R 0 ϭ 37 Å) and optimal tethered pair ( y ϭ 20.8 Å; R 0 ϭ 40.4 Å) yield a ⌬Q of 51 and 60% of the theoretical limit, respectively (Fig.…”

“…Most of these reporters combine nonconducting mutants of voltage-gated cation channels with derivatives of the green fluorescent protein (GFP). For example, FlaSh and its variants (Siegel and Isacoff, 1997;Guerrero et al, 2002) are based on the Shaker potassium channel, whereas SPARC (sodium channel protein-based activity reporting construct) (Ataka and Pieribone, 2002) is a mutant Na v 1.4 sodium channel, and VSFP1 (voltage-sensitive fluorescent protein 1) (Sakai et al, 2001) uses an isolated voltage sensor domain of the Kv2.1 potassium channel.…”

Imaging membrane potential in dendrites and axons of single neurons

“…An additional issue is that the response is not linearly related to the change in membrane potential, but rather follows the sigmoidal voltage dependence of channel activation. Improvements in speed have been obtained by using fluorescence resonance energy transfer (FRET) between a pair of GFP variants linked to the same potassium channel [47] and by inserting GFP at a site closer to the voltage sensor of voltage-activated sodium channels [6]. In a recent development, a hybrid approach, which links GFP to a synthetic voltage-sensing molecule using FRET, has been shown to offer increased signal to noise, as well as a relatively linear change in fluorescence with membrane potential, and fast (sub-millisecond) response time [9].…”

Uses of GFP in Transgenic Vertebrates

“…pH, Ca ++ , and voltage sensitive variants of GFP generated through protein fusions can be used for monitoring neural activity non-invasively (Miesenbock et al, 1998;Miyawaki et al, 1999;Sakai et al, 2001). A novel GFP fusion protein can be used for mapping neural connectivity: a fusion of GFP to a non-toxic fragment of tetanus toxin is transferred across synapses in a retrograde direction in transgenic mice (Maskos et al, 2002 …”