The first generation FP voltage sensors were developed by molecular fusion of a GFP-based fluorescent reporter to voltage-gated ion channels or components thereof. There are three prototypes obtained from three different laboratories, each of which constitutes a proof of principle and provided valuable insights ().
Fig. 1 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 (more ...)
The very first prototype, FlaSh, was generated in the Isacoff laboratory and obtained by fusing wtGFP to the C-terminus of the Drosophila
Shaker potassium channel (Siegel and Isacoff, 1997
). When expressed in oocytes, an 80-mV depolarization of the plasma membrane resulted in a 5% decrease in fluorescence (), this was designated FlaSh, for “fluorescent Shaker”.
To reduce unwanted effects on the cell's physiology, FlaSh was rendered non-conducting by introducing a W434F mutation, preventing ions from moving through the pore while maintaining voltage-dependent rearrangements (Perozo et al., 1993
). In order to resolve action potentials, an ideal sensor would generate a robust signal on a millisecond timescale. The signal strength from FlaSh is comparable to that of the voltage-sensitive organic dye, di4-ANEPPS; however, the on and off rates are rather slow (τ-on ~100 ms; τ-off ~60 ms). In an effort to improve the kinetics of FlaSh, Guerrero et al. (2002)
replaced the wtGFP with several different FPs. The change in optical characteristics mediated by the different FPs was substantial. The signal intensity, the direction of the fluorescent change, and the speed of FlaSh were all altered by the chromophore in an unsystematic way. Variants involving wtGFP and uvGFP both have reduced fluorescence in response to depolarization steps, while Ecliptic variants of GFP, YFP, and CFP exhibit increased fluorescence in response to depolarization. Remarkably, upon depolarization, an increase in fluorescence is seen when eGFP is excited at 450 nm, but there is a decrease in fluorescence when excited at 480 nm. The speed of the response is also governed by the chromophore, with the Ecliptic variant of GFP generating the fastest response (τ-on ~5 ms). While the mechanism underlying the fluorescence change that results from an alteration in membrane potential remains poorly understood, it is clear that the fluorescent reporter contributes significantly to the kinetics of the optical response and is an important parameter to vary in an attempt to improve genetically encoded voltage sensors.
The second prototypic design realized in the Knöpfel laboratory and termed VSFP1 exploits the voltage-dependent conformational changes around the fourth transmembrane segment (S4) of the voltage-gated potassium channel Kv2.1 and uses either fluorescence resonance energy transfer (FRET) (; Sakai et al., 2001
) or a permuted FP (Knöpfel et al., 2003
). The third prototype, SPARC (Pieribone laboratory; ), was generated by inserting a FP between domains I and II of the rat skeletal muscle Na+
channel (Ataka and Pieribone, 2002
These first generation voltage sensors are capable of optically reporting changes in membrane potential, but their use in mammalian systems is significantly hindered by their poor plasma membrane expression. When expressed in HEK 293 cells, the expression of these constructs is primarily intracellular and little if any of these first generation FP voltage sensors are co-localized at the cell surface with di8-ANEPPS (). shows the profiles of the FP in green and that of di8-ANEPPS in red along the red lines in the right column of . Much better co-localization occurs with Kv1.4 with GFP at the N-terminus and with the cation/chloride cotransporter, NKCC1, with YFP fused near the carboxyl terminus. There does seem to be some plasma membrane expression for Flare (a Kv1.4 variant of FlaSh), but the best-case scenario still exhibits fluorescence of a predominantly intracellular origin. More importantly, no functional optical signals could be detected using Flare, VSFP1, or SPARC (Baker et al., 2007
Fig. 2 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 (more ...)
The absence of a signal from the first generation probes is in part due to their low membrane expression and in part due to a large, nonresponsive background fluorescence that would mask any voltage-dependent signal. Because Kv1.4 with an N-terminal GFP exhibits excellent membrane expression, several strategies to release Flare or its Kv2.1-based homologues from the ER have been tried. Unfortunately, mutagenesis of potential ER retention signals, addition of ER release motifs, and expression in hippocampal neurons that endogenously express trafficking partners all failed to significantly improve the plasma membrane expression (Ray et al., unpublished observations; Baker and Cohen, unpublished observations).
For the second generation of FP voltage sensors, we hoped to overcome this issue of poor targeting to neuronal membranes.