Lee EEL and Bezanilla F (2017), Biophysical Characterization of Genetically Enc...

XB-ART-54249

Biophys J 2017 Nov 21;11310:2178-2181. doi: 10.1016/j.bpj.2017.10.018.

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Biophysical Characterization of Genetically Encoded Voltage Sensor ASAP1: Dynamic Range Improvement.

Lee EEL , Bezanilla F .

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Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1. Gating charge movement and fluorescence kinetics track closely with each other, although ASAP1 gating currents are significantly faster than those of Ciona intestinalis voltage-sensitive phosphatase. Altering the residue before the first gating charge removes a split in the ASAP1 QV curve, but preserves the accelerated kinetics that allow for the faithful tracking of action potentials in neurons.

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Species referenced: Xenopus laevis
Genes referenced: asap1 map9

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	Figure 1. Biophysical characterization of ASAP1. (A) A cartoon representation of the design of ASAP1. (B) Representative gating current recordings of ASAP1. The inset describes the pulse protocol. From a holding potential of −60 mV, a 50 ms prepulse to −160 mV is followed by a 30 ms variable voltage pulse from −180 to 180 mV in increments of 20 mV, with a 50 ms postpulse at −160 mV. (C) Representative traces of ASAP1 fluorescence, with the same pulse procedure. (D) Normalized QV and 1-FVnormalized (inverted fluorescence) curves for ASAP1. (E) A comparison of the activation τfast of the gating charge and the fluorescence of ASAP1. Gating as circles and fluorescence as squares; n = 11. To see this figure in color, go online.
	Figure 2. Small fluorescence lag in ASAP1. A comparison of fluorescence lags, calculated from double exponential fits (see inset), of different GEVIs as a function voltage with ASAP1 as circles, ArcLight as inverted triangles, and ArcLightning in triangles shows a very short lag for ASAP1. Inset: Data are the small dots, the fitted curve is the solid line, and the fluorescence lag is measured as the distance from the intersection of the fitted curve to the x axis, as shown with the dashed line. ASAP1: n = 11; ArcLight: n = 5; ArcLightning: n = 5. To see this figure in color, go online.
	Figure 3. Biophysical characterization of GgVSD. (A) A comparison of the QV curves of GgVSD (square) versus ASAP1 (circle). (B) A comparison of the fast activation τ of GgVSD and ASAP1. Error bars are within the symbols. ASAP1: n = 11; GgVSD: n = 6. To see this figure in color, go online.
	Figure 4. Biophysical characterization of ASAP-Y and comparison to ASAP1. (A) Normalized QV curves for ASAP1 (circle) and ASAP-Y (square). (B) A comparison of the activation τfast of the gating charge for ASAP1 and ASAP-Y. (C) Normalized QV (circle) and 1-FVnormalized (square) curves for ASAP-Y. (D) A comparison of the ΔF/F % in a physiological range of ASAP1 (circle) and ASAP-Y (square). (ASAP1: n = 8; ASAP-Y: n = 6). To see this figure in color, go online.
	Figure 5. ASAP-Y can follow action potentials in rat dorsal root ganglion neurons. (A) Representative traces of action potentials from dorsal root ganglion neurons transfected with ASAP1 (left) and ASAP-Y (right). Corresponding fluorescence traces are shown below as −ΔF/F (%). (B) A table comparing the −ΔF/F per 100 mV and deactivation τ of ASAP1 and ASAP-Y fluorescence. ASAP1: n = 11; ASAP-Y: n = 13.

References [+] :

Baker, Genetically encoded fluorescent sensors of membrane potential. 2008, Pubmed