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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.
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 â60 mV, a 50 ms prepulse to â160 mV is followed by a 30 ms variable voltage pulse from â180 to 180 mV in increments of 20 mV, with a 50 ms postpulse at â160 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 Ïfast 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 Ï 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 Ïfast 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 ÎF/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 âÎF/F (%). (B) A table comparing the âÎF/F per 100 mV and deactivation Ï of ASAP1 and ASAP-Y fluorescence. ASAP1: n = 11; ASAP-Y: n = 13.
Baker,
Genetically encoded fluorescent sensors of membrane potential.
2008, Pubmed
Baker,
Genetically encoded fluorescent sensors of membrane potential.
2008,
Pubmed
Hochbaum,
All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.
2014,
Pubmed
Jin,
Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.
2012,
Pubmed
Lacroix,
Tuning the voltage-sensor motion with a single residue.
2012,
Pubmed
Li,
Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain.
2014,
Pubmed
,
Xenbase
Lundby,
Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP.
2010,
Pubmed
Murata,
Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor.
2005,
Pubmed
,
Xenbase
Pédelacq,
Engineering and characterization of a superfolder green fluorescent protein.
2006,
Pubmed
Stefani,
Cut-open oocyte voltage-clamp technique.
1998,
Pubmed
,
Xenbase
St-Pierre,
High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor.
2014,
Pubmed
,
Xenbase
Villalba-Galea,
Charge movement of a voltage-sensitive fluorescent protein.
2009,
Pubmed
,
Xenbase
Yang,
Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo.
2016,
Pubmed
