Single-molecule fluorimetry and gating currents inspire an improved optical voltage indicator
- University of Chicago, United States
Figures
Figure 1
ArcLight fluorescence responds to voltage sensor movements.
(A) Family of gating currents from an oocyte injected with ArcLight R217Q. The holding potential is -80 mV, and the pulses range from -160 mV (darkest red) to +140 mV (darkest blue) in 20 mV …
Figure 2 with 1 supplement
Voltage-sensitive fluorescence can be recorded from single ArcLight' molecules.
(A) An image of the surface of an oocyte expressing ArcLight' in single-molecule concentrations. Square image field is 50 μm × 50 μm. Height in the z-axis corresponds to fluorescence intensity. This …
Figure 2—figure supplement 1
A polymer cushion improves clamp speed under a peeled oocyte.
The speed of the voltage clamp on the bottom side of an oocyte is observed by monitoring the fluorescence of di-8-ANEPPS in the oocyte membrane. When a peeled oocyte is placed directly on a clean …
Figure 3
Fluorescence from an isolated ArcLight' GFP domain and eGFP show significant fluctuation noise.
(A) Fluorescence from a single isolated GFP domain from ArcLight' with no attached voltage sensor. The fluorescence displays fluctuations between different levels and does not appear to be …
Figure 4 with 3 supplements
ArcLight fluorescence partially follows the kinetics of voltage sensor movement.
(A) ArcLight fluorescence response to an 'on' pulse protocol with a holding potential of -120 mV with 200 ms pulses ranging from +120 mV to -140 mV by 20 mV intervals. (B) As in A, but in response …
Figure 4—figure supplement 1
ArcLight fluorescence changes are slower than integrated gating charge kinetics.
(A) Both gating charge movement kinetics (blue, obtained by integrating gating currents) and fluorescence changes (green) of ArcLight are well-fit by a double-exponential function (circles – faster …
Figure 4—figure supplement 2
The onset of ArcLight fluorescence change lags behind voltage change onset.
(A) Following the onset of the voltage change (vertical red line), a measurable lag occurs prior to the onset of ArcLight fluorescence change. Traces are shown from a holding potential of -80 mV and …
Figure 4—figure supplement 3
The R217R and R217E ArcLight mutants show similar behaviors to R217Q, but with shifted voltage dependence.
(A) Gating current weighted time constants (blue), fluorescence lags (red), and the fast time constants of the fluorescence response (green) were calculated for ArcLight R217R as described above for …
Figure 5 with 1 supplement
Accelerated gating kinetics speed up fluorescence, up to a point.
(A) The voltage-dependence of fluorescence change of ArcLight I126F Q217R occurs over a physiological voltage range, as taken from an on protocol identical to that in Figure 4. (B) The gating …
Figure 5—figure supplement 1
Substitution of I126F and Q217R into ArcLight induces a roughly constant acceleration of gating current.
(A) Ratio of ArcLight gating current kinetics to I126F Q217R gating current kinetics across all voltages in response to an 'on' protocol. (B) As in A, but in response to an 'off' protocol. The on …
Figure 6 with 1 supplement
ArcLightning displays improved fluorescence response to repetitive pulses in mammalian cells.
(A) ArcLightning (pink) expressed in HEK cells displays moderately faster kinetics in response to depolarization and much faster kinetics in response to hyperpolarization than ArcLight (blue) at …
Figure 6—figure supplement 1
ArcLightning is faster than ArcLight in mammalian cells.
(A) The on kinetics of ArcLightning (pink squares) are faster than those of ArcLight (blue circles) at physiological voltages. As seen in oocytes, kinetics became similar at highly depolarized …
Figure 7 with 2 supplements
A minimal four-state model of ArcLight kinetics.
The first transition carries the gating charge movement while the third transition carries the fluorescence change and the second transition carries no observable changes. The kinetics of the first …
Figure 7—figure supplement 1
A minimal three-state model of ArcLight kinetics.
The first transition carries the gating charge movement while the second transition carries the fluorescence change. If , the fluorescence would be slower than the gating currents, as observed. …
Figure 7—figure supplement 2
ArcLight fluorescence may be influenced by the relaxed state.
(A) The Q-V curve obtained from a protocol of hyperpolarizing pulses from a +40 mV holding potential (red squares) is shifted to more negative potentials compared to a Q-V curve obtained from …
