An Ultrasensitive Genetically Encoded Voltage Indicator Uncovers the Electrical Activity of Non‐Excitable Cells

Abstract Most animal cell types are classified as non‐excitable because they do not generate action potentials observed in excitable cells, such as neurons and muscle cells. Thus, resolving voltage signals in non‐excitable cells demands sensors with exceptionally high voltage sensitivity. In this study, the ultrabright, ultrasensitive, and calibratable genetically encoded voltage sensor rEstus is developed using structure‐guided engineering. rEstus is most sensitive in the resting voltage range of non‐excitable cells and offers a 3.6‐fold improvement in brightness change for fast voltage spikes over its precursor ASAP3. Using rEstus, it is uncovered that the membrane voltage in several non‐excitable cell lines (A375, HEK293T, MCF7) undergoes spontaneous endogenous alterations on a second to millisecond timescale. Correlation analysis of these optically recorded voltage alterations provides a direct, real‐time readout of electrical cell–cell coupling, showing that visually connected A375 and HEK293T cells are also largely electrically connected, while MCF7 cells are only weakly coupled. The presented work provides enhanced tools and methods for non‐invasive voltage imaging in living cells and demonstrates that spontaneous endogenous membrane voltage alterations are not limited to excitable cells but also occur in a variety of non‐excitable cell types.

A) Imaging protocol for all molecular brightness-voltage recordings.Light of 470 nm and 530 nm wavelength was applied as indicated.Excitation with 470 nm started 1.5 seconds before image acquisition to measure fluorescence after the internal photoswitching of cpGFP.Images for cpGFP and EGFP were acquired with 470 nm illumination, and for mKate2, 530 nm illumination was used.The acquisition was performed using an EGFP-mCherry filter set (Chroma).B-L) Molecular brightness-voltage relationships of all mKate2-ASAP3:Q396R:T134 mutants with sufficient plasma membrane expression.The molecular brightness values were obtained by normalizing the recorded FcpGFP/FmKate2 signals to the average FEGFP/FmKate2 of mKate2-EGFP (4.01 ± 0.05, n = 168 cells) expressed in HEK293T cells and recorded under the same illumination conditions.The mean fits of the mKate2-ASAP3 (black) and mKate2-ASAP3:Q396R (red) brightness-voltage relationships are shown as dashed lines.The data are presented as means with standard error of the mean (SEM) and superimposed fits according to Equation 1.For n and fit parameters, see Table 1.A-O) Molecular brightness-voltage relationships of all mKate2-ASAP3:Q396R:N138 mutants with sufficient plasma membrane expression.Data were acquired and analyzed as described in Figure S1.For n and fit parameters, see Table 1.The mean fits of the mKate2-ASAP3 (black) and mKate2-ASAP3:Q396R (red) brightness-voltage relationships are shown as dashed lines.

Figure S3. Molecular brightness-voltage relationship of ASAP3:Q396R:Y141 variants.
A-P) Molecular brightness-voltage relationships of all mKate2-ASAP3:Q396R:Y141 mutants with sufficient plasma membrane expression.Data were acquired and analyzed as described in Supplementary Figure 1.For n and fit parameters, see Table 1.The mean fits of the mKate2-ASAP3 (black) and mKate2-ASAP3:Q396R (red) brightnessvoltage relationships are shown as dashed lines.A) Molecular brightness-voltage relationships of mKate2-rEstus excited at 480 nm, recorded from HEK293T cells at varying extracellular pH.Data are presented as means ± SEM with superimposed fits (Equation 1), and n values are shown in parentheses.Extracellular solutions were buffered with 10 mM MES (pH 5.9, 6.4), HEPES (pH 6.9, 7.4, 7.9), or TRIS (pH 8.4, 8.9) and adjusted to the respective pH with NaOH or HCl.B) Same as in A but for mKate2-rEstus-A318S (mKate2-ASAP3-N138G-Y141T-Q396R).C-F) Parameters of the fits from A as a function of pH for mKate2-rEstus and mKate2-rEstus-A318S.Brightness-pH relationships in C and D were fitted according to Equation 5; pKa and Hill coefficients (nh) are depicted in the graphs.F480 image of HEK293T cells stably expressing rEstus (from Figure 5D) with superimposed ROIs (orange).ROIs were drawn across the cell body and excluded membrane areas that separate two connected cells.This approach avoids unwanted correlation by fluorescence crosstalk from neighboring cells.

Figure S4 .
Figure S4.Mutation S318A in cpGFP increases the molecular brightness of ASAP3 derivatives by up to 60%.A) 3D model of ASAP3:Q396R.Mutated residues in the cpGFP moiety are numbered with respect to ASAP3 and, in parentheses, with respect to GFP.B) Molecular brightness-voltage relationships of mKate2-ASAP3:Q396R variants S318A (red), S178G (magenta), and V170T (cyan), recorded from HEK293T cells.The fit curve of mKate2-ASAP3:Q396R from Figure 1C is shown for comparison (dashed line).C) Excitation spectra of cpGFP of mKate2-ASAP3:Q396R (S318, n = 5) and the S318A variant (S318A, n = 4) in HEK293T cells voltage-clamped at -100 mV; data are mean ± SEM.The spectra were normalized to the mKate2 signal and scaled to reach the same maximum B(%EGFP) for S318 as in B. D) Absolute green fluorescence intensity (FcpGFP) of HEK293T cells expressing ASAP3:Q396R and the S318A variant with and without N-terminally fused mKate2.Individual green symbols represent medians of 3700-7400 cells, and each type of symbol represents measurements from one day.Black rhombs represent mean values of the individual medians.D) B(Vm) relationship of mutant mKate2-ASAP3:N138G:Y141T:S318A:Q396R, termed mKate2-rEstus.For reference, B(Vm) for the construct with S318 is shown as a dashed line.F) Absolute value of the first derivative (from E) as a function of Vm.For reference, |dB/dVm| is shown for mKate2-ASAP3:N138G:Y141T:Q396R and mKate2-ASAP3 (black) as dashed lines.G) Absolute change in molecular brightness (ΔB) across the resting Vm (yellow) and overshoot (red) range for mKate2-ASAP3 and mKate2-rEstus.Numbers indicate the gain in molecular brightness change in the indicated ranges of mKate2-rEstus with respect to mKate2-ASAP3.All data were obtained from voltage-clamped HEK293T cells and are presented as means ± SEM.For more details, such as n values and fit parameters, refer to Table1.

Figure S5 .
Figure S5.S318A enhances the brightness of F480 at physiological pH values by shifting the pKa.

Figure S6 .
Figure S6.Voltage-sensor kinetics.A,B) FcpGFP responses of voltage-clamped HEK293T cells expressing mKate2-ASAP3 or mKate2-ASAP3:Q396R recorded with a photodiode system.Voltage steps of the same amplitude (-80 mV holding potential to 40 mV), but with varying durations (ranging from 2 ms to 512 ms), were applied to cells expressing the specified constructs.The traces are the means of the indicated number of recordings from independent cells.The traces were linearly corrected for bleaching, and normalized to the average FcpGFP signal between 430 and 480 ms of the 512-ms pulse.C-O) As in A and B but for cells expressing derivatives of mKate2-ASAP3:Q396R.

Figure S7 .
Figure S7.Voltage-sensor kinetics for ASAP3-Q396R:N138 variants.A-O) FcpGFP responses of voltage-clamped HEK293T cells expressing N138 derivatives of mKate2-ASAP3:Q396R recorded with a photodiode system.Voltage steps of the same amplitude (-80 mV holding potential to 40 mV), but with varying durations (ranging from 2 ms to 512 ms), were applied to cells expressing the specified constructs.The traces are the means of the indicated number of recordings from independent cells.The traces were linearly corrected for bleaching, and normalized to the average FcpGFP signal between 430 and 480 ms of the 512-ms pulse.

Figure S8 .
Figure S8.Voltage-sensor kinetics for ASAP3-Q396R:Y141 variants.A-O) FcpGFP responses of voltage-clamped HEK293T cells expressing Y141 derivatives of mKate2-ASAP3:Q396R recorded with a photodiode system.Voltage steps of the same amplitude (-80 mV holding potential to 40 mV), but with varying durations (ranging from 2 ms to 512 ms), were applied to cells expressing the specified constructs.The traces are the means of the indicated number of recordings from independent cells.The traces were linearly corrected bleaching, and normalized to the average FcpGFP signal between 430 and 480 ms of the 512-ms pulse.

Figure S9 .
Figure S9.Voltage dependence of rEstus and ASAP3 kinetics.A) Normalized FcpGFP responses of voltage-clamped HEK293T cells expressing mKate2-ASAP3 or mKate2-rEstus to the indicated depolarizing voltage steps recorded with a photodiode system at 23°C; all traces were fit with a doubleexponential function (Equation 3, shown in red for a step to 60 mV).B) The fast and slow time constants from the time-course fits (from A) plotted as a function of Vm.The means ± SEM of the relative amplitudes of the fast component (Afast) are also indicated; straight lines connect points for clarity; n = 4 for mKate2-ASAP3 and n = 5 for mKate2-rEstus.C,D) As in A and B but for repolarizing voltage steps; fit curves in C are shown for a step to -100 mV.

Figure S10 .
Figure S10.Simultaneous whole-cell patch-clamp and fluorescence recordings.A) Endogenous membrane potential (Vm) fluctuations of a HEK293T cell stably transfected with rEstus in whole-cell current clamp mode were recorded using either the electrode (black) or fluorescence imaging (green).The galvanic recording was conducted at 1 kHz and binned by a factor of 100 to match the sampling rate of the fluorescence recording.B) Fluorescence plotted as a function of Vm (from A).For small changes in Vm, the fluorescence signal behaves nearly linearly (red line).Voltage fluctuations recorded using the electrode and the fluorescence signal from rEstus show a strong correlation (r 2 of 0.95).C) Fluorescence signal of the same cell as in A but in voltage clamp mode, with a holding Vm of -15 mV.The majority of the observed fluctuations in current clamp mode disappear.

Figure S11 .
Figure S11.Endogenous Vm fluctuations of HEK293T cells with and without 400-nm light exposure.A) Normalized F480 traces of a HEK293T cell stably transfected with rEstus.To test whether the endogenous fluctuations were induced by 400-nm light exposure, fluorescence was recorded with excitation at 480 nm (100 ms exposure every 200 ms) without (left, right) and with intercalated 400-nm exposure (100 ms, middle).Fluorescence traces were corrected for photobleaching and switching using a single-exponential function.B,C) Relative standard deviation (B) and relative peak-to-peak amplitudes (C) of the normalized F480 traces within a 30-s time interval for the same cells before, during, and after 400 nm illumination, n = 52 cells.Rhombs denote mean values.

Figure S12 .
Figure S12.ROI selection for fluorescence fluctuation correlation analysis.

Figure S13 .
Figure S13.Bleaching correction for fluorescence fluctuation correlation analysis.A) Non-corrected normalized F480 traces from Figure5Bwith superimposed linear fits.B) Linear correlation of the endogenous F480 fluctuations between connected cells (cell 1 and 2) without linear bleaching correction.C,D) As in A and B but after linear bleaching correction.E-H) As in A-D but for cells without direct electrical connection (cell 1 and cell 3).Traces of connected cells (cell 1 and cell 2) exhibit a strong correlation regardless of the correction.The correction reduces erroneous correlation arising from bleaching between cells that are not electrically connected (cell 1 and cell 3).