Anap as a fluorescent probe in hHV1. A) Ribbon representation of transmembrane segments S1-S4 of closed hHV1 based on the model of Randolph et al. (41). S1-S3 are in grey whereas S4 is in light blue. S4 positively charged arginine residues are shown as cyan sticks, whereas the residues where Anap was incorporated individually in the S4 segment are depicted as green sticks and with green arrow heads in the S3-S4 sequence below; positively charged arginine residues are indicated in marine blue. B) Structure of non-canonical amino acid Anap (left), and a schematic representation (right) that shows the incorporation of Anap (green star) into the hHV1 dimer expressed in HEK293 cells. An mCherry fluorescent protein (magenta cylinder) was fused to the C-terminal end of hHV1 as an Anap incorporation reporter. C) Images of a representative Patch-clamp Fluorometry (PCF) experiment, showing the voltage-clamped cell and the co-localization of Anap and mCherry fluorescence in the cell membrane for Anap incorporated at position Q191 of hHV1. D) G-V curves obtained from currents produced by each hHV1 mutant rescued by Anap incorporation. All G-V s were obtained at ΔpH=1 and compared with hHV1 WT. Continuous lines are the fit of the conductance data to equation 1; fit parameters are summarized in Supplemental table I. The incorporation of Anap at the I202 site sifts the G-V ~65 mV to more negative potentials. Data shown are mean ± s.e.m. E) Normalized mean emission spectrum of Anap (continuous lines) and mCherry (dashed lines) at each incorporation site (color code from D). Q191(n=15); A197(n=10); L198(n=3); G199(n=5); L200(n=1); L201(n=4); I202(n=5). The vertical blue line indicates the peak emission of Anap in water (486 nm). A second emission peak can be distinguished in every position inside S4 where Anap was incorporated, except Q191Anap. This peak is located around 610 nm which coincides with the peak emission of mCherry.

The fluorescence of incorporated Anap is stable to external acidity and local pH changes. A) Mean spectra of Anap fluorescence in the hHV1-Q191Anap mutant at each external pH tested (pHo). The emission peak of spectra of Anap remained inside the wavelength range of 475-480 nm. B) Percentage of fluorescence intensity change normalized to fluorescence at pHo 7 in hHV1-Q191Anap mutant (n=13). The intensity was measured from the peak of emission spectra. C) Representative PCF experiments with the hHV1-V62Anap mutant. Currents (upper panel, orange traces) and fluorescent signal (lower panel, gray traces) were elicited in response to voltage pulses from −100 mV to 120 mV in steps of 20 mV. D) F-V and G-V relationships from the experiments shown in C. Relative fluorescence changes at the end of voltage test pulses are shown in gray triangles, and conductance is shown in orange circles. The orange continuous line is the fit to equation 1 of G-V data (fit parameters: V0.5 = 24.4 ± 1.6 mV; q = 1.5 ± 0.1 e0). Data in B and

D are Mean ± s.e.m.

Anap incorporation in position A197 reveals that the movement of S4 is modulated by ΔpH. A-B) Representative PCF experiment with A197Anap at ΔpH=0 and ΔpH=2, respectively. Proton current families (upper panels) are shown in blue traces and fluorescent Anap signal (lower panel) in lemon traces. C) Activation time constant of current (blue) and fluorescent (lemon) signals at ΔpH=0 obtained by fitting Eq. 3. The dark blue curve shows the exponential fit to Eq. 4. The fit parameters were: τ(0) = 935 ms and q = −0.06 e0 for fluorescence and 678 ms and −0.09 e0 for current. D) F-V (empty triangles) and G-V (filled diamonds) curves and different ΔpH values (ΔpH=0 in blue; ΔpH=1 in red; ΔpH=2 in black). The data were fit to equation 1(G-V, continuous curves; F-V, discontinuous curves) with the following parameters: ΔpH=0; F-V: V0.5 = 72.3 ± 6 mV; q = 1.0 ± 0.1 e0. G-V: V0.5 = 69.6 ± 1.5 mV; q = 1.1 ± 0.1 e0. ΔpH =1; F-V: V0.5 = 26.6 ± 1.5 mV; q = 1.3± 0.1 e0. G-V: V0.5 = 23.4 ± 1.3 mV; q = 1.5 ± 0.1 e0. ΔpH = 2; F-V: V0.5 = −6.1 ± 1.8 mV; q = 1.0 ± 0.1 e0. G-V: V0.5 = −9.2 ± 2.3 mV; q = 1.2 ± 2.4 e0. E) Normalized spectra of Anap in A197Anap mutant obtained in steady-state (300 ms at the end of holding potential and the end of the test pulse, green bars in the inset) in response to different voltages (color code indicates the test pulse in mV: purple, −60; dark blue, −40; light blue, 20; cyan, 0; light green, 20; dark green, 40; olive, 60; yellow, 80; orange, 100; dark red, 120; red, 140). Data shown in C and D are mean ± s.e.m.

The charge transfer center (F150) is an Anap quencher. A) Cartoon showing the presence of aromatic residues in hHV1 (rendered as space-filling dots, main chain in light blue, S3 was removed for illustration). F150 in yellow and Anap in pink. B) Averages of spectra of Anap incorporated in both mutants (HV1-A197Anap, red; HV1-F150A-A197Anap, green) normalized to the fluorescence of mCherry (black). The double mutant’s brightness is approximately 60% higher. Shadows represent s.e.m. C) Comparison of the intensity of the emission spectrum peak of Anap normalized to the intensity of the fluorescent protein mCherry between the mutant HV1-A197Anap-Cherry (0.49 ± 0.03) and double mutant HV1-F150A-A197Anap-Cherry (0.79 ± 0.05), taken at 48 hours post-transfection. Each point indicates an individual spectrum measured from a single cell; n = 41 and 49, respectively. Black horizontal lines are the mean ± s.e.m. T-test value p <0.001. D) Representative fluorescence traces from PCF experiments of the double mutant HV1-F150A-A197Anap at ΔpH=1 (upper panel) and ΔpH=0 (lower panel). E) Comparison of G-V (diamonds) and F-V (triangles) relationship between both ΔpH conditions (ΔpH=1 in green; ΔpH=0 in purple) of the double mutant HV1-F150A-A197Anap. F-V curve of HV1-F150A-A197Anap at ΔpH=0 is shifted negatively around 58 mV compared to ΔpH=1. Boltzmann fit parameters of HV1-F150A-A197Anap were: ΔpH=1 F-V: V0.5=-19.8 ± 2.7 mV; q =1.2 ± 0.1 e0; G-V: V0.5 = 22.7 ± 2.3 mV; q = 0.9 ± 0.1 e0. ΔpH=0 F-V: V0.5=38.0 ± 3.0 mV; q =0.9 ± 0.1 e0; G-V: V0.5 = 42.6 ± 3.8 mV; q = 1.0 ± 0.1 e0. Data shown in B, C and E are mean ± s.e.m.

The kinetics of fluorescent signal during deactivation is strongly modulated by pH. Representative PCF experiments with the hHV1-L201Anap mutant at A) ΔpH=0 [5.5int-5.5ext]. B) ΔpH=1, and C) ΔpH=2. Current families are shown in the upper panel (purple traces) and fluorescent signals in the lower panel (black and gray traces). D) G-V (filled diamonds) and F-V (empty triangles) relationships at ΔpH=0 (purple markers, n = 3), ΔpH=1 (orange markers, n=4) and ΔpH=2 (black markers, n=5) of mutant hHV1-L201Anap. Data are mean ± s.e.m. Note that the difference between the activation at ΔpH=1 and ΔpH=0 is around 77 mV/ΔpH unit. Boltzmann fit parameters: ΔpH=0, F-V; V0.5= 84.6 ± 2.1 mV, q =1.0 e0 ± 0.1. G-V; V0.5= 79.7 ± 1.8 mV, q = 1.4 ± 0.1 e0. ΔpH =1, F-V; V0.5= 7.7 ± 1.6 mV, q =1.2 ± 0.1 e0. G-V: V0.5= 6.3 ± 2.2 mV; q = 1.2 ± 0.1 e0. ΔpH = 2, F-V: V0.5= −21.1 ± 2.3 mV; q =1.1 ± 0.1 e0. G-V: V0.5= −30.7 ± 1.9 mV; q = 1.2 ± 0.1 e0. E) Comparison of the current and fluorescence at two values of ΔpH with the predictions of the sequential activation model in Scheme I. Experimental current and fluorescence traces are color coded as in A). Simulated current traces are orange and fluorescence traces are lemon. Simulation parameters can be found in Supplementary Table II.

Absolute pH values are gating determinants in hHV1. A) Representative PCF experiment at ΔpH=0 (5.5o-5.5i). Currents are purple and fluorescence black. B) Similar experiment to A) with ΔpH=0 (7o-7i). Current and fluorescence traces color coded as in A). C) G-V (filled diamonds) and F-V (empty triangles) curves at ΔpH=0 but with different absolute pH values (pHo/pHi =5.5/5.5 in blue; pHo/pHi =7/7 in orange, n= 4). Boltzmann fit parameters were pHo/pHi =7/7 F-V: V0.5= 75.3 ± 2.2 mV; q =0.8 ± 0.04 e0. G-V: V0.5= 39.6 ± 1.3 mV; q = 1.2 ± 0.1 e0. pHo/pHi =5.5/5.5 F-V: V0.5= 84.6 ± 2.1 mV; q =1.0 e0 ± 0.1. G-V: V0.5= 79.7 ± 1.8 mV; q = 1.4 ± 0.1 e0. Data are mean ± s.e.m.