Supplementary Figure 5: The slow component of the fluorescence signal of Alexa-labeled K173C channels tracks Hv1 gating transition.

(a) Cartoon and sequence alignment of CiHv1, hHv1, and mHv1 with Alexa488-maleimide attached to K173C. (b) VCF current and fluorescence traces (as indicated in cartoon inset) of K173C-Alexa for steps from −80 mV to (in mV): grey, −100; blue, −40; green, +20; yellow, +60; red, +100. (c) Magnification of current and fluorescence traces for a step to +20 mV at the onset of the voltage step (activation, inset 1 in (b)) and at the end of the voltage step (deactivation, inset 2 in (b)), highlighting the two fluorescence components of 173C-Alexa fluorescence signal, both during activation and deactivation: a fast fluorescence component (F_fast) occurring during the capacitive transient, and a slow fluorescence component (F_slow) developing after the capacitive transient has relaxed. (d) G-V (black), F-V (red) and F_slow-V (pink) curves of 173C-Alexa. Similarly to 175C*, the G-V curve was measured from the tail currents (black arrowhead in (b)) and the F-V curve from the ΔFs at the end of the depolarization steps (red arrowhead in (b)). The F_slow-V curve was measured during repolarization to −80 mV, at the same time point as the G-V curve, after F_fast has decayed (pink arrowhead in (b)). All curves were fitted with single Boltzmann equations with V_1/2 = 33 ± 5 mV, kT/ze₀ = 19 ± 1 mV (G-V); V_1/2 = 14 ± 12 mV, kT/ze₀ = 26 ± 4 mV (F-V); V_1/2 = 34 ± 8 mV, kT/ze₀ = 19 ± 2 mV (F_slow-V) (n = 8 oocytes). Note the close match between the G-V and F_slow-V curves. (e-f) The rate of F_slow onset matches the rate of channel opening during depolarization. (e) Protocol used to measure the rate of F_slow onset. Because the fluorescence signal during depolarization is largely dominated by the fast fluorescence component F_fast, direct comparison between F_slow and current kinetics during a voltage step was not possible. The rate of F_slow onset was determined by measuring the tail fluorescence amplitude 60 ms (red arrowhead) after the end of voltage steps to +40 mV of different time durations (10 ms to 1.1 s). At this time point, F_fast has already decayed. Rate of channel opening (G) was measured from the tail current amplitudes at the same time points as F_slow (black arrowhead). (f) Left, F_slow (red) and G (black) onset curves of an example cell fitted with double exponential functions (red and black solid curves, respectively) and normalized to their maximal amplitude. Right, rise times (time necessary to rise from 10 to 90 % of the maximal amplitude) of F_slow and G onset curves. Note the close match between G and F_slow rise times. p = 0.07, two-tailed paired t-test (n = 7 oocytes). (g-h) F_slow and current have close kinetics during deactivation. (g) VCF current and fluorescence traces of 173C-Alexa during repolarizations to various potentials (–140 to −40 mV in 20 mV increments), from a voltage step to +80 mV. (h) Superposition of the normalized current (black) and slow component of fluorescence (red) during repolarization from +80 to −80 mV. (i) Decay times of current (black) and F_slow (red) following repolarization from +80 mV to various potentials (n = 8 oocytes). Note the close match between current and fluorescence decay times at all potentials. Error bars, s.e.m.