Supplementary Figure 6: Separation of voltage dependences of the F-V and the G-V curves at S4 labeling sites.
From: A specialized molecular motion opens the Hv1 voltage-gated proton channel
(a) Representative VCF current and fluorescence traces of S242C channels labeled with TAMRA-MTS (S242C*) for a voltage step to +100 mV. Similarly to S242C labeled with Alexa488-maleimide2,3, fluorescence of TAMRA-labeled S242C channels had two components: an early negative ΔF component (F1) and a later positive ΔF component (F2). (b) G-V (closed circles) and F-V (open circles) of S242C (black) and G249S S242C channels (blue) labeled with TAMRA-MTS (S242C* and G249S S242C*). G-V curves were fitted with single Boltzmann functions, while F-V curves were fitted with double Boltzmann functions and normalized to the maximal amplitude of their F1 component. 242C* (n = 6 oocytes): V1/2 = 55 ± 7 mV, kT/ze0 = 22.9 ± 0.3 mV (G-V); V1 = −19 ± 4 mV, (kT/ze0)1 = 19 ± 3 mV, V2 = 40 ± 8 mV, (kT/ze0)2 = 17 ± 6 mV (F-V). The first ΔF component F1, whose voltage-dependence is left-shifted compared to the voltage-dependence of opening, has been proposed to track the voltage-sensing S4 motion, while the second component F2, whose voltage-dependence matches the voltage-dependence of opening, was proposed to represent a motion associated with gating2,3. To verify that the voltage shift between the F-V and the G-V of 249C* channels is not due to a decoupling between voltage-sensing and gating induced by the G249C mutation, we mutated G249 into a serine (similar to Cys, but not modifiable by the fluorophore) in the 242C background and measured the F-V and G-V of G249S S242C* channels. G249S 242C* (n = 8 oocytes): V1/2 = 68 ± 5 mV, kT/ze0 = 25 ± 1 mV (G-V); V1 = −2 ± 4 mV, (kT/ze0)1 = 15 ± 2 mV, V2 = 72 ± 19 mV, (kT/ze0)2 = 56 ± 34 mV (F-V). G249S mutants induced a ~ 13 mV rightward shift of the G-V and a similar ~ 17 mV shift of the first component of the F-V that tracks voltage sensing, thus keeping the voltage shift between the F-V and the G-V close to the shift in WT-S242C* channels. Mutation of G249 is thus not likely to uncouple the voltage sensor and the gate. Error bars, s.e.m.
2. Gonzalez, C., Koch, H.P., Drum, B.M. & Larsson, H.P. Strong cooperativity between subunits in voltage-gated proton channels. Nat Struct Mol Biol 17, 51-6 (2010).
3. Qiu, F., Rebolledo, S., Gonzalez, C. & Larsson, H.P. Subunit Interactions during Cooperative Opening of Voltage-Gated Proton Channels. Neuron 77, 288-98 (2013).
