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A specialized molecular motion opens the Hv1 voltage-gated proton channel

Nature Structural & Molecular Biology volume 22, pages 283–290 (2015)Cite this article

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Abstract

The Hv1 proton channel is unique among voltage-gated channels for containing the pore and gate within its voltage-sensing domain. Pore opening has been proposed to include assembly of the selectivity filter between an arginine (R3) of segment S4 and an aspartate (D1) of segment S1. We determined whether gating involves motion of S1, using Ciona intestinalis Hv1. We found that channel opening is concomitant with solution access to the pore-lining face of S1, from the cytoplasm to deep inside the pore. Voltage- and patch-clamp fluorometry showed that this involves a motion of S1 relative to its surroundings. S1 motion and the S4 motion that precedes it are each influenced by residues on the other helix, thus suggesting a dynamic interaction between S1 and S4. Our findings suggest that the S1 of Hv1 has specialized to function as part of the channel's gate.

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Figure 1: Weak voltage dependence of accessibility at the external end of S1.
Figure 2: Strong voltage dependence of accessibility at the internal end of S1.
Figure 3: Environmental changes around S1 are concomitant with gating.
Figure 4: PCF fluorescence changes at S1 track gating.
Figure 5: S1 undergoes voltage-dependent motion.
Figure 6: PCF fluorescence changes at S4 track the voltage-sensing step.
Figure 7: Neutralization of D1 and R3 has differential effects on S1 and S4 ΔFs.

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Acknowledgements

We are grateful to Y. Okamura (Osaka University) for the cDNA of CiHv1 and to Y. Okamura and A. Nakagawa (Osaka University) for very generously sharing their crystal-structure data. We would like to thank H. Otsuki-Okada (University of California, Berkeley) for help with the cloning and the members of the Isacoff laboratory for discussion. This work was supported by a postdoctoral fellowship for advanced researchers from the Swiss National Science Foundation (SNSF; PA00P3_134163 to T.K.B.) and by a grant from the US National Institutes of Health (R01 NS35549 to E.Y.I.).

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Author notes

  1. Laetitia Mony and Thomas K Berger: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.,

    Laetitia Mony, Thomas K Berger & Ehud Y Isacoff

  2. Department of Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany

    Thomas K Berger

  3. Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, California, USA.,

    Ehud Y Isacoff

  4. Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Ehud Y Isacoff

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  1. Laetitia Mony
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Contributions

L.M. and T.K.B. performed the experiments and analyzed data. L.M., T.K.B. and E.Y.I. designed the experiments and wrote the manuscript.

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Correspondence to Ehud Y Isacoff.

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Integrated supplementary information

Supplementary Figure 1 State-dependent modification of S1 residues.

(a) WT channels undergo current rundown after successive depolarizations to +60 mV in TEVC. (i) Cartoon of Hv1. (ii) WT TEVC currents elicited by a +60 mV voltage step at the beginning (grey) and at the end (black) of protocols used to measure external MTS effects with 10% and 60% of the time at +60 mV. (iii) Average current rundown of WT channels spending either 10% or 60% of the time at +60 mV. Rundown is expressed as the ratio of current (arrowhead in (ii)) at the end of the protocol on current at the beginning of the protocol (n = 4 oocytes for each protocol).

(b-c) Extracellular MTSES (b) or MTSET (c) have no effect on WT channels. (i) Cartoon of Hv1 with bath application of MTSES or MTSET (red). (ii) WT TEVC currents elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSES or MTSET. (iii) Steady-state MTS-induced inhibition measured at the arrowhead in (ii) for WT channels spending either 10% or 60% of the time at +60 mV (MTSES, n = 4 oocytes for each protocol; MTSET, n = 5 oocytes for each protocol). Note that the amount of current decrease after MTS treatment and washout corresponds to the amount of natural current rundown during the same protocol (Supplementary Fig. 1a).

(d) D171C is more rapidly modified by external MTSET at positive voltage. (i) Cartoon shows D171C modified by extracellular MTSET. (ii) D171C currents in response to a voltage step to +60 mV before (grey trace) and after (black trace) application of 1 mM MTSET and washout. (iii) Steady-state change in current amplitude measured at arrowhead in (ii) after MTSET application and washout with 10 or 60% of the time at +60 mV. (iv) Rate constants of D171C modification with 10% (kMTSET = 37 ± 6 M−1 s−1, n = 9 oocytes) or 60% (kMTSET = 83 ± 13 M−1 s−1, n = 9 oocytes) of the time at +60 mV. p < 0.01, two-tailed Student’s t-test.

(e) Intracellular MTSET has no effect on WT channels (n = 4 patches). (i) Cartoon of Hv1 with bath application of MTSET. (ii) WT currents from inside-out patches elicited by a +80 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSET. (iii) Steady-state inhibition of wt channels after application and washout of 1 mM MTSET, measured at arrowhead in (ii).

(f) Intracellular MTSET slows down the rate of deactivation of I150C. (i) Cartoon shows I150C modified by intracellular MTSET. (ii) I150C currents elicited by a +80 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSET.

(g) Intracellular MTSET accelerates opening of I154C at positive voltage (n = 4 patches). (i) Cartoon shows I154C modified by intracellular MTSET. (ii) Currents before and after application of 1 mM MTSET, either at −80 mV, or during repeated steps to +80 mV (chronological color code from violet to red), followed by washout at −80 mV. Inset, magnification of the current traces at stimulus onset. (iii) Steady-state increase in current amplitude at arrowhead in (ii) after application and washout of MTSET at −80 or +80 mV (p < 0.05, two-tailed Student’s t-test). Dashed line represents absence of effect. (iv) Time course of MTSET effect at arrowhead in (ii), at −80 (linear fit) or +80 mV (single exponential fit). (v) Rate constants of I154C modification at −80 mV (kMTSET = 0.79 ± 0.16 M−1 s−1) or +80 mV (kMTSET = 56 ± 18 M−1 s−1) measured from the fits in (iv).

(h) V157C is modified by internal MTSET at positive voltage (n = 3 patches). (i) Cartoon shows V157C modified by intracellular MTSET. (ii) Currents before and after application of 0.5 mM MTSET, either at −80 mV, or during repeated steps to +80 mV (chronological color code from violet to red), followed by washout at −80 mV. Inset, magnification of the current traces at stimulus onset. (iii) Steady-state increase in current amplitude at arrowhead in (ii) after application and washout of MTSET at −80 or +80 mV. Dashed line represents absence of effect. (iv) Time course of MTSET effect at arrowhead in (ii), at −80 or +80 mV. (v) Rate constants of V157C modification at −80 mV (kMTSET = 0.74 ± 0.06 M−1 s−1) or +80 mV (kMTSET = 150 ± 72 M−1 s−1).

Error bars, s.e.m.

Supplementary Figure 2 Mapping of the positions showing modification by MTS compounds.

Mapping of the positions showing modification by MTS compounds onto the mHv1cc crystal structure1, viewed from the interior of the VSD. S2 and S3 segments were removed for clarity reasons. Positions of the α-carbons of S1 residues showing state-dependent and state-independent MTS modification are depicted as green and orange spheres, respectively. Except for V151 and K173, residues showing MTS modification are located on the same helix face as D1 (D160, pink sphere), and are thus likely to face the channel pore. S4 arginine side chains are shown as sticks.

1. Takeshita, K. et al. X-ray crystal structure of voltage-gated proton channel. Nat Struct Mol Biol (2014).

Supplementary Figure 3 Wild-type channels treated with TAMRA-MTS do not show any fluorescence signal.

(a) VCF current (black) and fluorescence (red) traces for WT channels treated with 50 µM TAMRA-MTS in response to a voltage step to +160 mV. (b) PCF current (black) and fluorescence (red) traces for WT channels treated with 50 µM TAMRA-MTS in response to a voltage step to +40 mV.

Supplementary Figure 4 Fluorescence changes of 175C* channels track Hv1 gating transition.

(a-c) Fluorescence changes of 175C* are not due to proton flux. (a) Cartoon depicting N264C I175C* channels modified by intracellular MTSET. (b) Current (I) and fluorescence (F) traces of N264C 175C* in response to a voltage step from −95 to +80 mV, before application (grey) and at the end (black) of the application of 1 mM MTSET. (c) Ratio of steady-state fluorescence and current amplitudes before and after modification by MTSET measured at arrowheads in (b) (p < 0.001, two-tailed Student’s t-test; n = 6 patches). (d-f) S1 fluorescence signal tracks opening and closing kinetics in gating mutant. (d) Normalized VCF current and fluorescence traces of 175C* (grey) and D1E 175C* (black) for a depolarizing step from −80 mV to +100 mV. (e,f) Left, superposition of normalized current (black) and fluorescence (red) traces of D1E 175C* upon depolarization to +100 mV (activation) (e) and back to −80 mV (deactivation) (f). Right, average fast and slow time constants (activation) (e) and decay times (deactivation) (f) of current and fluorescence changes for 175C* (n = 16 and 13 oocytes, respectively) and D1E 175C* (n = 7 oocytes).

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 (Ffast) occurring during the capacitive transient, and a slow fluorescence component (Fslow) developing after the capacitive transient has relaxed. (d) G-V (black), F-V (red) and Fslow-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 Fslow-V curve was measured during repolarization to −80 mV, at the same time point as the G-V curve, after Ffast has decayed (pink arrowhead in (b)). All curves were fitted with single Boltzmann equations with V1/2 = 33 ± 5 mV, kT/ze0 = 19 ± 1 mV (G-V); V1/2 = 14 ± 12 mV, kT/ze0 = 26 ± 4 mV (F-V); V1/2 = 34 ± 8 mV, kT/ze0 = 19 ± 2 mV (Fslow-V) (n = 8 oocytes). Note the close match between the G-V and Fslow-V curves. (e-f) The rate of Fslow onset matches the rate of channel opening during depolarization. (e) Protocol used to measure the rate of Fslow onset. Because the fluorescence signal during depolarization is largely dominated by the fast fluorescence component Ffast, direct comparison between Fslow and current kinetics during a voltage step was not possible. The rate of Fslow 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, Ffast has already decayed. Rate of channel opening (G) was measured from the tail current amplitudes at the same time points as Fslow (black arrowhead). (f) Left, Fslow (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 Fslow and G onset curves. Note the close match between G and Fslow rise times. p = 0.07, two-tailed paired t-test (n = 7 oocytes). (g-h) Fslow 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 Fslow (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.

Supplementary Figure 6 Separation of voltage dependences of the F-V and the G-V curves at S4 labeling sites.

(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).

Supplementary Figure 7 S1 and S4 interact during gating.

(a-b) Voltage-dependence of channel conductance and ΔFs of 175C* and 249C* channels containing different combinations of D1 and R3 neutralizations. (a) G-V (closed circles) and F-V (open circles) of 175C* mutants. Note the good match between the G-V and F-V curves of each mutant. No current was observed for D1N-175C* channels, so only the F-V curve is provided for this construct. Curves were fitted with single Boltzmann. 175C* (n = 28 oocytes): V1/2 = 53 ± 3 mV, kT/ze0 = 19.6 ± 0.7 mV (G-V), V1/2 = 44 ± 3 mV, kT/ze0 = 23.0 ± 0.6 mV (F-V); R3S 175C* (n = 11 oocytes): V1/2 = 68 ± 2 mV, kT/ze0 = 27 ± 1 mV (G-V), V1/2 = 75 ± 6 mV, kT/ze0 = 26 ± 2 mV (F-V); D1N 175C* (n = 10 oocytes): V1/2 = 52 ± 3 mV, kT/ze0 = 15 ± 1 mV (F-V); and D1N R3S 175C* (n = 5 oocytes): V1/2 = 66 ± 11 mV, kT/ze0 = 25 ± 3 mV (G-V), V1/2 = 72 ± 9 mV, kT/ze0 = 21 ± 2 mV (F-V). (b) G-V (closed circles) and F-V (open circles) of 249C* mutants. No current was observed for D1N G249C* channels, so only the F-V curve is provided for this construct. Curves were fitted with single Boltzmann. 249C* (n = 10 oocytes): V1/2 = 55 ± 4 mV, kT/ze0 = 24 ± 1 mV (G-V), V1/2 = 3 ± 3 mV, kT/ze0 = 26 ± 2 mV (F-V); D1N 249C* (n = 1 oocyte): V1/2 = 21 mV, kT/ze0 = 15 mV (F-V); and D1N R3S 249C* (n = 6 oocytes): V1/2 = 97 ± 5 mV, kT/ze0 = 40 ± 3 mV (G-V), V1/2 = −18 ± 8 mV, kT/ze0 = 45 ± 2 mV (F-V). (c,d) Superposition of current (black) to inverted fluorescence (purple) or fluorescence (red) following repolarization from +100 to −80 mV for 175C* and D1N R3S 175C* channels (c), and 249C* and D1N R3S 249C* channels (d). (e,f) S4’s third arginine (R261, referred to as R3) influences S1 movement. (e) VCF recordings of 175C* (black trace) and 175C* with the R3S (red trace) or D1N (blue trace) mutation, with superimposed normalized fluorescence tails of 175C*, R3S 175C* and D1N 175C* following repolarization from +100 to −80 mV on the right. (f) Time constants of fluorescence recovery (τoff) of 175C* (n = 14 oocytes), R3S 175C* (n = 10 oocytes) and D1N 175C* (n = 10 oocytes) following repolarization from +100 to −80 mV. **, p < 0.01, two-tailed Student’s t-test. (g,h) S1’s aspartate D1 influences S4 movement. (g) VCF recordings of 249C* with (blue trace) or without (black trace) the D1N mutation, with superimposed normalized fluorescence tails of G249C* and D1N G249C* following repolarization from +100 to −80 mV on the right. (h) Time constants of the fast component of fluorescence recovery (fast τoff) of G249C* (n = 9 oocytes) and D1N G249C* (n = 7 oocytes) following repolarization from +100 to −80 mV. p < 0.01, two-tailed Student’s t-test. Error bars, s.e.m.

Supplementary Figure 8 Putative model of Hv1 gating.

(a) Putative sequence of molecular motions leading to channel opening. Arrangement of the S1 and S4 segments in the resting state was taken from the mHv1cc crystal structure1 and modified to obtain putative activated and open states. The S2 and S3 segments have been removed for clarity. The beige shape represents the solvent-inaccessible volume of the S1-S4 region of the mHv1cc chimera generated with a probe radius of 1.4 Å (~ radius of a water molecule). Water-accessible cavities inside the channel are represented in blue. α-carbon positions of key residues are shown as spheres: D1 (pink), S4’s arginines (blue) and the four internal S1 residues with state-dependent accessibility, V151, I153, I154, and V157 (green). Voltage-sensing step: outward motion and rotation of S4 to place R3 in register with D1. Opening step: putative rearrangement of the internal side of S1, which opens a wide intracellular cavity (in blue) up to the selectivity filter residues D1 and R3 and exposes S1 internal residues (green spheres) to the solvent. (b-d) mHv1cc chimera internal vestibule is too narrow to allow access of MTSET to S1 internal residues in the closed state. (b-c) Water-accessible surface of residues from the mHv1cc crystal structure1 viewed (b) from the side, S1 and S3 in the foreground; and (c) from the entrance of the intracellular vestibule (arrow in (b)). Surface was created with a probe of 1.4 Å radius and color-coded based on residue hydrophobicity, from brown to blue (hydrophobic to hydrophilic). Solvent accessible surfaces of residues equivalent to CiHv1 internal residues whose MTSET accessibility changes during gating, namely I153, I154, and V157 (I101, I102, and V105, respectively, in mHv1cc), are colored in red, green and yellow, respectively. Residues blocking access of internal MTSET to I153 and I154, S1’s H150 (Q98 in mHv1cc), S2’s I206 (I154 in mHv1cc), and S4’s I262 (I208 in mHv1cc) are shown in purple in Corey-Pauling-Koltun (CPK) representation. (d) Close-up view, from the same orientation as in (c), on residues blocking MTSET access to I153 and I154, depicted in purple CPK (CiHv1 numbering in purple, mHv1cc numbering in black). I153 and I154 depicted as red and green sticks, respectively. The three residues define a passage to I153 and I154 of 2.7 Å radius (yellow sphere), smaller than the radius of MTSET, which is approx. 3 Å.

1. Takeshita, K. et al. X-ray crystal structure of voltage-gated proton channel. Nat Struct Mol Biol (2014).

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Mony, L., Berger, T. & Isacoff, E. A specialized molecular motion opens the Hv1 voltage-gated proton channel. Nat Struct Mol Biol 22, 283–290 (2015). https://doi.org/10.1038/nsmb.2978

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