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

Mony L, Berger TK, Isacoff EY - Nat. Struct. Mol. Biol. (2015)

Bottom Line: We determined whether gating involves motion of S1, using Ciona intestinalis Hv1.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.

View Article: PubMed Central - PubMed

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

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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Environment changes around S1 are concomitant with gating(a) Internal end: comparison of the voltage-dependence of the modification rate of I153C by MTSET (accessibility, green curve) with the voltage-dependence of I153C conductance before MTS modification (G, black curve). A-V curve (n = 3 for –20 and –10 mV data points, n = 2 for other data points), V1/2 = –27 ± 2 mV, kT/ze0 = 14 ± 3 mV; G-V curve (n = 6 patches), V1/2 = –30 ± 4 mV, kT/ze0 = 7.61 ± 0.25 mV. (b-e) VCF ΔFs at outer end of S1 track opening and closing kinetics. (b) Cartoon and sequence alignment of CiHv1, hHv1, and mHv1 with TAMRA-MTS attached to I175C. (c) VCF current and fluorescence traces (as indicated in cartoon inset) of 175C* for steps from –80 mV to (in mV): grey, –100; blue, –40; green, +20; yellow, +60; red, +100. (d) Left, superposition of normalized current (black) and fluorescence (red) for a voltage step to +100 mV (activation). Right, average fast and slow activation time constants (τon) of current and fluorescence (double exponential fit; n = 14 oocytes; p = 0.39 for fast τon and 0.12 for slow τon, two-tailed paired t-tests). (e) Left, superposition of normalized current (black), fluorescence (red), and inverted fluorescence (pink) during repolarization from +100 to – 80 mV (deactivation). Right, average deactivation-time constants (τoff) of current and fluorescence (single exponential fit; n = 8 oocytes; p = 0.08, two-tailed paired t-test). Error bars, s.e.m.
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Figure 3: Environment changes around S1 are concomitant with gating(a) Internal end: comparison of the voltage-dependence of the modification rate of I153C by MTSET (accessibility, green curve) with the voltage-dependence of I153C conductance before MTS modification (G, black curve). A-V curve (n = 3 for –20 and –10 mV data points, n = 2 for other data points), V1/2 = –27 ± 2 mV, kT/ze0 = 14 ± 3 mV; G-V curve (n = 6 patches), V1/2 = –30 ± 4 mV, kT/ze0 = 7.61 ± 0.25 mV. (b-e) VCF ΔFs at outer end of S1 track opening and closing kinetics. (b) Cartoon and sequence alignment of CiHv1, hHv1, and mHv1 with TAMRA-MTS attached to I175C. (c) VCF current and fluorescence traces (as indicated in cartoon inset) of 175C* for steps from –80 mV to (in mV): grey, –100; blue, –40; green, +20; yellow, +60; red, +100. (d) Left, superposition of normalized current (black) and fluorescence (red) for a voltage step to +100 mV (activation). Right, average fast and slow activation time constants (τon) of current and fluorescence (double exponential fit; n = 14 oocytes; p = 0.39 for fast τon and 0.12 for slow τon, two-tailed paired t-tests). (e) Left, superposition of normalized current (black), fluorescence (red), and inverted fluorescence (pink) during repolarization from +100 to – 80 mV (deactivation). Right, average deactivation-time constants (τoff) of current and fluorescence (single exponential fit; n = 8 oocytes; p = 0.08, two-tailed paired t-test). Error bars, s.e.m.

Mentions: To identify the functional transition associated with the voltage-dependent change in S1 accessibility, we took advantage of the sharp state-dependence of the 153C modification by MTSET and measured the modification rate at a range of voltages (see Online Methods). The voltage-dependence of the I153C modification rate by MTSET (accessibility-voltage relationship, A-V) was very close to the conductance-voltage relationship (G-V) of the channel before MTS modification (Fig. 3a), suggesting that the increase of accessibility of the internal end of S1 occurs in the opening transition.


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

Mony L, Berger TK, Isacoff EY - Nat. Struct. Mol. Biol. (2015)

Environment changes around S1 are concomitant with gating(a) Internal end: comparison of the voltage-dependence of the modification rate of I153C by MTSET (accessibility, green curve) with the voltage-dependence of I153C conductance before MTS modification (G, black curve). A-V curve (n = 3 for –20 and –10 mV data points, n = 2 for other data points), V1/2 = –27 ± 2 mV, kT/ze0 = 14 ± 3 mV; G-V curve (n = 6 patches), V1/2 = –30 ± 4 mV, kT/ze0 = 7.61 ± 0.25 mV. (b-e) VCF ΔFs at outer end of S1 track opening and closing kinetics. (b) Cartoon and sequence alignment of CiHv1, hHv1, and mHv1 with TAMRA-MTS attached to I175C. (c) VCF current and fluorescence traces (as indicated in cartoon inset) of 175C* for steps from –80 mV to (in mV): grey, –100; blue, –40; green, +20; yellow, +60; red, +100. (d) Left, superposition of normalized current (black) and fluorescence (red) for a voltage step to +100 mV (activation). Right, average fast and slow activation time constants (τon) of current and fluorescence (double exponential fit; n = 14 oocytes; p = 0.39 for fast τon and 0.12 for slow τon, two-tailed paired t-tests). (e) Left, superposition of normalized current (black), fluorescence (red), and inverted fluorescence (pink) during repolarization from +100 to – 80 mV (deactivation). Right, average deactivation-time constants (τoff) of current and fluorescence (single exponential fit; n = 8 oocytes; p = 0.08, two-tailed paired t-test). Error bars, s.e.m.
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Related In: Results  -  Collection

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Figure 3: Environment changes around S1 are concomitant with gating(a) Internal end: comparison of the voltage-dependence of the modification rate of I153C by MTSET (accessibility, green curve) with the voltage-dependence of I153C conductance before MTS modification (G, black curve). A-V curve (n = 3 for –20 and –10 mV data points, n = 2 for other data points), V1/2 = –27 ± 2 mV, kT/ze0 = 14 ± 3 mV; G-V curve (n = 6 patches), V1/2 = –30 ± 4 mV, kT/ze0 = 7.61 ± 0.25 mV. (b-e) VCF ΔFs at outer end of S1 track opening and closing kinetics. (b) Cartoon and sequence alignment of CiHv1, hHv1, and mHv1 with TAMRA-MTS attached to I175C. (c) VCF current and fluorescence traces (as indicated in cartoon inset) of 175C* for steps from –80 mV to (in mV): grey, –100; blue, –40; green, +20; yellow, +60; red, +100. (d) Left, superposition of normalized current (black) and fluorescence (red) for a voltage step to +100 mV (activation). Right, average fast and slow activation time constants (τon) of current and fluorescence (double exponential fit; n = 14 oocytes; p = 0.39 for fast τon and 0.12 for slow τon, two-tailed paired t-tests). (e) Left, superposition of normalized current (black), fluorescence (red), and inverted fluorescence (pink) during repolarization from +100 to – 80 mV (deactivation). Right, average deactivation-time constants (τoff) of current and fluorescence (single exponential fit; n = 8 oocytes; p = 0.08, two-tailed paired t-test). Error bars, s.e.m.
Mentions: To identify the functional transition associated with the voltage-dependent change in S1 accessibility, we took advantage of the sharp state-dependence of the 153C modification by MTSET and measured the modification rate at a range of voltages (see Online Methods). The voltage-dependence of the I153C modification rate by MTSET (accessibility-voltage relationship, A-V) was very close to the conductance-voltage relationship (G-V) of the channel before MTS modification (Fig. 3a), suggesting that the increase of accessibility of the internal end of S1 occurs in the opening transition.

Bottom Line: We determined whether gating involves motion of S1, using Ciona intestinalis Hv1.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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus