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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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Neutralization of D1 and R3 has differential effects on S1 and S4 ΔFs(a-b) VCF current and fluorescence traces of D1N-R3S-175C* (a, S1 motion) and D1N-R3S-249C* (b, S4 motion). Insets, superposition of normalized deactivation current and fluorescence of 175C* (a) or 249C* (b) with (black) or without (grey) D1N-R3S mutations. (c) No significant changes in deactivation time constants (τoff) between 175C* (n = 8 oocytes) and D1N-R3S-175C* (n = 5 oocytes). Current, p = 0.34; fluorescence, p = 0.63; two-tailed Student's t-tests. No significant changes in τoff between current and fluorescence (175C*, p = 0.08; D1N-R3S-175C*, p = 0.49; two-tailed paired t-tests). (d) Significant changes in fast deactivation time constants (fast τoff) of fluorescence (p = 0.002) but not current (p = 0.63) between 249C* (n = 9 oocytes) and D1N-R3S-249C* (n = 7 oocytes). Two-tailed Student's t-tests. Significant changes in fast τoff between current and fluorescence in D1N-R3S-249C* (p = 0.02) but not in 249C* (p = 0.12). Two-tailed paired t-tests. (e) Proposed molecular motions that gate Hv1.Arrangement of S1 and S4 segments from the mHv1cc crystal structure31 (putative resting state), with marked key residues D1 (pink), S4's arginines (blue), and two S1 residues, D171 and I153 (green). Arrows, putative S4 and S1 motions during gating. 1, voltage-sensing: outward motion and rotation of S4 to place R3 in register with D1. 2, opening: rearrangement of S1, which opens a wide intracellular cavity, allowing access to the D1-R3 selectivity filter. Error bars, s.e.m.
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Figure 7: Neutralization of D1 and R3 has differential effects on S1 and S4 ΔFs(a-b) VCF current and fluorescence traces of D1N-R3S-175C* (a, S1 motion) and D1N-R3S-249C* (b, S4 motion). Insets, superposition of normalized deactivation current and fluorescence of 175C* (a) or 249C* (b) with (black) or without (grey) D1N-R3S mutations. (c) No significant changes in deactivation time constants (τoff) between 175C* (n = 8 oocytes) and D1N-R3S-175C* (n = 5 oocytes). Current, p = 0.34; fluorescence, p = 0.63; two-tailed Student's t-tests. No significant changes in τoff between current and fluorescence (175C*, p = 0.08; D1N-R3S-175C*, p = 0.49; two-tailed paired t-tests). (d) Significant changes in fast deactivation time constants (fast τoff) of fluorescence (p = 0.002) but not current (p = 0.63) between 249C* (n = 9 oocytes) and D1N-R3S-249C* (n = 7 oocytes). Two-tailed Student's t-tests. Significant changes in fast τoff between current and fluorescence in D1N-R3S-249C* (p = 0.02) but not in 249C* (p = 0.12). Two-tailed paired t-tests. (e) Proposed molecular motions that gate Hv1.Arrangement of S1 and S4 segments from the mHv1cc crystal structure31 (putative resting state), with marked key residues D1 (pink), S4's arginines (blue), and two S1 residues, D171 and I153 (green). Arrows, putative S4 and S1 motions during gating. 1, voltage-sensing: outward motion and rotation of S4 to place R3 in register with D1. 2, opening: rearrangement of S1, which opens a wide intracellular cavity, allowing access to the D1-R3 selectivity filter. Error bars, s.e.m.

Mentions: Mutants of selectivity-filter residues D1 and R3 further distinguished S1 and S4 motions. In VCF, double-mutant neutralization D1N-R3S slowed ΔF deactivation kinetics only at 249C* but not at 175C* (Fig. 7a-d). In addition, D1N-R3S-175C* showed a rightward shift of both F-V and G-V relationships compared to 175C*, preserving a close match between the F-V and G-V in the mutant (a further confirmation that 175C* fluorescence tracks gating, Supplementary Fig. 7a). In contrast, the discrepancy between the voltage-dependences channel opening and S4 motion increased for D1N-R3S-249C* as compared to 249C* (Supplementary Fig. 7b). These results further support the interpretation that the ΔF observed at 175C* does not reflect a motion of S4, but a distinct conformational change of S1 that is associated with gating.


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

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

Neutralization of D1 and R3 has differential effects on S1 and S4 ΔFs(a-b) VCF current and fluorescence traces of D1N-R3S-175C* (a, S1 motion) and D1N-R3S-249C* (b, S4 motion). Insets, superposition of normalized deactivation current and fluorescence of 175C* (a) or 249C* (b) with (black) or without (grey) D1N-R3S mutations. (c) No significant changes in deactivation time constants (τoff) between 175C* (n = 8 oocytes) and D1N-R3S-175C* (n = 5 oocytes). Current, p = 0.34; fluorescence, p = 0.63; two-tailed Student's t-tests. No significant changes in τoff between current and fluorescence (175C*, p = 0.08; D1N-R3S-175C*, p = 0.49; two-tailed paired t-tests). (d) Significant changes in fast deactivation time constants (fast τoff) of fluorescence (p = 0.002) but not current (p = 0.63) between 249C* (n = 9 oocytes) and D1N-R3S-249C* (n = 7 oocytes). Two-tailed Student's t-tests. Significant changes in fast τoff between current and fluorescence in D1N-R3S-249C* (p = 0.02) but not in 249C* (p = 0.12). Two-tailed paired t-tests. (e) Proposed molecular motions that gate Hv1.Arrangement of S1 and S4 segments from the mHv1cc crystal structure31 (putative resting state), with marked key residues D1 (pink), S4's arginines (blue), and two S1 residues, D171 and I153 (green). Arrows, putative S4 and S1 motions during gating. 1, voltage-sensing: outward motion and rotation of S4 to place R3 in register with D1. 2, opening: rearrangement of S1, which opens a wide intracellular cavity, allowing access to the D1-R3 selectivity filter. Error bars, s.e.m.
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Figure 7: Neutralization of D1 and R3 has differential effects on S1 and S4 ΔFs(a-b) VCF current and fluorescence traces of D1N-R3S-175C* (a, S1 motion) and D1N-R3S-249C* (b, S4 motion). Insets, superposition of normalized deactivation current and fluorescence of 175C* (a) or 249C* (b) with (black) or without (grey) D1N-R3S mutations. (c) No significant changes in deactivation time constants (τoff) between 175C* (n = 8 oocytes) and D1N-R3S-175C* (n = 5 oocytes). Current, p = 0.34; fluorescence, p = 0.63; two-tailed Student's t-tests. No significant changes in τoff between current and fluorescence (175C*, p = 0.08; D1N-R3S-175C*, p = 0.49; two-tailed paired t-tests). (d) Significant changes in fast deactivation time constants (fast τoff) of fluorescence (p = 0.002) but not current (p = 0.63) between 249C* (n = 9 oocytes) and D1N-R3S-249C* (n = 7 oocytes). Two-tailed Student's t-tests. Significant changes in fast τoff between current and fluorescence in D1N-R3S-249C* (p = 0.02) but not in 249C* (p = 0.12). Two-tailed paired t-tests. (e) Proposed molecular motions that gate Hv1.Arrangement of S1 and S4 segments from the mHv1cc crystal structure31 (putative resting state), with marked key residues D1 (pink), S4's arginines (blue), and two S1 residues, D171 and I153 (green). Arrows, putative S4 and S1 motions during gating. 1, voltage-sensing: outward motion and rotation of S4 to place R3 in register with D1. 2, opening: rearrangement of S1, which opens a wide intracellular cavity, allowing access to the D1-R3 selectivity filter. Error bars, s.e.m.
Mentions: Mutants of selectivity-filter residues D1 and R3 further distinguished S1 and S4 motions. In VCF, double-mutant neutralization D1N-R3S slowed ΔF deactivation kinetics only at 249C* but not at 175C* (Fig. 7a-d). In addition, D1N-R3S-175C* showed a rightward shift of both F-V and G-V relationships compared to 175C*, preserving a close match between the F-V and G-V in the mutant (a further confirmation that 175C* fluorescence tracks gating, Supplementary Fig. 7a). In contrast, the discrepancy between the voltage-dependences channel opening and S4 motion increased for D1N-R3S-249C* as compared to 249C* (Supplementary Fig. 7b). These results further support the interpretation that the ΔF observed at 175C* does not reflect a motion of S4, but a distinct conformational change of S1 that is associated with gating.

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