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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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Strong voltage-dependence of accessibility at internal end of S1(a-e) V151C modified by internal MTSET only at positive voltage (n = 4 patches). (b) V151C currents before (control), during application, first at –80 mV, then during repeated steps to +80 mV (chronological color code from violet to red), and after washout (at –80 mV) of 200 μM MTSET. Inset, magnification of the current traces at stimulus onset. (c) MTSET-induced steady-state current increase at arrowhead in (b), at –80 or +80 mV (p < 0.001, two-tailed Student's t-test). Dashed line, absence of effect. (d) Outward-current change at –80 or +80 mV, fitted with exponential (+80 mV) or linear (–80mV). (e) Rate constants of modification at –80 mV (kMTSET = 1.6 ± 0.4 M–1 s–1) and +80 mV (kMTSET = 558 ± 60 M–1 s–1). (f-j) I153C modified by internal MTSET only at positive voltage. (g) Currents before (control), during application, first at –80 mV, then at +80 mV, and after washout (at –80 mV) of 1 mM MTSET. (h) MTSET-induced steady-state current inhibition at –80 mV (n = 5 patches) or +80 mV (n = 6 patches), measured at arrowhead in (g) (p < 0.001, two-tailed Student's t-test). Dashed line, absence of effect. (i) Current in response to a +80 mV voltage step in presence of MTSET, with single exponential fit (grey trace). (j) Rate constant of modification at +80 mV (kMTSET = 920 ± 170 M–1 s–1). Error bars, s.e.m.
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Figure 2: Strong voltage-dependence of accessibility at internal end of S1(a-e) V151C modified by internal MTSET only at positive voltage (n = 4 patches). (b) V151C currents before (control), during application, first at –80 mV, then during repeated steps to +80 mV (chronological color code from violet to red), and after washout (at –80 mV) of 200 μM MTSET. Inset, magnification of the current traces at stimulus onset. (c) MTSET-induced steady-state current increase at arrowhead in (b), at –80 or +80 mV (p < 0.001, two-tailed Student's t-test). Dashed line, absence of effect. (d) Outward-current change at –80 or +80 mV, fitted with exponential (+80 mV) or linear (–80mV). (e) Rate constants of modification at –80 mV (kMTSET = 1.6 ± 0.4 M–1 s–1) and +80 mV (kMTSET = 558 ± 60 M–1 s–1). (f-j) I153C modified by internal MTSET only at positive voltage. (g) Currents before (control), during application, first at –80 mV, then at +80 mV, and after washout (at –80 mV) of 1 mM MTSET. (h) MTSET-induced steady-state current inhibition at –80 mV (n = 5 patches) or +80 mV (n = 6 patches), measured at arrowhead in (g) (p < 0.001, two-tailed Student's t-test). Dashed line, absence of effect. (i) Current in response to a +80 mV voltage step in presence of MTSET, with single exponential fit (grey trace). (j) Rate constant of modification at +80 mV (kMTSET = 920 ± 170 M–1 s–1). Error bars, s.e.m.

Mentions: We next turned to residues located at the intracellular side of S1 (146–159). In inside-out patches, MTSET had no effect on wt channels (Supplementary Fig. 1e), but we detected modification of five cysteine mutants (Fig. 1a). One mutant (H150C) was too inconsistent in behavior to assess state-dependence quantitatively (Supplementary Fig. 1f). MTSET accelerated opening of V151C, I154C, and V157C (Fig. 2a-e and Supplementary Fig. 1g,h) and inhibited I153C (by approximately 70 %, Fig. 2f-h). All four positions showed a very sharp state-dependency, with no or very little modification in the resting state at –80 mV and rapid modification in the open state at +80 mV (Fig. 2 and Supplementary Fig. 1).


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

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

Strong voltage-dependence of accessibility at internal end of S1(a-e) V151C modified by internal MTSET only at positive voltage (n = 4 patches). (b) V151C currents before (control), during application, first at –80 mV, then during repeated steps to +80 mV (chronological color code from violet to red), and after washout (at –80 mV) of 200 μM MTSET. Inset, magnification of the current traces at stimulus onset. (c) MTSET-induced steady-state current increase at arrowhead in (b), at –80 or +80 mV (p < 0.001, two-tailed Student's t-test). Dashed line, absence of effect. (d) Outward-current change at –80 or +80 mV, fitted with exponential (+80 mV) or linear (–80mV). (e) Rate constants of modification at –80 mV (kMTSET = 1.6 ± 0.4 M–1 s–1) and +80 mV (kMTSET = 558 ± 60 M–1 s–1). (f-j) I153C modified by internal MTSET only at positive voltage. (g) Currents before (control), during application, first at –80 mV, then at +80 mV, and after washout (at –80 mV) of 1 mM MTSET. (h) MTSET-induced steady-state current inhibition at –80 mV (n = 5 patches) or +80 mV (n = 6 patches), measured at arrowhead in (g) (p < 0.001, two-tailed Student's t-test). Dashed line, absence of effect. (i) Current in response to a +80 mV voltage step in presence of MTSET, with single exponential fit (grey trace). (j) Rate constant of modification at +80 mV (kMTSET = 920 ± 170 M–1 s–1). Error bars, s.e.m.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385474&req=5

Figure 2: Strong voltage-dependence of accessibility at internal end of S1(a-e) V151C modified by internal MTSET only at positive voltage (n = 4 patches). (b) V151C currents before (control), during application, first at –80 mV, then during repeated steps to +80 mV (chronological color code from violet to red), and after washout (at –80 mV) of 200 μM MTSET. Inset, magnification of the current traces at stimulus onset. (c) MTSET-induced steady-state current increase at arrowhead in (b), at –80 or +80 mV (p < 0.001, two-tailed Student's t-test). Dashed line, absence of effect. (d) Outward-current change at –80 or +80 mV, fitted with exponential (+80 mV) or linear (–80mV). (e) Rate constants of modification at –80 mV (kMTSET = 1.6 ± 0.4 M–1 s–1) and +80 mV (kMTSET = 558 ± 60 M–1 s–1). (f-j) I153C modified by internal MTSET only at positive voltage. (g) Currents before (control), during application, first at –80 mV, then at +80 mV, and after washout (at –80 mV) of 1 mM MTSET. (h) MTSET-induced steady-state current inhibition at –80 mV (n = 5 patches) or +80 mV (n = 6 patches), measured at arrowhead in (g) (p < 0.001, two-tailed Student's t-test). Dashed line, absence of effect. (i) Current in response to a +80 mV voltage step in presence of MTSET, with single exponential fit (grey trace). (j) Rate constant of modification at +80 mV (kMTSET = 920 ± 170 M–1 s–1). Error bars, s.e.m.
Mentions: We next turned to residues located at the intracellular side of S1 (146–159). In inside-out patches, MTSET had no effect on wt channels (Supplementary Fig. 1e), but we detected modification of five cysteine mutants (Fig. 1a). One mutant (H150C) was too inconsistent in behavior to assess state-dependence quantitatively (Supplementary Fig. 1f). MTSET accelerated opening of V151C, I154C, and V157C (Fig. 2a-e and Supplementary Fig. 1g,h) and inhibited I153C (by approximately 70 %, Fig. 2f-h). All four positions showed a very sharp state-dependency, with no or very little modification in the resting state at –80 mV and rapid modification in the open state at +80 mV (Fig. 2 and Supplementary Fig. 1).

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