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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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Weak voltage-dependence of accessibility at external end of S1(a) S1 sequence alignment of CiHv1, human Hv1 (hHv1), and mouse Hv1 (mHv1). Color-coded letters, residues tested for MTS modification. (b-f) K173C equally accessible to external MTSET at negative and positive voltages (n = 11 and 10 oocytes for 10 % and 60 % of the time at +60 mV, respectively). (c) Current elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 100 μM MTSET. (d) MTSET-induced steady-state current decrease at arrowhead in (c) (grey dashed line, wt). (e) Tail-current changes, fitted with single exponentials. (f) Rate constants of modification (10%, kMTSET = 870 ± 80 M–1 s–1; 60 %, kMTSET = 1160 ± 180 M–1 s–1; p = 0.17, two-tailed Student's t-test). (g-k) D171C more rapidly modified by external MTSES at positive voltage (n = 6 oocytes). (h) Current elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSES. (i) MTSES-induced steady-state current inhibition at arrowhead in (h). (j) Outward-current changes, fitted with single exponentials. (k) Rate constants of modification (10%, kMTSES = 82 ± 12 M–1 s–1; 60 %, kMTSES = 15.0 ± 0.8 M–1 s–1; p < 0.01, two-tailed Student's t-test). Error bars, s.e.m.
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Figure 1: Weak voltage-dependence of accessibility at external end of S1(a) S1 sequence alignment of CiHv1, human Hv1 (hHv1), and mouse Hv1 (mHv1). Color-coded letters, residues tested for MTS modification. (b-f) K173C equally accessible to external MTSET at negative and positive voltages (n = 11 and 10 oocytes for 10 % and 60 % of the time at +60 mV, respectively). (c) Current elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 100 μM MTSET. (d) MTSET-induced steady-state current decrease at arrowhead in (c) (grey dashed line, wt). (e) Tail-current changes, fitted with single exponentials. (f) Rate constants of modification (10%, kMTSET = 870 ± 80 M–1 s–1; 60 %, kMTSET = 1160 ± 180 M–1 s–1; p = 0.17, two-tailed Student's t-test). (g-k) D171C more rapidly modified by external MTSES at positive voltage (n = 6 oocytes). (h) Current elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSES. (i) MTSES-induced steady-state current inhibition at arrowhead in (h). (j) Outward-current changes, fitted with single exponentials. (k) Rate constants of modification (10%, kMTSES = 82 ± 12 M–1 s–1; 60 %, kMTSES = 15.0 ± 0.8 M–1 s–1; p < 0.01, two-tailed Student's t-test). Error bars, s.e.m.

Mentions: We tested for voltage-dependent changes of solvent accessibility of S1-cysteine mutants by measuring their rate of modification by membrane-impermeable methane-thiosulfonate (MTS) reagents. The substituted-cysteine accessibility method (SCAM), which assumes that the modification rate by MTS compounds is directly proportional to the solvent accessibility of the introduced cysteine, was previously used to demonstrate that S4 translocates through the membrane during the gating of voltage-dependent ion channels, including Hv119,27-29. In total, we made 29 S1-cysteine mutants of Ciona intestinalis Hv1 (CiHv1) (Fig. 1a). We tested external accessibility to MTSET of residues C-terminal of D1 in two-electrode voltage clamp (TEVC) and internal accessibility of residues N-terminal of D1 in excised inside-out patch-clamp recordings.


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

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

Weak voltage-dependence of accessibility at external end of S1(a) S1 sequence alignment of CiHv1, human Hv1 (hHv1), and mouse Hv1 (mHv1). Color-coded letters, residues tested for MTS modification. (b-f) K173C equally accessible to external MTSET at negative and positive voltages (n = 11 and 10 oocytes for 10 % and 60 % of the time at +60 mV, respectively). (c) Current elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 100 μM MTSET. (d) MTSET-induced steady-state current decrease at arrowhead in (c) (grey dashed line, wt). (e) Tail-current changes, fitted with single exponentials. (f) Rate constants of modification (10%, kMTSET = 870 ± 80 M–1 s–1; 60 %, kMTSET = 1160 ± 180 M–1 s–1; p = 0.17, two-tailed Student's t-test). (g-k) D171C more rapidly modified by external MTSES at positive voltage (n = 6 oocytes). (h) Current elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSES. (i) MTSES-induced steady-state current inhibition at arrowhead in (h). (j) Outward-current changes, fitted with single exponentials. (k) Rate constants of modification (10%, kMTSES = 82 ± 12 M–1 s–1; 60 %, kMTSES = 15.0 ± 0.8 M–1 s–1; p < 0.01, two-tailed Student's t-test). Error bars, s.e.m.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Weak voltage-dependence of accessibility at external end of S1(a) S1 sequence alignment of CiHv1, human Hv1 (hHv1), and mouse Hv1 (mHv1). Color-coded letters, residues tested for MTS modification. (b-f) K173C equally accessible to external MTSET at negative and positive voltages (n = 11 and 10 oocytes for 10 % and 60 % of the time at +60 mV, respectively). (c) Current elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 100 μM MTSET. (d) MTSET-induced steady-state current decrease at arrowhead in (c) (grey dashed line, wt). (e) Tail-current changes, fitted with single exponentials. (f) Rate constants of modification (10%, kMTSET = 870 ± 80 M–1 s–1; 60 %, kMTSET = 1160 ± 180 M–1 s–1; p = 0.17, two-tailed Student's t-test). (g-k) D171C more rapidly modified by external MTSES at positive voltage (n = 6 oocytes). (h) Current elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSES. (i) MTSES-induced steady-state current inhibition at arrowhead in (h). (j) Outward-current changes, fitted with single exponentials. (k) Rate constants of modification (10%, kMTSES = 82 ± 12 M–1 s–1; 60 %, kMTSES = 15.0 ± 0.8 M–1 s–1; p < 0.01, two-tailed Student's t-test). Error bars, s.e.m.
Mentions: We tested for voltage-dependent changes of solvent accessibility of S1-cysteine mutants by measuring their rate of modification by membrane-impermeable methane-thiosulfonate (MTS) reagents. The substituted-cysteine accessibility method (SCAM), which assumes that the modification rate by MTS compounds is directly proportional to the solvent accessibility of the introduced cysteine, was previously used to demonstrate that S4 translocates through the membrane during the gating of voltage-dependent ion channels, including Hv119,27-29. In total, we made 29 S1-cysteine mutants of Ciona intestinalis Hv1 (CiHv1) (Fig. 1a). We tested external accessibility to MTSET of residues C-terminal of D1 in two-electrode voltage clamp (TEVC) and internal accessibility of residues N-terminal of D1 in excised inside-out patch-clamp recordings.

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