Trp207 regulation of voltage-dependent activation of human Hv1 proton channel

In voltage-gated Na+ and K+ channels, the hydrophobicity of noncharged residues in the S4 helix has been shown to regulate the S4 movement underlying the process of voltage-sensing domain (VSD) activation. In voltage-gated proton channel Hv1, there is a bulky noncharged tryptophan residue located at the S4 transmembrane segment. This tryptophan remains entirely conserved across all Hv1 members but is not seen in other voltage-gated ion channels, indicating that the tryptophan contributes different roles in VSD activation. The conserved tryptophan of human voltage-gated proton channel Hv1 is Trp207 (W207). Here, we showed that W207 modifies human Hv1 voltage-dependent activation, and small residues replacement at position 207 strongly perturbs Hv1 channel opening and closing, and the size of the side chain instead of the hydrophobic group of W207 regulates the transition between closed and open states of the channel. We conclude that the large side chain of tryptophan controls the energy barrier during the Hv1 VSD transition.

In voltage-gated Na + and K + channels, the hydrophobicity of noncharged residues in the S4 helix has been shown to regulate the S4 movement underlying the process of voltage-sensing domain (VSD) activation.In voltage-gated proton channel H v 1, there is a bulky noncharged tryptophan residue located at the S4 transmembrane segment.This tryptophan remains entirely conserved across all H v 1 members but is not seen in other voltage-gated ion channels, indicating that the tryptophan contributes different roles in VSD activation.The conserved tryptophan of human voltage-gated proton channel H v 1 is Trp207 (W207).Here, we showed that W207 modifies human H v 1 voltage-dependent activation, and small residues replacement at position 207 strongly perturbs H v 1 channel opening and closing, and the size of the side chain instead of the hydrophobic group of W207 regulates the transition between closed and open states of the channel.We conclude that the large side chain of tryptophan controls the energy barrier during the H v 1 VSD transition.
The voltage-gated ion channels, including Na v , K v , Ca v , and H v 1 proton channels, all contain voltage-sensing domains (VSDs) that are responsible for detecting changes in membrane potential in the cells (1)(2)(3).The VSDs of voltage-gated ion channels are made of four transmembrane segments (S1 through S4).The S4 helix contains several positively charged residues located at every third position.To respond to the changes in membrane potential, the S4 helix undergoes transmembrane movement that is mediated by these charged residues (4)(5)(6).Moreover, other noncharged residues in the S4 helix have been shown to modify the S4 movement, and the hydrophobicity of noncharged residues regulates the process of voltage-dependent activation of the channels (7)(8)(9).In the Shaker K v channel, replacements of hydrophilic residues at position 361 (L361K and L361R) in the S4 helix profoundly shifted the G-V curve toward negative voltages, and hydrophilic replacements at this position produced faster activation kinetics (8).In the Na v 1.4 channel, substitution of hydrophobic residues in the S4 helix of domains I, II, and III has been shown to regulate the voltage sensor movement and alter steady-state activation and inactivation curves (7).
In the voltage-gated proton channel family, there is a bulky noncharged residue tryptophan located at the S4 helix, which remains highly conserved across all H v 1 channels (Fig. 1A) (10,11).Despite progress in the functional characterization of noncharged residues in the S4 helix of Na v and K v channels (7)(8)(9), the role of S4 noncharged residues in H v 1 channel voltage-dependent activation remains unclear.In addition, the bulky tryptophan is highly conserved in the H v 1 family (Fig. 1A) but is not seen in other voltage-gated ion channels (Fig. S1), indicating that it might contribute different roles in VSD activation.
The highly conserved tryptophan of the human H v 1 channel (hH v 1) is W207.In Na v and K v channels, the hydrophobic group of noncharged residues in S4 helix significantly influences the VSD activation (7)(8)(9).To assess whether the hydrophobic group of W207 regulates hH v 1 VSD activation as well, we introduced mutations in the W207 by substituting the other 19 amino acids.In contrast to findings in other channels, we showed that the size of the side chain instead of the hydrophobic group at position W207 is essential for the H v 1 proton channel voltage-dependent activation.We further showed that small residues replacement at position 207 exhibited faster activation and deactivation kinetics, suggesting that the presence of the natively bulky tryptophan slows the gating process and controls the transition between closed and open states of the channel.We proposed a simple-state model of H v 1 VSD transition and discussed the conserved Trp regulation of H v 1 VSD activation.

Effects of W207 mutations on the voltage-dependent activation of human H v 1 channel
The H v 1 channel has been shown to form dimers in which two VSD subunits are held together by a C-terminal coiled-coil domain (12)(13)(14), and deletion of the coiled-coil domain results in a monomeric form of the channel (15)(16)(17).H v 1 VSD subunits were reported to be allosterically coupled and work cooperatively (18,19).To eliminate the influence of cooperation between subunits, we generated monomeric hH v 1 by deletion of coiled-coil domain.
We introduced mutations at position 207 in monomeric H v 1 to investigate effects of the conserved tryptophan on the H v 1 voltage-dependent activation.The representative currents recorded in HEK293 cells expressing monomer human H v 1 (hH v 1) 207W (wildtype) and 207 mutation channels are shown in Figure 2. In the recording, proton currents were measured from a holding potential of −60 mV to a first prestep and a second test-step with rest intervals at −60 mV.The proton channel conductance is determined by the equation: G(V test ) = (I test -I tail )/(V test -V tail ), where I test is the current at V test , measured at the end of the depolarization step, and I tail is the current at V tail , measured at the beginning of the repolarization step.Currents after depolarization in the prestep were used to correct for current rundown.
It was shown that all W207 mutations produced left-shifted G-V relationships (Fig. 1B, Table S1), suggesting that mutations at position 207 perturb the channel voltage-dependent gating and that the conserved tryptophan is essential for H v 1 VSD activation.To investigate the relationships between the H v 1 voltage dependence of activation and amino acid mutations' physicochemical properties, we conducted a linear correlation analysis.We determined the midpoint voltage (V 1/2 ) from the fitted G-V curve to measure the voltage dependence of activation parameter.The physicochemical properties we considered for amino acids were side chain size and hydrophobicity (20)(21)(22).
Our analysis revealed that the hydrophobicity of the side chain had no significant correlation with V 1/2 (Fig. 3).However, we observed a robust linear relationship between the voltage dependence of H v 1 VSD and the side chain surface area or volume (Fig. 1, C and D).These results indicated that the size of the side chain at position 207 was closely associated with V 1/2 , with a bulky residue at position W207 being critical for channel function.
W207 mutations have been shown to perturb ΔpH-dependent gating at lower pH o (23).Our results showed that, in monomer WT H v 1 channel, the G-V shifts between pH o = 7/ pH i = 6 and pH o = 6/pH i = 6 is 38 ± 5 mV, whereas the shift is 21 ± 6 mV in W207A and 19 ± 5 mV in W207G (Fig. S3).The results are consistent with previous findings that W207 mutants compromise the ΔpH-dependent gating of the H v 1 channels (23).

Effects of small side chain substitutions of W207 on the open and closed states of H v 1
To investigate how the large side chain of W207 affects the H v 1 voltage-dependent activation, we substituted the tryptophan with a small residue alanine at position 207 (207A).Mutation 207A exhibited significantly faster activation kinetics compared with wildtype channel (207W), suggesting that 207A mutation favors the open state of the channel (Fig. 4A).We then introduced two more small residues at position 207 (207G and 207S), and both were shown to facilitate channel opening with faster activation kinetics (Fig. 4B).These results indicated that small residue replacements at position 207 are likely to reduce an energy barrier for the channel activation and make the channel easier to open, accounting for faster activations in the mutated channels (207A, 207G, and 207S).
The effects of a small side chain at position 207 on the closed state of the channel were further investigated.The 207A mutation showed faster deactivation kinetics (Fig. 5A).The channel closing time constant Ʈ deact values were determined, and 207A took less time to close compared with WT (Fig. 5B).Similar results have been observed in the other two small residues replacement at position 207 (207G, 207S), and both  207G and 207S altered the closing time constant Ʈ deact decreasing the values at given membrane potentials (Fig. 5B).This indicates that small residue replacements at position 207 also reduce an energy barrier for channel deactivation and facilitate channel closing.
We further evaluated dimer hH v 1 channels.It was shown that the bulky side chain at position 207 regulates dimer hH v 1 voltage-dependent activation as well, and smaller side chain substitutions of Trp consistently produce left-shifted G-V relationships (Fig. 6).Moreover, dimer channel W207 mutations exhibited much faster activation and deactivation kinetics compared with dimer wildtype channel (Fig. 7), suggesting that small side chain substitutions of Trp favor the open and closed state of the dimer channel.In other words, small residue replacement reduces an energy barrier during the H v 1 channel voltage-dependent activation.
Taken together, these results suggest that the endogenous residue tryptophan at position 207 increases the energy barrier that makes the channel neither easily open nor easily closed.

State model of W207 mutation regulation of H v 1 VSD transitions
To describe the effects of small residue replacements at position 207 on the H v 1 VSD voltage-dependent activation, we proposed a simple-state model of H v 1 VSD transitions.In the model, the small residue replacements (W207X) decreased the energy barrier underlying the transition between closed and open states of the channel (Fig. 8).The effects of W207X mutations on the closed state were satisfied by decreasing the relative depth of the energy well for the "Close" state and decreasing the energy barrier for the Close -> Open transition to allow for the faster deactivation (Figs.5A and 8).W207X mutations' influences on the open state could also be satisfied by decreasing the relative depth of the energy well for the "Open" state, which is achieved by the reduced energy barrier for the Open -> Close transition to interpret the faster activation kinetics (Figs.4A and 8).A smaller ΔG 0 (which is a free energy difference between the closed and open states of the activation, Table S1) for W207X was consistent with decreased activation E act (W207X) (Fig. 4B) and reduced deactivation kinetics E deact (W207X) (Fig. 5B).

Discussion
In the H v 1 family, there is a bulky residue tryptophan located at the S4 segment, which remains entirely conserved across all H v 1 members.This highly conserved Trp has been shown to play important roles in regulating dimer coupling  Ʈ act was obtained from exponential fit to rising currents.Ʈ act between W207 mutation and WT were compared statistically using two-tailed test (***p < 0.001).C, representative tail currents recorded from dimer WT and 207S channels.The tail currents were elicited by a prepulse to 120 mV, in 10 mV decrements from 0 to −60 mV.D, the deactivation (channel closing) time constant Ʈ deact in dimer H v 1 channels.Ʈ deact was obtained from exponential fit to the tail currents, n = 4 to 6 for each group.Ʈ deact between W207 mutation and WT were compared statistically using two-tailed test (***p < 0.001).
and pH gradient-dependent gating (18,23,24).Here, we showed that the Trp strongly modifies the process of H v 1 voltage-dependent activation.The large side chain of the Trp controls the transition between closed and open states of the H v 1 channel and regulates the energy barrier during the H v 1 VSD transition.
To determine the effects of Trp on H v 1 VSD voltagedependent activation, we introduced other 19 residues at position 207 in monomer hH v 1 channel.We determined the G-V curves of W207 mutations and analyzed the effects of physicochemical properties of W207 mutations on the voltagedependent activation.In contrast to the findings that the hydrophobicity of noncharged residues in the S4 helix of Na v and K v channels play roles in regulation of VSD activation (7, 8), there were no significant correlations between the hydrophobicity of side chain and the V 1/2 in H v 1.However, replacement of smaller residues at position 207 consistently shifts the G-V curve toward more negative voltages, indicating that large size of side chain at position 207 is crucial for the H v 1 channel voltage-dependent activation.Additionally, we notice that some amino acids containing rigid rings (e.g., 207P, 207W, 207F, and 207Y) generate relatively higher V 1/2 (Table S1).Although the leucine (L), which does not have rigid rings, also generates a greater V 1/2 , we cannot exclude the possible contributions of rigid rings to the channel activation, which might increase the energy cost.
We replaced the tryptophan (W) with the three smallest residues (A, S, G) to determine how the largest residue (W) at position 207 affects hH v 1 VSD activation, and all small residues exhibited faster activation and deactivation kinetics, indicating that small residues replacement facilitate channel opening and closing.These results suggest that the endogenous residue tryptophan increases the energy barrier that makes the channel neither easily open nor easily closed.We observed that the whole cell currents carried by W207 mutations are less than wildtype monomer channel (Fig. 4).We considered an explanation that W207-mutated channel might produce either relatively smaller single channel conductance and/or lower expression of the cell membrane that overcomes the advantage of open probability to influence the whole cell current amplitude.
H v 1 is mainly expressed in nonexcitable cells such as epithelial cells, sperm cells, and white blood cells, in which H v 1 is implicated in pH regulation in airway epithelium and sperm cells and reactive oxygen species production in phagocytes (2,3).The pH is determined by the H + concentration, and an alteration of H + concentration causes abnormal local pH that influences cell physiology.Overactivity of the H v 1 channel can result in low concentration of intracellular H + and lead to cellular acid-base imbalances (2).The presence of large tryptophan in the H v 1 channel can prevent frequent transitions between closed and open states of the channel and protect against the overactivity of H v 1, and this function might manipulate H v 1 activity to coordinate the regulation of pH homeostasis in white blood cells, sperm cells, epithelial cells, and other cells where H v 1 expressed and maintain normal physiological functions in these cells.
The H v 1 channel function is regulated by pH gradient (ΔpH).It was reported that the G-V of H v 1 channel shifts around 40 mV/unit change in ΔpH, regardless of whether pH o or pH i is changed (25).W207 mutations have been shown to influence ΔpH-dependent gating at lower pH o , supporting the existence of distinct internal and external pH sensors (23).In the present study, W207 mutations exhibited less G-V shifts compared with wildtype channel in the same pH gradient, which is consistent with previous findings that W207 mutations compromise the ΔpH-dependent gating of the H v 1 channels (23).W207 mutations might generate allosterically conformational effects on the pH sensors to perturb the ΔpHdependent gating of the channel.
The H v 1 channel has been shown to form dimers in which two VSD subunits are held together by a C-terminal coiled-coil domain (12)(13)(14).Studies have found that the two H v 1 subunits gate cooperatively, and it was shown that the opening of one subunit substantially increases the probability of the other subunit to open (19).Okuda et al. discovered a molecular mechanism by which two H v 1 subunits cooperate with each other (18).It was shown that the two H v 1 S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment.To delineate the interaction between two H v 1 subunits, Okuda et al. performed a scanning mutagenesis in the S4 transmembrane helices of the H v 1 channel and showed that the aromatic-aromatic interaction mediated by two Trps plays a role in regulation of the dimer's cooperativity.Okuda et al. showed that replacement of Trp in some positions of the S4 helix (e.g., 250W, 253W, 257W in Ciona Hv1) produced stronger effects for deactivation.The analysis of interaction energies for the positions 250, 253, and 257 in the S4 helix showed strong interaction of the two Trp residues in two subunits during the deactivation phase, suggesting that positions 250, 253, and 257 play crucial roles in regulating two Hv1 subunits interaction during gating cooperativity (18).

Trp207 regulation of H v 1 channel
In the present study, we focus on the role of Trp in regulating H v 1 voltage sensor transition in each VSD subunit.Our results showed that Trp modifies the process of H v 1 voltage-dependent activation (i.e., a process by which the channel transitions between its closed and open states).We performed mutagenesis at position Trp207 in human H v 1 channel and found that replacement of small residues at position 207 exhibited faster gating kinetics, in other words, the endogenous Trp at the position 207 presented much slower deactivation and activation compared to the ones of small residues substitution, indicating that the Trp at position 207 plays a role in the regulation of Hv1 channel gating.To date, although several models of voltage sensing, including transporter model, helical screw model, and paddle model, were proposed to describe the movement of VSD S4 helix in voltagegated Na + , Ca + , and K + channels (26), so far, the way of the movement of S4 helix in H v 1 VSD subunit is not yet clear.Our results indicated that the bulky side chain of Trp mediates the movement of H v 1 VSD S4 helix that might undergo rotating and translate along its axis to move across the transmembrane electric field, similar with helical screw model in other ion channels.The small side chain at position 207 reduces the barrier during the process of H v 1 S4 helix rotation and translation in H v 1 VSD, accounting for fast activation and deactivation of the channel when W207 mutated to small residues.
We previously found that a highly conserved residue F150 is important for the hH v 1 channel function and that the hydrophobic group of F150 stabilizes the resting H v 1 VSD (27).Here, we showed that another highly conserved residue W207 regulates the energy barrier during the H v 1 VSD transition.In contrast to the contributions of hydrophobicity of F150 to the channel function, the large size of side chain of W207 is essential for the H v 1 VSD activation, in which it controls the transition between closed and open states of the channel (Fig. 9).The H v 1 proton channel is known to play a role in reactive oxygen species generation and the regulation of pH homeostasis (25,(28)(29)(30)(31)(32).Exploring how the conserved residues including W207 regulates the process of H v 1 voltage-dependent activation will provide insights into the development of targeted reagents modulating H v 1 activity (33).

Mutagenesis
Recombinant human H v 1 channels were subcloned in the pNICE vector.Monomeric hH v 1 was achieved by introducing a stop codon at position 224 (S224 stop ), which deletes coiledcoil domains essential for H v 1 dimer formation (15).Singlepoint mutations were introduced with standard PCR techniques (34,35).PCR primers were purchased from IDT DNA Technologies.Custom-designed primers for mutagenesis of hH v 1 constructs were included in Table S2.H v 1 mutation was introduced to the template plasmid using primers in a PCR protocol.The PCR cycles were initiated at 98 C for 1.5 min, followed by 25 amplification cycles.Each amplification cycle consisted of 98 C (20 s), 60 C (30 s), and 72 C (5 min).The cycles were finished with an extension step at 72 C for 15 min, followed by 4 C for 30 min.After template plasmid was removed by DpnI (NEB, R0176S), the Stbl2 competent cells (Invitrogen,10268019) were transformed with the PCR product.Plasmids were isolated from Stbl2 cells with resulting colonies using QIAprep Spin Miniprep Kit (Qiagen, 27106).Mutation confirmed by the DNA sequencing was used for subsequent transfection.
Cell culture and H v 1 transfection HEK293 cells (Sigma-Aldrich) were reseeded on coverslips containing Dulbecco modified Eagle's medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 C under 5% CO 2 .Cells were transiently transfected with human H v 1 and GFP cDNA plasmids after growth to 70% confluence.The HEK-293 cells were transiently transfected with 2 μg of the cDNA encoding H v 1 construct and 0.25 μg of a plasmid encoding GFP using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's protocol.The mixture was then added to the culture dish, and the cells were incubated at 37 C for 24 h before the electrophysiology studies were conducted.A coverslip with HEK293 cells was placed in a recording chamber containing bath solution on the stage of a fluorescence microscope (Olympus), and the transfected cells detected by the fluorescent signal emitted from GFP were applied for electrophysiological measurements.Patch clamp experiments were conducted 13 days after transfection.EGTA, adjusted to pH i 6.0 with methanesulfonic acid.We performed recording in constant perfusion of the bath compartment to increase buffer turnover by convection, and the solution (pH i = pH o = 6.0) is used to minimize the effects of H + accumulation on the local H + concentration during the recording, and the V rev of H v 1 channels are closed to theoretical equilibrium Nernst potential (0 mV) for reversal potential (V rev ) (Fig. S2 and Table S1).All measurements were performed at 22 ± 3 C. Pipettes had 2 to 5 MΩ access resistance.Current traces were filtered at 1 kHz and analyzed with Clampfit11 (Molecular Devices) and Origin 2019 (OriginLab).

Electrophysiological measurements and analysis
In the recording, proton currents were measured from a holding potential of −60 mV to a first prestep and a second test-step with rest intervals at −60 mV (37,38).The proton channel conductance is determined by the equation: G(V test ) = (I test -I tail )/(V test -V tail ), where I test is the current at V test , measured at the end of the depolarization step, and I tail is the current at V tail , measured at the beginning of the repolarization step.Currents after depolarization in the prestep were used to correct for current rundown.Steady-state activation G-V curves were fitted by the Boltzmann equation ( 39): G/G max = 1/(1 + exp(V 1/2 -V)/k), where G/G max is the relative conductance normalized by the maximal conductance, V 1/2 is the potential of half activation, V is the test pulse, and k is the slope factor.k is equal to RT/ɀF, where ɀ is the equivalent charge, R is the gas constant, F is Faraday's constant, and T is temperature in Kelvin.Reported V 1/2 and k values derived from the Boltzmann fits to data from multiple cells were used to assess the free energy difference at 0 mV (ΔG 0 ) between the closed and open states of the activation, the ΔG 0 was calculated according to the following: ΔG 0 = ɀFV 1/2 .The channel opening time constant Ʈ act and closing time constant Ʈ deact values were calculated by fitting current traces with the singleexponential equation according to the following: I(t) = I 0 + A(1-exp(-t/Ʈ)), where I(t) represents the current at time point t, I 0 is the initial current amplitude, Ʈ is the time constant.

Data and statistical analysis
All data were presented as mean ± standard deviation.Significance between means was determined by Student's t test.Electrophysiological parameters (V 1/2 , Ʈ deact , Ʈ deact , etc) were determined from each individual cell and used for comparison with two-tailed t test.p < 0.05 was considered to indicate a statistically significant difference and were indicated by *. p < 0.01 and p < 0.001 are signified by ** and ***, respectively.

Figure 1 .
Figure 1.The bulky side chain at position 207 regulates H v 1 voltage-dependent activation.A, sequence alignment of the S4 transmembrane segment of H v 1 proton channels from human (hH v 1), mouse (mH v 1), zebrafish (DrH v 1), Ciona intestinalis (CiH v 1), Emiliana (EhH v 1), and Suillus luteus (SlH v 1).Highly conserved tryptophan (W) is highlighted in purple and bold font.W corresponds to Trp207 in hH v 1.The positive S4 residues are highlighted in blue.B, G-V curves for the monomer hH v 1 proton channel W207 mutations colored from blue to red when the side chain of the substituted side chain decreases, and structures of substituted residues are shown in inset.Only several W207 mutations are shown for clarity, and Table S1 summarized all W207 mutations, n = 4 to 8 for each mutation.C and D, the V 1/2 values obtained from G-V curves are plotted with size of the substituted side chain at position W207, using either side chain volume (C) or side chain surface area (D).Black lines indicate fits of the data to a linear function in (C and D), and r values are presented in red in each panel.

Figure 3 .
Figure 3.The hydrophobic group at position 207 does not correlate with H v 1 voltage-dependent activation.A and B, the V 1/2 values obtained from G-V curves are plotted as a function of hydrophobicity of the substituted side chain at position W207, using either Kyte-Doolittle hydrophobicity scale (A) or Goldman-Engelman-Steitz hydrophobicity scale (B).Black lines indicate fits of the data to a linear function, r values are presented in red in each panel.

Figure 2 .
Figure 2. G-V measurements from monomer H v 1 channel currents.A, representative currents were recorded in HEK293 cells expressing monomer WT (207W) H v 1 channel, pH i = pH o = 6.0.The corresponding pulse protocols are shown above the current traces.Currents were measured from a holding potential of −60 mV to a first prestep (+120 mV) and a second test-step (ranging between +150 and −50 mV in 10 mV steps) with rest intervals at −60 mV.Currents after depolarization in the prestep to +120 mV were used to correct for current rundown.For clarity, only the first and last traces elicited by the depolarization prestep are shown.The blue arrows indicate the parameters used for the channel conductance analysis.B, the channel conductance is determined by the equation: G(V test ) = (I test -I tail )/(V test -V tail ), where I test is the current at V test , measured at the end of the depolarization step, and I tail is the current at V tail , measured at the beginning of the repolarization step.C and D, representative currents were recorded in HEK293 cells expressing monomer H v 1 mutations 207S (C) and 207G (D), pH i = pH o = 6.0.The corresponding pulse protocols are shown above the current traces.

Figure 5 .
Figure 5. Effects of small side chain substitutions of W207 on the closed state of the channel.A, representative tail currents recorded from WT (207W), 207S, 207A, and 207G.The tail currents were elicited by a prepulse to 120 mV, in 10 mV decrements from 0 to −60 mV.Red arrows indicate faster deactivation of W207 mutations.B, the deactivation (channel closing) time constant Ʈ deact in WT or W207 mutations.Ʈ deact was obtained from exponential fit to the tail currents, n = 3 to 6 for each group.Ʈ deact between W207 mutation and WT were compared statistically using two-tailed test (***p < 0.001).

Figure 4 .
Figure 4. Effects of small side chain substitutions of W207 on the open state of the monomer H v 1 channel.A, representative rising currents recorded from WT (207W), 207S, 207A, and 207G.Currents were measured from a holding potential of −60 mV to test potentials ranging between −60 and +120 mV in 10 mV steps.B, the channel opening time constant Ʈ act in WT or W207 mutations.Ʈ act was obtained from exponential fit to rising currents, n = 3 to 6 for each group.Ʈ act between W207 mutation and WT were compared statistically using two-tailed test (***p < 0.001).

Figure 6 .
Figure 6.Effects of side chain at position 207 on dimer H v 1 channel voltage-dependent activation.A, representative currents were recorded in HEK293 cells expressing dimer WT H v 1 (207W) channel, pH i = pH o = 6.0.For clarity, only the first and last traces elicited by the depolarization prestep are shown.The corresponding pulse protocols are shown above the current traces.B, G-V curves for the dimer H v 1 proton channel W207 mutations colored from blue to red when the side chain of the substituted side chain decreases, n = 4 to 7 for each group.

Figure 7 .
Figure 7. Effects of small side chain substitutions of W207 on the open and closed state of the dimer H v 1 channel.A, representative rising currents recorded from dimer WT and 207S channels.Currents were measured from a holding potential of −60 mV to test potentials ranging between −60 and +120 mV in 10 mV steps.B, the channel opening time constant Ʈ act in dimer H v 1 channels.Ʈ act was obtained from exponential fit to rising currents.Ʈ act between W207 mutation and WT were compared statistically using two-tailed test (***p < 0.001).C, representative tail currents recorded from dimer WT and 207S channels.The tail currents were elicited by a prepulse to 120 mV, in 10 mV decrements from 0 to −60 mV.D, the deactivation (channel closing) time constant Ʈ deact in dimer H v 1 channels.Ʈ deact was obtained from exponential fit to the tail currents, n = 4 to 6 for each group.Ʈ deact between W207 mutation and WT were compared statistically using two-tailed test (***p < 0.001).

Figure 8 .
Figure 8. State model of W207 mutation regulation of H v 1 VSD transitions.The black lines represent the voltage-dependent activation for bulky side chain Trp (W) at position 207 (i.e., wildtype H v 1 channel), and red lines are changes mediated by the small side chain (X) substitutions at position 207 (W207X).The reduced energy barrier caused by W207X is satisfied by decreased activation kinetic E act (W207X) and decreased deactivation kinetic E deact (W207X).

Figure 9 .
Figure 9. Contributions of conserved residues to the H v 1 VSD energy landscape.Previous study showed that increasing the hydrophobicity of residue at position F150 (green arrow) stabilizes the resting state relative to active state.Present study indicates that increasing the size of the side chain at position W207 (blue arrow) is essential to control H v 1 VSD energy barrier.VSD, voltage-sensing domain.