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The role of Phe150 in human voltage-gated proton channel

Summary The voltage-gated proton channel Hv1 is a member of voltage-gated ion channels containing voltage-sensing domains (VSDs). The VSDs are made of four membrane-spanning segments (S1 through S4), and their function is to detect changes in membrane potential in the cells. A highly conserved phenylalanine 150 (F150) is located in the S2 segment of human voltage-gated proton channels. We previously discovered that the F150 is a binding site for the open channel blocker 2GBI. Here, we show that the Hv1 VSD voltage-dependent activation requires a hydrophobic group at position F150. We perform double-mutant cycle analysis to probe interactions between F150 and positively charged arginines in the S4 segment of the channel. Our results indicate that F150 interacts with two arginines (R2 and R3) in the S4 segment and catalyzes the transfer of the S4 arginines in the process of voltage-dependent activation.


INTRODUCTION
The voltage-gated proton channel H v 1 is a member of the family of voltage-gated ion channels (Ramsey et al., 2006;Sasaki et al., 2006). H v 1 channel plays roles in the regulation of reactive oxygen species (ROS) production by the NADPH oxidase and cellular pH homeostasis (DeCoursey, 2016;Ma et al., 2022;Seredenina et al., 2015;Taylor et al., 2011;Wu et al., 2022); it was found in many cell types including airway epithelia cells DeCoursey and Cherny, 1995;Fischer, 2012;Murphy et al., 2005), sperm cells (Lishko et al., 2010;Zhao et al., 2018Zhao et al., , 2021b), brain microglia (Peng et al., 2021;Wu et al., 2012), and cancer cells (Fernandez et al., 2016;Ribeiro-Silva et al., 2016;Wang et al., 2011Wang et al., , 2012Wang et al., , 2013, in which H v 1 activity has been implicated in the pathology of asthma, sperm capacitation, ischemic stroke, and tumor metastasis. The voltage-gated sodium, potassium, and calcium channels are all made of a pore domain (PD) responsible for ion permeation and four non-permeable voltage-sensing domains (VSDs) detecting changes in membrane potential in the cells. The H v 1 proton channel does not have a PD and forms as a dimer consisting of two permeable VSDs held together by the coiled-coil domain (Koch et al., 2008;Lee et al., 2008;Tombola et al., 2008).
The VSDs of voltage-gated ion channels are made of four transmembrane segments (S1 through S4). A highly conserved phenylalanine is located in the S2 segment of the VSD and acts as a part of the ''gating charge transfer center'' ( Figure 1A) (Tao et al., 2010). In the voltage-gated Shaker K + channel, the conserved phenylalanine (F290) was shown to interact with the positively charged residues of the S4 segment and catalyze the sequential movement of the gating charges across the electric field (Lacroix and Bezanilla, 2011;Lacroix et al., 2014;Pless et al., 2011;Schwaiger et al., 2013;Tao et al., 2010). A state model of voltage sensor transitions in the Shaker channel indicated that F290 interacts with the first positively charged residue in the fully closed VSD, and with the fifth positively charged residue when the VSD is in its depolarized conformation, suggesting a gating process consists of four transitions from the resting state to the final pore opening in the Shaker potassium channel (Tao et al., 2010). Additionally, the size of the conserved phenylalanine is important for the channel function, and the large side chain at position F290 is required to stabilize the Shaker channel depolarized VSD conformation (Lacroix et al., 2014;Tao et al., 2010).
Despite progress in the functional characterization of F290 in the Shaker K + channel, the role of the highly conserved phenylalanine in H v 1 VSD activation remains unclear. The H v 1 channel has a more positive halfactivation voltage (V 1/2 ) and less number of positively charged residues in S4 segment compared with the Shaker channel, indicating that the conserved phenylalanine contributes different roles in voltage-dependent activation and regulation of the VSD in specific conformations.
The highly conserved phenylalanine of the human H v 1 channel (hH v 1) is F150. We previously discovered that the side chain of F150 affects the strength of the interaction between the H v 1 channel and the blocker 2GBI (Hong et al., 2013(Hong et al., , 2014, and there was a negative correlation between the side chain size at position 150 and the blocker binding affinity (Zhao et al., 2021a), suggesting that the size of F150 plays a role in 2GBI's binding.
To assess whether the side chain size of F150 regulates hH v 1 VSD activation as well, we introduced mutations in the F150 by substituting other 19 amino acids. We found that the hydrophobic group instead of the size of the side chain at position F150 is essential for the H v 1 voltage-dependent activation. We then investigated interactions between F150 and arginines in the S4 segment of the H v 1 channel, and the doublemutant cycle analysis characterized significantly thermodynamic couplings between F150 and R2 or R3 when the channel transits from closed state to open state. Moreover, substitutions of charge-conserving mutations at R2 or R3 destabilize the closed state or open state of the channel, respectively. We discussed a simple model consists of two states connected by one transition.

RESULTS
The hydrophobicity of phenylalanine at position 150 is essential for the voltage-dependent activation of the hH v 1 channel We first introduced a small residue replacement (alanine) at position 150 of the human H v 1 channel (F150A) and observed that the F150A mutation shifted the conductance versus voltage relationship (G-V curve) to Figure 1. The hydrophobic group at position 150 is essential for H v 1 voltage-dependent activation (A) Sequence alignment of the S2 transmembrane segment of hH v 1 proton channel, Shaker potassium channel, NachBac sodium channel, Ca v 1.1 calcium channel, and CiVSP. Highly conserved phenylalanine (F) in the S2 segments is highlighted. F corresponds to Phe150 in hH v 1. (B) G-V curves for the hH v 1 proton channel F150 mutations colored from red to blue when the hydrophobicity of the substituted side chain decreases, only several F150 mutations are shown for clarity. Table S1 summarized  To comprehensively explore the effects of F150 on the voltage dependence of activation, we characterized all 19 mutations at position 150 (Table S1). It was shown that all mutations produced left-shifted G-V relationships that increased open state stability relative to the closed state ( Figure 1B, Table S1), indicating that the nature of the side chain of phenylalanine at position 150 is required to stabilize the resting state of the channel. We next performed linear correlation analysis for the relationship between the H v 1 voltage dependence of activation and physicochemical properties of amino acid mutations. The voltage dependence of activation parameter V 1/2 was measured as the midpoint voltage obtained from the fitted G-V curve, and the physicochemical properties of amino acids included side chain volume and hydrophobicity (Engelman et al., 1986;Kyte and Doolittle, 1982;Zamyatnin, 1972) (Table S2).
There were no significant correlations between the V 1/2 and the size of the side chain ( Figure S2). In contrast, it showed a strong linear dependence between the H v 1 VSD voltage dependence and hydrophobicity ( Figures 1C and 1D). We tested two hydrophobicity scales Kyte-Doolittle (Kyte and Doolittle, 1982) and Goldman-Engelman-Steitz (Engelman et al., 1986), and the V 1/2 is correlated with both of hydrophobicity scales. The data showed that the V 1/2 is associated with the hydrophobicity at position 150, and a hydrophobic residue at position F150 is important for the channel function.
Effects of mutations at position 150 depend on the positive residues in the S4 transmembrane segment The highly conserved phenylalanine has been reported to interact with S4 charges to control the VSD gating ( Figure 2A). In the Shaker channel, the effect of phenylalanine to tryptophan mutation (F290W) was associated with charged residue at position 5 (K5 in the Shaker S4) when the Shaker K + channel VSD . Since F150 mutations have strong effects on the voltagedependent activation of the H v 1 channel, we wondered whether we could identify interactions similar to those between F290 and S4 charges in the Shaker channel that also occur in the H v 1 channel.
A double-mutant cycle analysis has been used to probe residues' interaction between transmembrane segments in the channel (Chamberlin et al., 2014). Similar approaches were introduced to investigate the effects of combining the F150W and charge-conserving S4 mutations ( Figure 2). The H v 1 channel has 3 arginines (R1, R2, and R3) in the S4 segment. An asparagine (N4) takes the fourth position of positive S4 residues in the VSDs of other channels (Figure 2A), and we generated a mutation at this position as well in the human H v 1 channel.
The G-V curves of the F150 mutation (F150W) with or without S4 segment mutations (R1K, R2K, R3K, and N4K) were summarized in Figures 2C-2F. For each mutation, the G-V curve was fitted with the Boltzmann equation, and V 1/2 and k slope factors were then determined and used to calculate the DG 0 , which is the free energy difference between the closed and open states of the activation. The |DDG 0 | (i.e., coupling DG 0 value) between two position mutations was determined by the thermodynamic coupling analysis as previously explained (Li-Smerin et al., 2000). The significant interaction between two residues was defined as a |DDG 0 | > 4.2 kJ/mol (1 kcal/mol) (Cheng et al., 2013;Zhang et al., 2005).
The |DDG 0 | values determined by the double-mutant cycle analysis were reported in Figure 2B. We observed that the combinations F150W-R1K and F150W-N4K had |DDG 0 | values smaller than 4.2 kJ/mol, indicating that there are no thermodynamic couplings between F150 and R1 or N4. In contrast, the combinations F150W-R2K and F150W-R3K dramatically increased the |DDG 0 |, and the values were 10.1 and 12.1 kJ/mol, respectively, both were substantially higher than the ones for F150W-R1K and F150W-N4K ( Figure 2B, Table S3). We then investigated residues near the F150 (F149 and M151), and the combinations F149W-R2K, F149W-R3K, M151W-R2K, and M151W-R3K all showed smaller |DDG 0 | compared with F150W-R2K and F150W-R3K ( Figure S3). This strongly suggests that F150 is able to form functional interactions with R2 or R3 during the voltage-dependent activation, and serve as a part of the gating charge transfer center in the H v 1 channel.
We previously used an open channel blocker to probe that the F150 and D112, a residue crucial for the proton selectivity of the channel, are located close to each other (Hong et al., 2014). We next investigated if the D112 affects the interactions between F150 and S4 arginines. The combination F150 and D112 mutation (F150W-D112E) generated a |DDG 0 | value (6.0 kJ/mol) larger than 4.2 kJ/mol, indicating an interaction between F150 and D112 ( Figure S4). The significantly thermodynamic coupling between D112 and F150 was consistent with the results that D112E mutation affected F150 interactions with R2 or R3 ( Figure S5).

F150 interacts with R2 and R3 to stabilize the closed and open states of the H v 1 channel
The VSD activation is a process that S4 segment positively charged amino acids move from inside to outside of the membrane field (Tombola et al., 2006). As the mutant cycle analysis showed that F150 interacts with both R2 and R3 during the channel activation (a process by which the channel transitions between its closed and open states), we propose that F150 interacts with R2 to stabilize the closed state of the channel, and when the channel is depolarized, the R3 reaches the binding site and interacts with F150 to stabilize the open state of the H v 1 VSD.
A charge-conserving mutation at R2 (R2K) was introduced to the H v 1 channel ( Figure 3). The R2K mutation shifted the G-V curve to the hyperpolarized direction with faster activation kinetics ( Figures 3C and S6). The channel closing time constant Ʈ deact values were determined, and R2K mutation took longer time to close compared with WT ( Figures 3B and 3D). The hyperpolarized-shifted G-V curve, decreased DG 0 , and the slower rate of closure indicate that the replacement of lysine at position 2 (R2K) destabilizes the closed state of the channel. This suggests that the endogenous residue (arginine) at position 2 increases the relative stability of the closed conformation.
The effects of charge-conserving mutation at the R3 (R3K) were further investigated (Figure 4). In contrast to R2K, the R3K shifted the G-V relationship to the depolarized direction ( Figure 4C) iScience Article depolarized-shifted G-V relationship, increased DG 0 , and faster deactivation kinetics ( Figure S7), which is indicative of a destabilized open state relative to the closed state. This is consistent with the results that R3K mutation significantly delayed the activation kinetics ( Figure 4B), and it altered the opening time constant Ʈ act increasing the values at given membrane potentials ( Figure 4D). These results indicated that the function of native arginine at position 3 is to stabilize the open channel, and mutation at R3 (R3K) strongly perturbs the stability of the open state. The spatial proximity between F150 and R3 was further assessed by cysteine pairs crosslinking; an approach that used Cd 2+ to probe for cysteine-cysteine distances has been previously applied on other channel (Campos et al., 2007). Here, we introduced double cysteine mutations (F150C-R3C) in the H v 1 channel expressing in Xenopus oocytes. The current carried by F150C-R3C cysteine pairs was remarkably decreased by intracellular Cd 2+ compared with the currents carried by single cysteine mutation F150C or R3C ( Figure S8), indicating a formation of Cd 2+ bridge between the two residue pairs.  Figure 5).

Model of H v 1 VSD transitions
In the model, the effect of the R2K mutation on the channel, which is closed state destabilization, was satisfied by decreasing the relative depth of the energy well for the ''Close'' state and increasing the energy barrier for the Open -> Close transition to allow for the slower deactivation (orange lines in Figure 5A). iScience Article A smaller DG 0 for R2K was consistent with decreased activation kinetics (E act (R2K)) ( Figure S6B) and increased deactivation kinetics (E deact (R2K)) ( Figure 3D).
The effects of the R3K mutation on the open state of the channel could also be described using this model, and R3K destabilizing the open state could be satisfied by decreasing the relative depth of the energy well for the ''Open'' state and increasing the energy barrier for the Close -> Open transition to interpret the slower activation (purple lines in Figure 5B). A larger DG 0 for R3K was consistent with increasing the height of the energy well (increased E act (R3K), Figure 4D) and decreasing the depth of the energy well for the open state (decreased E deact (R3K)) ( Figure S7B).

DISCUSSION
In the Shaker potassium channel, the large side chain at position F290 is required to stabilize the depolarized VSD conformation (Lacroix et al., 2014); the role of the side chain size at position F290 in Shaker channel was supported by discoveries that the size of unnatural amino acid analogs of phenylalanine at position 290 was negatively correlated with the voltage-dependent channel activation curves (Pless et al., 2011;Tao et al., 2010).
In contrast to the Shaker channel, the highly conserved phenylalanine of the H v 1 channel (F150) exhibited distinctive features. We determined the G-V curves of F150 mutations and analyzed the effects of physicochemical properties of F150 mutations on the voltage-dependent activation. There were no significant correlations between the size of side chain and the midpoint V 1/2 values in H v 1. Strikingly, replacements of hydrophilic residues at position 150 consistently shift the G-V curve toward more negative voltages, indicating that stabilization of the resting H v 1 VSD conformation requires a hydrophobic group at position F150. A recent finding that increasing the hydrophilicity of the H v 1 hydrophobic gasket region including iScience Article F150 facilitated channel opening supports this conclusion (Banh et al., 2019). The presence of the hydrophobic side chain at position 150 might play an essential role in preventing protons permeating the VSD when the channel is in closed conformation.
Although the side chain size at position F150 is not correlated with the H v 1 VSD voltage-dependent activation, it strongly affects the H v 1 pharmacology. Mutations at position F150 have been shown to influence the strength of the interaction between the channel and the H v 1 blocker 2GBI (Hong et al., 2013(Hong et al., , 2014. A replacement of small amino acid alanine (F150A) generated over 350-fold increase in binding affinity for the blocker, whereas substitution of bulky residue at position 150 (F150W) reduced the binding affinity of the 2GBI (Hong et al., 2013). These suggest that the mechanism by which F150 mutations alter the blocker binding is distinct from the mechanism by which they alter the movement of the voltage sensor. We conclude that the nature of the side chain of phenylalanine at position 150 has differential effects on the channel function: the size of phenylalanine plays a key role in blocker binding, while the hydrophobicity of phenylalanine is essential for the channel voltage-dependent activation.
Previous studies on the VSDs of voltage-gated potassium channels revealed that the conserved phenylalanine interacts with different S4 arginines in different conformations (Henrion et al., 2012). In the Shaker channel, F290 was proposed to interact with the first positively charged residue in the closed state, and with the fifth positively charged residue in the channel open conformation, suggesting a gating process consists of four transitions from the resting state to the final pore opening in the Shaker potassium channel (Henrion et al., 2012;Tao et al., 2010).
Compared with the Shaker channel, the S4 movement in H v 1 channel is shorter. Two different models of H v 1 voltage-dependent activation have been proposed (DeCoursey, 2015;Larsson, 2020). In the ''three-click'' model, the S4 segment moves three turns of the helix upward and three arginines all move outward past the hydrophobic gasket in the VSD (Gonzalez et al., 2010). In the ''one-click'' model, there is only one transition and the S4 moves one turn of the helix upward (Li et al., 2015).
We applied double-mutant cycle analysis to probe interactions between the conserved phenylalanine F150 and arginines in the S4 segment, and found that there are significantly thermodynamic couplings between F150 and R2 or R3 when the channel transits between closed and open conformations. Destabilization of the open state caused by R3K is achieved by decreasing the relative depth of the energy well for the ''Open'' state and increasing the energy barrier for the Close -> Open transition to account for the slower activation. The larger DG 0 for R3K is consistent with increased activation kinetics E act (R3K) and decreased deactivation kinetics E deact (R3K).

OPEN ACCESS
iScience 25, 105420, November 18, 2022 iScience Article Our findings indicated that F150 is close to the arginine at position 2 (R2) in the resting state, which is consistent with the crystal structure of the closed conformation of the H v 1-VSP chimera (Takeshita et al., 2014). Substitution of a charge-conserving mutation at R2 (R2K) produced hyperpolarized shifts of the G-V curve (Figure 3), and this mutation took less time to activate and much longer time to close, indicating that the R2K strongly destabilizes the closed state relative to the open state of the channel. This might be due to the fact that the charged side chain of lysine (K) is shorter than arginine (R), and F150 could not effectively interact with the lysine at position 2 (R2K) in order to stabilize the closed conformation.
To date, the crystal structure of the open state of the H v 1 channel has not yet been achieved. We previously used an open channel blocker 2GBI to probe a locally structural information when the H v 1 channel opens (Hong et al., 2014); we characterized that the binding environment of 2GBI is a cluster of tightly packed residues including F150 and R3 within the H v 1 VSD. This suggested that in the H v 1 open conformation, F150 and R3 are packed in space and likely interact with each other. The results in the present study were consistent with this conclusion. The R3K mutation markedly slowed the process of channel opening (Figure 4), suggesting that it perturbed the open conformation of the channel. The depolarized shift of the G-V produced by R3K showed that more energies are required to push the lysine (K) at position 3 into the binding site to interact with F150, suggesting that the native arginine (R) binds more tightly relative to lysine (K) in the presence of phenylalanine at position 150, which made the voltage sensor to be stabilized in its open conformation. Moreover, the interaction between F150 and R3 was supported by the results of cysteine pairs cross-links ( Figure S8).
We propose a simple model consists of two states (closed and open) connected by one transition: The F150 interacting with R2 stabilizes the closed state of the channel. When depolarized, the R3 moves sequentially upward to interact with F150 stabilizing the open state of the channel ( Figure 6).
Although our results indicated that F150 does not interact with R1, the R1 mutations have been shown to modulate the H v 1 channel voltage-dependent activation and facilitate channel opening with faster activation kinetics (Ramsey et al., 2006;Sasaki et al., 2006). Previous studies in NaChBac channel revealed that the positive residue R1 of the S4 segment interacted with a negative residue E43 at the extracellular end of the S1 segment to form charge-charge interaction stabilizing the channel in resting state (DeCaen et al., 2011;Yarov-Yarovoy et al., 2012). The E43 in NaChBac corresponds to the E119 in human H v 1 channel. Mutations at R1 in the H v 1 proton channel likely generated a perturbation of the charge-charge interaction between R1 and E119 and destabilized the resting state of the channel, reducing an energy barrier for the channel activation, accounting for faster activations in the R1 mutations of the H v 1 channel.
The physicochemical basis for interaction between F150 and S4 arginines in the H v 1 channel is potentially associated with cation-p binding. In Shaker channel, cation-p interaction was found to stabilize the interaction between S4 positive-charged residue K374 (K5) and F290W. Fluorine modifications at F290W produced a stepwise right-shift in the G-V of the Shaker channel, indicating that the electronegative surface potential of aromatic residue contributes to a cation-p interaction with positive charged residue (Pless et al., 2011). In the H v 1 channel, the aromatic side chain of F150 has been previously shown to contribute a cation-p interaction between the F150 and blocker 2GBI (Zhao et al., 2021a). A similar interaction might iScience Article be present in the H v 1 channel when F150 interacts with S4 positive-charged residues during the channel activation. The aromatic ring of F150 catalyzes the S4 movement potentially through the electrostatic cation-p interaction with the R2 or R3. A detailed mechanism underlying the interaction remains to be explored in future work.
Taken together, our results identify a role for F150 in the regulation of H v 1 channel function. We show that the highly conserved phenylalanine of the H v 1 channel exhibits different features and that the hydrophobicity of phenylalanine is essential for channel voltage-dependent activation. The F150 functionally interacts with arginines at positions 2 and 3 in the S4 segment and catalyzes the transfer of the S4 arginines in the process of voltage sensing. The voltage-gated proton channel H v 1 has been associated with ROS generation and regulation of pH homeostasis. Understanding how H v 1 proton channel activity is manipulated by transmembrane potential will shed light on the instructions in creation of reagents targeting this channel, and therapeutics to rationally modulation of H v 1 activity.

Limitations of the study
The study showed that the conserved phenylalanine F150 in the human H v 1 channel interacts with two S4 arginines when the channel transits between closed and open conformations. Although the data in our study supported that in the H v 1 open conformation, R3 and F150 interact with each other to stabilize the open state, so far, the structure of the activated state of the H v 1 channel is not available to justify the model. Additionally, the detailed mechanism underlying the interaction between the hydrophobic phenylalanine and positively charged arginines in the H v 1 channel remains to be explored in future work.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: