Trapped Pore Waters in the Open Proton Channel H V 1

  1. Danila Boytsov
  2. Stefania Brescia
  3. Gustavo Chaves
  4. Sabina Koefler
  5. Christof Hannesschlaeger
  6. Christine Siligan
  7. Nikolaus Goessweiner‐Mohr
  8. Boris Musset
  9. Peter Pohl

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Abstract

The voltage‐gated proton channel, H V 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single‐channel water permeability 2.3 × 10 −12 cm 3 s −1 reflects the permeability‐governing number of hydrogen bonds between water molecules in single‐file configuration and pore residues. However, the measured unitary water permeability does not confirm the predicted value. Osmotic deflation of reconstituted lipid vesicles reveals negligible water permeability of the H V 1 wild‐type channel and the D174A mutant open at 0 mV. The conductance of 1400 H + s −1 per wild‐type channel agrees with the calculated diffusion limit for a ≈2 Å capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H + conductance by reducing the capture radius. At least one intervening amino acid contributes to H + conductance while interrupting the water wire across the membrane.

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  1. Version published to 10.1002/smll.202205968
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    Reply to the reviewers

    All the Reviewer’s comments are reproduced below, with our responses interspersed in [[brackets]]. Citations from the revised manuscript are included in “quotation marks”. The website accepts input only as plain text. Consequently, we had to transform the mathematical expressions into plain text. We apologize for the reduced readability.

    Reviewer #1

    1. The authors state that: "the conductance density mediated by the expression of the mutant was 2.5 times smaller than the wild type, although we transfected the same amount of plasmid DNA (Fig. 2E). Assuming that protein expression is independent of the mutation, the observation suggested that the unitary proton flux ratio RC of wild type to mutant channel was equal to 2.5" (lines 82‐85).

    Macroscopic conductance (G) depends on channel number (N), microscopic or unitary conductance (γ), and open probability (PO) by G=N γ PO. The authors assume that the level of WT and D174A mutant protein expression on plasma membrane, which determines N, are equal; however, this critical assumption does not appear to have been tested.

    The fact that conductance density (nS/pF) is plotted in Fig. 2E does not alter this caveat because this procedure normalizes the data only for cell surface area (i.e., size). The authors' conclude that "The conductance density relationship (Fig. 2E) compares the maximal conduction of both constructs; this is the fully open channel (open probability ≈ 1)"(lines 87‐88). However, neither raw currents nor G‐V data are shown. Typically, currents measured at large, near‐saturating PO are used to compare the relative conductances of WT and mutant ion channels. The currents shown in Fig. 2A and 2B exhibit prominent 'droop' at even modest depolarizing potentials (+10 mV for D174A and +30 mV for WT), indicating that the proton gradient has been substantially perturbed by the flow of ge depolarizing voltages needed to drive channels to near‐maximal PO. Furthermore, there is no evidence that maximal PO itself is also not different in WT and D174A channels. Indeed, maximal PO for native Hv1 channels measured using variance analysis is reported by significantly smaller than 1.0, and assuming that PO = 1.0 for either WT or D174A is therefore not well supported. Maximal could be altered by the D174A mutation, which has a clear and strong effect on channel gating evidenced by the large (‐70 mV) negative shift in threshold potential reported both here and previously in the literature. Effects of mutations on maximal PO due to altered gating behavior could be separate and distinct from any change in plasma membrane channel number (N). 3 Lastly, because D174A channels have a much higher PO than WT at 0 mV, the mutant will necessarily conduct inward proton currents at the physiological resting membrane potential (RMP) in tsa‐201 cells (perhaps ‐30 mV?). Inwardly directed proton currents will therefore cause intracellular acidification under resting conditions.

    The constitutive acid load in cells expressing D174A, but not WT, is likely to have a variety of physiological consequences, including decreased protein expression or plasma membrane targeting of D174A. There is evidence that another constitutively open Hv1 mutant (R205H) also generates smaller currents macroscopic conductance than WT, and this phenomenon is likely to result from decreased cell surface expression. To conclude that the microscopic conductances of WT and D174A are unequal, the authors must demonstrate that N is not different. The authors' conclusion that D174A "conducts protons at a lower rate" (line 89) is therefore not well supported by the experimental data.

    [[

    We toned down our conclusions from the experiments to accommodate the reviewer's criticism: (page 4): " Consequently, the mutant channel is nearly fully open (Fig. 2D), readily seen when the membrane potential is 0 mV and external voltage is absent. The high open probability of the D174 mutant under symmetrical pH conditions is readily seen in the tail current amplitude reaching a quasi-saturation (Fig. 2A). The resulting outward currents have a higher amplitude in the wild-type (Fig. 2A+B). Interestingly, the conductance density mediated by the expression of the mutant was 2.5 times smaller than the wild type, although we transfected the same amount of plasmid DNA (Fig. 2E). Our observation suggests a reduced flux through the mutant if we assume that protein abundance in the plasma membrane is independent of the mutation."]]

    1. The authors indirectly measure apparent proton flux rates (λD) in LUVs containing WT and D174A mutant Hv1 channels using a fluorescence‐based approach, and conclude that λD is 2.4 times smaller for D174A than WT. However, the method for estimating λD is not performed under voltage clamp, and the driving force for proton current is neither known nor measured.

    [[

    The reviewer is mistaken. The method for estimating λD is performed under voltage clamp, and the driving force for proton current is known.

    Page 6: “To obtain λD, we encapsulated c_k^i=150 mM KCl in the HV1 containing large unilamellar vesicles (LUVs) and exposed these vesicles to a buffer with a K+ concentration c_k^o= 3 mM. The addition of valinomycin facilitated K+ efflux, thereby inducing a membrane potential, ψ. ψ constituted the driving force for H+ uptake. It can be calculated according to the Goldman equation:

    ψ = -RT/F ln ((c_k^i+(P_H/P_K ) c_H^i)/(c_k^o+(P_H/P_K ) c_H^o ))

    (1)

    The ratio of the HV1 mediated proton permeability P_H to the valinomycin-mediated potassium permeability P_K is always smaller than 0.04. We base our conclusion on the observation that the CCCP-mediated proton permeability represents an upper limit for P_H since CCCP always induces a faster vesicular proton uptake than HV1 (Fig. 3). Accordingly, the maximum value of P_H/P_K can be estimated as the ratio of valinomycin to CCCP conductivities. The respective values are equal to 1.6 10-3 Ω-1 cm2 [1] and 4 10-6 Ω-1 cm-2 [2]. At pH 7.5, we find c_H^o=10^(-7.5) M, i.e., c_k^o ≫ (P_H/P_K )c_H^o. Similarily, c_k^I ≫ (P_H/P_K ) c_H^i for a broad range of intravesicular pH. With these simplifications, Eq. 1 transforms into the Nernst equation yielding:

    ψ = -RT/F ln (c_k^i)/(c_k^o )=-100 mV

    (2)

    ψ of such size may decrease intravesicular pH by nearly two units. Such acidification does not violate c_k^i ≫ (P_H/P_K ) c_H^i so that ψ remains constant throughout the experiment. That is, the vesicle experiments proceed under voltage clamp conditions. The simple explanation is that, due to the small proton concentration and the limited buffer capacity, the K+ conductance exceeds H+ conductance under all conditions. The conclusion is in line with simulations (32), confirming that the membrane potential is driven very near the Nernst potential for K+.”]]

    The authors state that "Transmembrane voltage constituted the driving force for proton uptake into LUVs (Figure M). It resulted from facilitated K+ efflux out of the vesicles (30)", (lines 261‐262), but this voltage is unknown and not likely to equal the Nernst equilibrium potential for K+ once Hv1 channels begin to open.

    [[

    The reviewer is mistaken. The voltage is known (see the equations above). The opening of the HV1 channels does not alter the potential because c_k^o ≫ (P_H/P_K ) c_H^o and c_k^i ≫ (P_H/P_K ) c_H^i for a broad range of intravesicular pH (see above).]]

    Once Hv1 channels begin to open, intra‐lumenal pH (pHi) will necessarily occur during the experiment. Such changes are likely exacerbated by a) the low proton buffering capacity of the system (5 mM HEPES) and b) the absence of any counter‐charge pathway to balance the effect of proton charge movement on the membrane potential.

    [[

    Vesicle acidification occurs. It signifies the presence of functional proton channels. Nevertheless, the membrane potential does not change (see Equation 1 above). The statement b) is not correct because the outward K+ movement counters the inward-directed proton charge movement.]]

    Given the small volume of LUVs, even a relatively modest difference in either membrane potential or pHi could substantially alter the driving force for proton movement. Together, these factors are highly likely to result in a rapid and potentially large change in the driving force for proton flux.

    [[

    As outlined above, membrane potential stays invariant. Vesicle acidification changes the driving force for proton flux. The steady state is reached when the electrochemical potentials for protons on the two sides of the membrane are equal to each other.]]

    Driving force changes may also be different for WT and D174A because their relative PO may be different under the experimental conditions used here. Because D174A activates at much more negative voltages, it is likely to open more quickly and to a higher PO than WT at early times after depolarization is initiated by addition of valinomycin (Fig. 3A). This fact will likely result in a larger initial inward current being carried by D174A than WT channels. The result would be a more rapid acidification of LUVs by D174A.

    [[

    The reviewer is mistaken. Assuming a transport rate of 20,000 potassium ions per second (G. Stark, B. Ketterer, R. Benz and P. Läuger; Biophys. J. 1971 Vol. 11 Pages 981-981) and a membrane capacity of 1 μF cm-2, it takes valinomycin about 10 ms to drive the vesicular potential to near Nernst values. Activation of the proton channel is at least 10 times slower. Thus, both mutant channel and wild type channel may open at roughly the same instant. The driving force is sufficient to open both channels to the same probability.]]

    The experimental data in Fig. 3A are consistent with the expectation that the proton gradient and driving force more rapidly approach equilibrium for D174A than WT channels: the apparent rate of AMCA fluorescence change is slower in D174A. Although the authors correctly interpret the experimental data to mean that the apparent λD is slower for D174A, they do not rule out the artifactual explanation for the measured differences. Indeed, the observation in Fig. 3A that AMCA fluorescence change eventually reaches a plateau and is not affected by CCCP means that the proton gradient has become exhausted during the experiment, and directly demonstrates that the proton driving force is uncontrolled under the current experimental conditions.

    [[

    The reviewer's interpretation of our results is flawed. Instead of becoming exhausted, the proton gradient builds up during the experiment. Initially, extravesicular and intravesicular pH values are equal to each other. Valinomycin-mediated K+ efflux results in a membrane potential that drives Hv1-mediated H+ influx.

    Page 8: “The number NC of reconstituted HV1 dimers per vesicle determines the acidification rate λ, i.e., the time that elapses before reaching the steady state. The final intraluminal pH is independent of NC. Similarly, CCCP addition in the steady state does not change the intraluminal pH of HV1-containing vesicles. But CCCP will affect the intraluminal pH of vesicles deprived of HV1 since H+ background permeability is too small to allow vesicle acidification within the time allotted for the experiment. Consequently, only HV1-free vesicles will acidify upon CCCP addition. That is, CCCP addition allows estimating the fraction of vesicles deprived of HV1.”]]

    In contrast to the authors' statement that "Our experiments with the purified and reconstituted channels corroborated the conclusion (Fig. 3A)", (lines 92‐93) it is not clear that unitary proton flux rates/unitary conductances are actually different in WT and D174A.

    [[

    The reviewer is mistaken. Since we measured under voltage clamp conditions, ensured rapid installment of the membrane potential, and selected a potential large enough to allow for the same open probability of wild-type and mutant channels, the measured transport rates, λ, are valid. Moreover, we determined the number of HV1 channels per vesicle and thus calculated the transport rate of an individual channel, λD. Since λD is different for WT and D174A, the unitary proton flux rates/unitary conductances are actually different in the wild type and mutant.]]

    1. The presumed differences in unitary conductances (i.e., 'transport rate') between WT and D174A are used to estimate Arrhenius activation energies (Ea): ("The difference in measures transport rates allows a rough estimation of the Arrhenius 128 activation energy Ea for HV1‐mediated proton flow. It amounts to 40 kJ/mol for the wild type and 23 kJ for the mutant. Thus, Ea exceeds the corresponding 15 kJ/mol barrier measured for gramicidin A (32, 33)", (lines 128‐130). The method for determining Ea in the current work is not well‐described. In Ref. 32, the authors estimate Arrhenius activation energy (Ea = 20 kJ/mol) for gramicidin D (not gramicidin A) from the slope of a line fit to measurements of currents at various temperatures. Here, the authors measure AMCA fluorescence decay rates at 4 °C and 23 °C and observe a similar temperature‐dependent difference in WT and D174A (Fig. S2). Given that the data indicate that WT and D174A are similarly temperature‐dependent, it is unclear how the authors arrive at different Ea values. The authors' conclusion that "The increment in Ea suggests that the transport mechanism may be different from a pure Grotthuss type, where the proton uses an uninterrupted water wire to cross the membrane", (lines 131‐133) therefore does not appear to be well‐supported.

    [[

    We removed both the calculation and discussion of activation energies. Knowledge and discussion of activation energies distract from the scope of the manuscript. We show the experiments at different temperatures solely to demonstrate that Hv1 and D174A facilitate proton transport at a decreased temperature where the background conductivity of the lipid bilayer to water is small.]]

    1. The authors report no difference in water permeability in WT vs. D174A (Fig. 5 and S1) and interpret the results to mean that proton currents are not associated with measurable bulk water flow. A similar conclusion was reached for native Hv1 channels using deuterium substitution (DeCoursey & Cherny, 1997).

    [[

    The comment of the reviewer is misleading:

    • Equal water permeabilities of WT and D174A would not exclude an association between proton currents and water flow. Accordingly, our manuscript does not contain the stipulated interpretation.
    • DeCoursey & Cherny (1997) did not evaluate bulk water flow through proton channels. They compared D+ and H+ currents across the plasma membrane of rat alveolar epithelial cells. Page 2: “Comparing deuterium ion and proton currents through the plasma membrane of rat alveolar epithelial cells, DeCoursey & Cherny (22) found an isotope effect exceeding that for hydrogen bond cleavage in bulk water. It suggested the involvement of an amino acid side chain in proton conduction (22). Alternatively, altered properties of confined water could have been responsible for the higher isotope effect.”]]

    However, the absence of bulk water flow does not itself rule out the possibility that 'trapped' waters within the Hv1 pore do not themselves carry the measured proton current. If intra‐pore water molecules are tethered by hydrogen bonds with protein atoms, they may not move when Hv1 channels open.

    [[

    The reviewer’s comment contains one misinterpretation and one unfounded statement:

    1. We never stated that 'trapped' waters within the Hv1 pore do not themselves carry the measured proton current. On the contrary, we envisioned the trapped waters delivering the protons to one or more titratable amino acid side chains and accepting the protons from them.
    2. The reviewer’s view that intra‐pore water molecules tethered by hydrogen bonds with protein atoms may not move when Hv1 channels open is a misconception. Page 12 bottom: “The contrasting opinion that instead of a channel obstruction hydrogen bonds may immobilize the pore water (19) is not convincing. First, the lifetime of a hydrogen bond is in the ps range while HV1’s mean open time exceeds 100 ms (41). Thus, hydrogen bonds may break more than 1011 times during the open state, rendering them unfit for tethering intraluminal water molecules. Second, the effect of hydrogen bonds between water molecules and pore residues is limited to decreased water mobility in narrow channels (23). Their number, NH, allows for predicting pf (26). Specifically, every H-bond donating or receiving pore-lining residue contributes an average increment ΔΔG╪ of 0.1 kcal/mol to the Gibbs free energy of activation ΔG╪ (24). Equation (1) allows the calculation of ΔG╪:

    ΔG╪= N_H ΔΔG╪ + ΔΔG╪_i (13)

    where ΔΔG╪_i = 2 kcal/mol (24). Since N_H = 6 (Fig. S1) in the open HV1 conformation, Eq. 1 predicts ΔG╪ = 2.6 kcal/mol. Eq. (2) allows calculating HV1’s pf from this value (42):

    p_f = v_0 v_w exp(-ΔG╪/RT) (14)

    where vw = 3 × 10−23 cm3 is the volume of one water molecule and ν0 is the universal attempt frequency, ν0 = kB∙T/h ≈ 6.2 × 1012 s−1 at room temperature (kB is Boltzmann’s and *h *is Planck’s constant).”]]

    Proton transfer through a hydrogen‐bonded network of waters requires only that the electronic structure of the network be rearranged during proton transfer; water is not required. As in the previous study (DeCoursey & Cherny, 1997), the lack of water flux reported here demonstrates seems to reinforce the notion that H+ moves separately from its waters of hydration (i.e., hydronium, H3O+, is not the permeant species) and does not necessarily imply information about the mechanism of proton transfer (i.e., side chain ionization vs. Grotthuss‐type transfer in a water‐wire).

    [[

    The reviewer is mixing two unrelated issues. Of course, proton transport may be separated from mass transfer. Yet, charge transfer may or may not include one or several titratable amino acid side chains. If proton side chain ionization is not involved in proton transfer, a water wire must exist that connects the aqueous solutions on both sides of the membrane. In this case, an osmotic gradient will drive water molecules through the open channel. Since we did not observe such water flux, we conclude that the water wire is interrupted by at least one side chain. Thus, our experiments imply information about the mechanism of proton transfer.]]

    The authors state that: 1) "every H‐bond donating or receiving pore‐lining residue would have contributed an increment ΔΔ𝐺‡ of 0.1 kcal/mol to the Gibbs free energy of activation Δ𝐺‡ (25)" (lines 145‐147), and 2) calculating NH from this Δ𝐺‡ allows estimation of the channel's unitary water permeability (Eqn. 2). Although hydrogen bonding patterns will undoubtedly alter the free energy for channel activation, this is not the same free energy change as that for proton transfer.

    [[

    The reviewer's remark is in line with the previous and the current versions of our manuscript.]]

    Hv1 gating involves conformational changes that are both voltage and Δ pH-dependent, and the D174A mutation is known to alter the voltage dependence of gating (Fig. 2 and previous studies). The effect of D174A on Hv1 unitary conductance, however, is speculated but not unambiguous (see above).

    [[

    Our experiments unambiguously demonstrate the effect of D174A on Hv1 unitary conductance. The interpretation of the experiments is straightforward – there is no speculation involved. The contrasting opinion of the reviewer rests on his misinterpretations of (i) our measurements of proton transport rate λD for wild-type and mutant (see above) and the CCCP-effect (see above).]]

    In the absence of definitive experimental data showing differences in the unitary conductance of WT vs. D174A, the authors' assumption that water permeability would be strongly temperature‐dependent (lines 154‐160) seems premature and their ensuing conclusion tenuous: "pore residues interrupt the HV1 spanning water wire, trapping the water molecules inside the HV1 channel. In contrast to water, protons cross the pore by hopping from one acidic residue to another through one or more bridging water molecules (Fig. 6)" (lines 161‐164).

    [[

    The reviewer chooses to misinterpret our lines. We did not assert that water permeability through the Hv1 channel would be strongly temperature‐dependent. We referred to the well-known fact that there is a strong temperature dependence of lipid bilayer water permeability - in contrast to the tiny effect of temperature on the water permeation across aqueous channels.

    Page 11, bottom: “Considering the stark dependence of the activation energy for background water flow across lipid bilayers (24), we repeated the experiments at a decreased temperature of 4°C. Thanks to the low background water permeability at 4°C, even tiny contributions of HV1 to Pf should be detectable. Yet, the channels did not contribute to the water flow through the vesicular membrane even though channel water permeability but weakly depends on temperature (24).”]]

    Furthermore, the authors calculate the number of hydrogen bonds (NH) that pore waters could form with pore lining residues based on an X‐ray structure of a chimeric proton channel protein (pdb: 3WKV) that is: a) manifests discontinuous transmembrane water density and is known to represent a non‐conductive conformation, b) contains residues from Ci‐VSP in the critical S2‐S3 linker that form part of the proton transfer pathway, and c) exhibits structural features (i.e., highly conserved ionizable residues such as D185 and R205, which like D174 are reported to dramatically alter Hv1 gating, are packed into a solvent‐free crevice) that are inconsistent with physiological function. Given that all Hv1 ionizable mutant combinations tested so far (the sole exception of D112V ‐ other nonionizable substitutions at D112 are tolerated) remain functional (Musset, Smith et al., 2011, Ramsey, Mokrab et al., 2010), the identities of water‐interacting residues speculative.

    [[

    We substituted the X‐ray structure of the chimeric proton channel protein for the AlphaFold structure. We now provide views of the open and closed conformations in the Supplement based on the homology structure (13). Microsecond-long molecular dynamics simulations have optimized the latter.

    The experimental observation of mutants’ functionality (with the sole exception of D112V) supports our view that proton transfer occurs through a hydrogen‐bonded network of waters that is only once (at D112) interrupted by an amino acid side chain. The nature of the amino acids interacting with the proton transferring water molecules is of little importance.]]

    Interpreting differences in the calculated NH based on pdb: 3WKV therefore seems unlikely to reveal fundamentally important insights into Hv1 function. The author's conclusion that "The observation rules out the formation of an uninterrupted water chain spanning the open channel from the aqueous solution at one side of the membrane to the other. NH would have governed water mobility if such a water wire had formed (24)", (lines 143‐145) therefore does not appear to be strongly supported.

    [[

    We did not base our conclusion of an obstructed water pathway on the analysis of structural models. In contrast, the conclusion is the result of our experiments. The structural models permitted the prediction of the expected water permeability. Depending on the model and the channel conformation, we find NH values between six and 16. All of these NH values translate into water permeabilities exceeding gramicidin’s water permeability. Thus, we would have been able to detect the water flux through an unobstructed proton channel.]]

    Reviewer #2:

    Summary: Voltage‐gated proton channels are peculiar members of the voltage‐gated ion channel family due to their absence of canonical pore. Instead, protons permeate through their voltage‐sensing domain. The mechanisms of proton permeation in Hv1 channels are still unclear, with currently two competing hypotheses: (i) hopping through titrable residues within the protein; or (ii) via Grotthuss mechanism involving proton jumping through a continuous water wire. So far, these hypotheses were only tackled by computation. The authors therefore aimed to experimentally test the two hypotheses. To do so, the authors measured the transport rates of protons and water through wild‐type and mutant D174A Hv1 reconstituted in lipid vesicles. Overall, the presented data are convincing and support their conclusion that proton conduction through the channel is not solely mediated by water transport. However, there are several aspects of the paper that I did not understand and would require clarification.

    [[

    We thank the reviewer for the positive evaluation.]]

    Major comments: My major concern is about the relevance of using the D174A mutant. The authors explain at the beginning of the paper that Hv1‐D174A is open at 0 mV, which allows measuring proton flux in systems in which voltage cannot be controlled. However, it seems from the proton flux experiments that wild‐type Hv1 can conduct protons perfectly well in the used experimental paradigm. So why test a mutant? It is actually not clear why wild‐type Hv1 can conduct protons in the proton conduction assay.

    [[

    We introduced the D174A mutation to measure water flux in a setting where the membrane potential is zero. We only performed the proton flux measurements to show that our reconstituted HV1 channels are functional. HV1 can conduct protons because we establish a transmembrane potential in the proton conduction assay. That is, only initially, extravesicular and intravesicular pH values are equal. Valinomycin addition results in a K+ efflux that, in turn, generates a membrane potential. This potential drives the HV1-mediated H+ influx.]]

    The authors should clearly state the trans‐membrane potential created by the K+ gradient across the vesicle, as well as the pH inside and outside the vesicle, and related these conditions to their electrophysiology data to give us an idea of the open probability of wild‐type Hv1 in the conditions used in the proton conduction assays. This is critical to be able to compare the relative rates of proton transport between the wild‐type and the mutant.

    [[Page 6, bottom:

    " ...we encapsulated c_k^i=150 mM KCl in the HV1 containing large unilamellar vesicles (LUVs) and exposed these vesicles to a buffer with a K+ concentration c_k^o= 3 mM. The addition of valinomycin facilitated K+ efflux, thereby inducing a membrane potential, ψ. ψ constituted the driving force for H+ uptake. It can be calculated according to the Goldman equation:

    ψ = -RT/F ln ((c_k^i+(P_H/P_K ) c_H^i)/(c_k^o+(P_H/P_K ) c_H^o ))

    (1)

    The ratio of the HV1 mediated proton permeability P_H to the valinomycin-mediated potassium permeability P_K is always smaller than 0.04. We base our conclusion on the observation that the CCCP-mediated proton permeability represents an upper limit for P_H since CCCP always induces a faster vesicular proton uptake than HV1 (Fig. 3). Accordingly, the maximum value of P_H/P_K can be estimated as the ratio of valinomycin to CCCP conductivities. The respective values are equal to 1.6 10-3 Ω-1 cm2 [1] and 4 10-6 Ω-1 cm-2 [2]. At pH 7.5, we find c_H^o=10^(-7.5) M, i.e., c_k^o ≫ (P_H/P_K )c_H^o. Similarily, c_k^I ≫ (P_H/P_K ) c_H^i for a broad range of intravesicular pH. With these simplifications, Eq. 1 transforms into the Nernst equation yielding:

    ψ = -RT/F ln (c_k^i)/(c_k^o )=-100 mV

    (2)

    ψ of such size may decrease intravesicular pH by nearly two units.

    Such acidification does not violate so that remains constant throughout the experiment. That is, the vesicle experiments proceed under voltage clamp conditions. The simple explanation is that, due to the small proton concentration and the limited buffer capacity, the K+ conductance exceeds H+ conductance under all conditions. The conclusion is in line with simulations (32), confirming that the membrane potential is driven very near the Nernst potential for K+.”]]

    Similarly, the buffers and pH used for the water transport assay are not explicitly mentioned. Are they the same as for the proton transport assay or are the buffers inside and outside the vesicle symmetrical?

    [[

    We added the information about buffers and pH used to the legend. Except for 150 mM sucrose, the internal and external solutions were identical: 150 mM KCl, 5 mM HEPES (pH 7.5), and 0.5 mM EGTA.]]

    Finally, in the introduction the authors base their assumptions about water transport on an X‐ray structure of Hv1 in a closed conformation (3WKV). I do not think it is relevant to study permeation, which in theory should only happen in an open state. If the authors want to make assumptions about the number of hydrogen bonds in the pore and how many water molecules are in the pore (and I don't think they need to do it), they should rather base their assumptions on the computational models of Hv1 open state.

    [[

    We thank the reviewer for the advice. We added a figure to the Supplement. It shows Hv1 models from long-timescale molecular dynamics simulations (Geragotelis et al, Proc Natl Acad Sci U S A 2020 Vol. 117 Issue 24 Pages 13490-13498). The open structure reveals NH=6. We used this value for our calculations.]]

    Minor comments:

    1. Figure 6: the authors should precise that the model of proton conduction through Hv1 is just an assumption. The structural features of Hv1 open state are indeed unknown.

    [[We modified the figure based on the simulation results of Geragotelis et al. We indicated in the legend that the scheme is based on HV1 homology models.]]

    1. Page 9, lines 170‐171 "Drastically prolonged tail current kinetics might reflect a decreased voltage‐dependence of the deactivation in the D174 mutant". Or rather the prolonged kinetics reflect the stabilization of the open state by the mutation (as stated by the authors just after).

    [[Page 14:

    “Drastically prolonged tail current kinetics might reflect (i) a decreased voltage dependence of the deactivation in the D174A mutant or (ii) a stabilized open state (14).”]]

    1. Supplementary figures are displayed in an odd fashion. Figure S3 should be placed before Figures S1 and S2.

    [[We added two more Supplementary Figures and displayed them in the order of text mentionings.]]

    1. In Figure 2, displaying the current trace corresponding to the 0 mV voltage step would improve readability of the figure, by showing that Hv1‐D174A mutants conduct protons at 0 mV and not wt Hv1.

    [[

    We show the current trace corresponding to the 0 mV voltage step for the D174A mutant in panel A and the trace for the wild-type in panel B of Fig. 2.]]

    1. Figure 2 legend "Pronounced inward H+ currents activate negatively to the reversal potential (here ‐70 mV)". I think the authors mean "Here 0 mV", ‐70 mV is the threshold potential. Panel (c), I guess the EH vs Vrev plot is for D174A mutants but it is not mentioned in the legend

    [[

    We corrected the legend. “Pronounced inward H+ currents activate negatively (here – 70 mV) to reversal potential (here – 8 mV), indicating a high open probability of the D174A mutant at 0 mV.” And “Comparison of calculated Nernst potential for protons (EH) and measured reversal potential (Vrev) for the D174A mutant.”]]

    1. Page 4, line 89: the fact that D174A conducts protons at a lower rate is, at this point, based on a lot on assumption. I would just correct the last sentence by saying "Thus, D174A, while opening with less depolarization, seems to conduct protons at a lower rate"

    [[We toned down our statement and inserted a phrase very close to the one suggested.

    Page 5: “Our observation suggests a reduced flux through the mutant if we assume that the protein expression level is independent of the mutation.”]]

    1. Page 6, line 107. The word "therefore" is not necessary

    [[ok]]

    1. Page 7, line 128: "of" in "measures of transport" is missing

    [[We deleted the paragraph.]]

    1. Page 12, lines 261‐262: "Figure M" ??

    [[“Inset of Figure 3A”]]

    CROSS‐CONSULTATION COMMENTS I agree with the two other reviewer's comments. I think our reviews more or less raise the same weaknesses in the study.

    Significance

    This paper addresses a single question with a clearly defined experimental paradigm. Once the issues addressed, the paper should bring important significance to the field of voltage‐gated ion channels since the nature of proton conduction in Hv1 was not known. It could help explain ion conduction in some channelopathies involving ion conduction through the voltage‐sensing domain. The audience is mainly the voltage‐gated ion channel community, as well as the community of membrane permeation mechanisms My field of expertise is in ion channel structure‐function and pharmacology. I have little expertise in the described proton and water flow assays. Therefore I do not have sufficient expertise to evaluate the detailed experimental protocol that led to the measurements.

    Reviewer #3:

    Summary: This study addresses a fundamental question about the mechanism of proton conduction in the voltage gated proton channel Hv1 i.e., whether protons hop through an uninterrupted water wire, or move by other means involving titratable channel residues. The authors argue that an uninterrupted water wire entails a certain rate of water movement through the open channel, which they estimate to be around 10‐12 cm3s‐1 based on a structural model of Hv1 and previous work on other channels. They then measure water permeability of LUVs containing a purified Hv1 mutant expected to be open at 0 mV via light scattering, and proton flux using a pH sensitive fluorescent dye. They calculate a water permeability much lower than predicted and conclude that the water in the conduction pathway does not form an uninterrupted water wire. The manuscript is written clearly, and the experimental measurements are convincing.

    [[We thank the reviewer for the positive evaluation.]]

    There are nonetheless some ambiguities in the way the formation of water wires is discussed.

    Major comments: A protein like Hv1 is larger and more complex than small peptides like gramicidin. In this context, transient water wires, frequently interrupted by titratable residues, or by steric hindrance from hydrophobic sidechains etc. are likely. Can the authors provide an estimate for the maximum frequency and lifetime of uninterrupted proton wires compatible with their measurements? This would be helpful to evaluate whether short‐lived uninterrupted water wires could contribute significantly to proton conduction or not. Trapping usually implies restricted movement. So, for how long do water molecules need to stay inside the channel in order to be considered trapped? Are the water molecules really trapped or simply forming broken wires?

    [[Page 13, bottom:

    “The question arises whether the obstacle in the water pathway is permanent. HV1’s titratable residues or steric hindrance from fluctuating sidechains may frequently interrupt otherwise intact water wires. Yet, our calculations (Eqs. 7 – 11) show that proton diffusion from the bulk solution to the pore mouth is the transport limiting step. Undoubtedly, transient closure would have caused a detectable pore resistance because part of the protons arriving at the pore mouth could not enter the pore. If the pore was closed longer than one ps, an arriving H+ may diffuse out of the capture zone and vanish into the bulk:

    t_c=(r_0^2)/6D = 10^(-16)/(6 × 8.65 × 10^(-5) ) s = 2 × 10^(-13) s

    (16)

    where tc denotes the time a proton requires to diffuse a distance equal to the capture radius r0. Since transient closures would give rise to experimentally undetected pore resistance, they must be ruled out. The observation agrees well with noise experiments, where Lorentzian time constants, albeit smaller than the time constants for H+ current activation but larger than 0.1 s were observed (41).

    We provided the calculations showing the diffusion limitations on page 9:

    “…we show that the transport limiting step is H+ diffusion to the pore (access resistance) and not transport through the pore. Therefore, we first calculate the maximum current Imax permitted by diffusion for a constantly open pore (35):

    I_max=2π F r_o D_H c_H

    (7)

    where F, r0, DH, and cH are Faraday's constant, the capture radius, the H+ diffusion constant, and the H+ concentration, respectively. The only unknown parameter is r0. Taking the gA estimate r0 = 0.87 Å (36), disregarding buffer effects and assuming DH = 8.65×105 cm2s-1, we find:

    I_max=2π (9.6 ×10^4 As)/mol × 0.87 × 10^(-8) cm × 8.65 x 10^(-5) (cm^2 s^(-1) × 4 × 10^(-7.5) mol)/(1000 cm^3 )

    (8)

    I_max=5.6 × 10^(-17) A

    (9)

    Eq. 8 considers that the approximately 25 % charged lipids in the bilayer induce an increase in surface proton concentration, i.e. it accounts for a surface potential of roughly -40 mV in 150 mM salt. The maximal unitary rate would then be equal to:

    q_max = 5.6 × 10^(-17) C/s/1.6 × 10^(-19) C =348 s^(-1)

    (10)

    Here we used the r0 value determined for gA (36). Acidic moieties at the entrance of HV1 and proton surface migration along the lipid bilayer could serve to increase that value (37, 38). The observation suggests transport limitations by poor proton availability. Calculation of the channel resistance, Rch (35), confirms the hypothesis:

    R_ch = R_pore+R_access =[l_ch+(π a_ch)/2] ρ/(π a_ch^2 )

    (11)

    where R_pore is the resistance of the pore proper and R_access is the access resistance. Assuming a channel radius, a_ch, of 0.15 nm, a length, l_ch of 4 nm and solution resistivity (H+ as the sole conducted ion at bulk pH of 7.5 and a surface potential of -40 mV), ρ, of 2×105 Ω cm, we find R_ch = 4×1013 Ω. Thus, the resulting current, Iρ, that we may expect for the vesicular membrane potential of 100 mV is equal to 3×10-15 A. Accordingly, Iρ exceeds Imax by more than one order of magnitude. Consequently, we may safely conclude that HV1 conductance is limited by proton availability under our conditions. ”]]

    The main conclusion of the paper rests on the negative results from the water permeability assay of Fig. 5. It is recommended to include a positive control (e.g., with gramicidin A), run under the same conditions and similar number of channels per LUV, to show how the results should look like in case of significant water permeability.

    [[We included the gramicidin measurements (Fig. 6) as requested.]]

    Figure 6 show a simplified scheme of proton transport with trapped water molecules in Hv1. Panel A represents a resting state (nonconductive); panel B represents an open state (conductive), favored by the D174A mutation. So, what makes B conductive and A nonconductive? Is it the presence of two salt bridges in B vs. three salt bridges in A? This should be clarified.

    [[

    We modified the figure based on the simulation results of Geragotelis et al. We indicate with arrows the parts of the channel where the proton is free to move and crosses the sites with insurmountable energy barriers.

    Legend to the figure (now Fig. 8): “In the region of the selectivity filter adjacent to D112, the channel is too narrow to let water molecules pass (see also Fig. S1). Yet, the proton may bypass the electrostatic barrier of the open channel at D112 (18), i.e., jump between the two neighboring water molecules. Removal of D174 shifts the voltage sensitivity so that most channels are already open at a transmembrane potential of 0 mV. B) The closed channel. It neither allows water nor proton transport. In its new location, D112 provides an insurmountable electrostatic barrier to proton passage.”]]

    Minor comments: The interpretation of Fig. 2E strongly depends on the assumption that the D174A mutation does not alter membrane trafficking. It is recommended to check the validity of this assumption, e.g., by colocalization with a plasma membrane marker. Images of SDS‐PAGE results for the studied Hv1 proteins should be provided to show preparation purity.

    [[

    We toned down the interpretation of Fig. 2E. As it stands now, Fig. 2 shows that the mutant (i) is functional and (ii) has a high open probability at 0 mV. These conclusions are independent on membrane trafficking. We included images of SDS page results for the studied HV1 proteins in the Supplement.]]

    CROSS‐CONSULTATION COMMENTS I agree with the comments from the other two reviewers. My major point is that refuting major water permeability in Hv1 is not the same thing as refuting that protons can be conducted by transient water wires, unless it is proved that the transient water wires cannot sustain enough proton movement to account for the single channel conductance. Reviewer #3 (Significance (Required)): The Hv1 channel plays important roles in the human body, including the immune, respiratory, and reproductive systems. Despite recent advances in understanding the mechanism of proton conduction by Hv1, whether or not protons hop within a continuous water wire in the open channel is a subject of debate (DeCoursey J. Physiol. 2017, Bennett & Ramsey J. Physiol. 2017). This work provides important insights on the debate by refuting the existence of a water wire that can sustain large water permeability. The findings reported here will be of interest to ion channel biophysicist like this reviewer, but also to biologists studying cellular pH homeostasis and the pathophysiology of Hv1.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This study addresses a fundamental question about the mechanism of proton conduction in the voltage gated proton channel Hv1 i.e., whether protons hop through an uninterrupted water wire, or move by other means involving titratable channel residues. The authors argue that an uninterrupted water wire entails a certain rate of water movement through the open channel, which they estimate to be around 10-12 cm3s-1 based on a structural model of Hv1 and previous work on other channels. They then measure water permeability of LUVs containing a purified Hv1 mutant expected to be open at 0 mV via light scattering, and proton flux using a pH sensitive fluorescent dye. They calculate a water permeability much lower than predicted and conclude that the water in the conduction pathway does not form an uninterrupted water wire. The manuscript is written clearly, and the experimental measurements are convincing. There are nonetheless some ambiguities in the way the formation of water wires is discussed.

    Major comments:

    A protein like Hv1 is larger and more complex than small peptides like gramicidin. In this context, transient water wires, frequently interrupted by titratable residues, or by steric hindrance from hydrophobic sidechains etc. are likely. Can the authors provide an estimate for the maximum frequency and lifetime of uninterrupted proton wires compatible with their measurements? This would be helpful to evaluate whether short-lived uninterrupted water wires could contribute significantly to proton conduction or not.

    Trapping usually implies restricted movement. So, for how long do water molecules need to stay inside the channel in order to be considered trapped? Are the water molecules really trapped or simply forming broken wires?

    The main conclusion of the paper rests on the negative results from the water permeability assay of Fig. 5. It is recommended to include a positive control (e.g., with gramicidin A), run under the same conditions and similar number of channels per LUV, to show how the results should look like in case of significant water permeability.

    Figure 6 show a simplified scheme of proton transport with trapped water molecules in Hv1. Panel A represents a resting state (nonconductive); panel B represents an open state (conductive), favored by the D174A mutation. So, what makes B conductive and A nonconductive? Is it the presence of two salt bridges in B vs. three salt bridges in A? This should be clarified.

    Minor comments:

    The interpretation of Fig. 2E strongly depends on the assumption that the D174A mutation does not alter membrane trafficking. It is recommended to check the validity of this assumption, e.g., by colocalization with a plasma membrane marker.

    Images of SDS-PAGE results for the studied Hv1 proteins should be provided to show preparation purity.

    Referees cross-commenting

    I agree with the comments from the other two reviewers. My major point is that refuting major water permeability in Hv1 is not the same thing as refuting that protons can be conducted by transient water wires, unless it is proved that the transient water wires cannot sustain enough proton movement to account for the single channel conductance.

    Significance

    The Hv1 channel plays important roles in the human body, including the immune, respiratory, and reproductive systems. Despite recent advances in understanding the mechanism of proton conduction by Hv1, whether or not protons hop within a continuous water wire in the open channel is a subject of debate (DeCoursey J. Physiol. 2017, Bennett & Ramsey J. Physiol. 2017). This work provides important insights on the debate by refuting the existence of a water wire that can sustain large water permeability. The findings reported here will be of interest to ion channel biophysicist like this reviewer, but also to biologists studying cellular pH homeostasis and the pathophysiology of Hv1.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Voltage-gated proton channels are peculiar members of the voltage-gated ion channel family due to their absence of canonical pore. Instead, protons permeate through their voltage-sensing domain. The mechanisms of proton permeation in Hv1 channels are still unclear, with currently two competing hypotheses: (i) hopping through titrable residues within the protein; or (ii) via Grotthuss mechanism involving proton jumping through a continuous water wire. So far, these hypotheses were only tackled by computation. The authors therefore aimed to experimentally test the two hypotheses. To do so, the authors measured the transport rates of protons and water through wild-type and mutant D174A Hv1 reconstituted in lipid vesicles. Overall, the presented data are convincing and support their conclusion that proton conduction through the channel is not solely mediated by water transport. However, there are several aspects of the paper that I did not understand and would require clarification.

    Major comments:

    My major concern is about the relevance of using the D174A mutant. The authors explain at the beginning of the paper that Hv1-D174A is open at 0 mV, which allows measuring proton flux in systems in which voltage cannot be controlled. However, it seems from the proton flux experiments that wild-typet Hv1 can conduct protons perfectly well in the used experimental paradigm. So why test a mutant? It is actually not clear why wild-type Hv1 can conduct protons in the proton conduction assay. The authors should clearly state the trans-membrane potential created by the K+ gradient across the vesicle, as well as the pH inside and outside the vesicle, and related these conditions to their electrophysiology data to give us an idea of the open probability of wild-type Hv1 in the conditions used in the proton conduction assays. This is critical to be able to compare the relative rates of proton transport between the wild-type and the mutant. Similarly, the buffers and pH used for the water transport assay are not explicitly mentioned. Are they the same as for the proton transport assay or are the buffers inside and outside the vesicle symmetrical? Finally, in the introduction the authors base their assumptions about water transport on an X-ray structure of Hv1 in a closed conformation (3WKV). I do not think it is relevant to study permeation, which in theory should only happen in an open state. If the authors want to make assumptions about the number of hydrogen bonds in the pore and how many water molecules are in the pore (and I don't think they need to do it), they should rather base their assumptions on the computational models of Hv1 open state.

    Minor comments:

    1. Figure 6: the authors should precise that the model of proton conduction through Hv1 is just an assumption. The structural features of Hv1 open state are indeed unknown.
    2. Page 9, lines 170-171 "Drastically prolonged tail current kinetics might reflect a decreased voltage-dependence of the deactivation in the D174 mutant". Or rather the prolonged kinetics reflect the stabilization of the open state by the mutation (as stated by the authors just after).
    3. Supplementary figures are displayed in an odd fashion. Figure S3 should be placed before Figures S1 and S2.
    4. In Figure 2, displaying the current trace corresponding to the 0 mV voltage step would improve readability of the figure, by showing that Hv1-D174A mutants conduct protons at 0 mV and not wt Hv1.
    5. Figure 2 legend "Pronounced inward H+ currents activate negatively to the reversal potential (here -70 mV)". I think the authors mean "Here 0 mV", -70 mV is the threshold potential. Panel (c), I guess the EH vs Vrev plot is for D174A mutants but it is not mentioned in the legend
    6. Page 4, line 89: the fact that D174A conducts protons at a lower rate is, at this point, based on a lot on assumption. I would just correct the last sentence by saying "Thus, D174A, while opening with less depolarization, seems to conduct protons at a lower rate"
    7. Page 6, line 107. The word "therefore" is not necessary
    8. Page 7, line 128: "of" in "measures of transport" is missing
    9. Page 12, lines 261-262: "Figure M" ??

    Referees cross-commenting

    I agree with the two other reviewer's comments. I think our reviews more or less raise the same weaknesses in the study.

    Significance

    This paper addresses a single question with a clearly defined experimental paradigm. Once the issues addressed, the paper should bring important significance to the field of voltage-gated ion channels since the nature of proton conduction in Hv1 was not known. It could help explain ion conduction in some channelopathies involving ion conduction through the voltage-sensing domain.

    The audience is mainly the voltage-gated ion channel community, as well as the community of membrane permeation mechanisms.

    My field of expertise is in ion channel structure-function and pharmacology. I have little expertise in the described proton and water flow assays. Therefore I do not have sufficient expertise to evaluate the detailed experimental protocol that led to the measurements.

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    Referee #1

    Evidence, reproducibility and clarity

    1. The authors state that: "the conductance density mediated by the expression of the mutant was 2.5 times smaller than the wild type, although we transfected the same amount of plasmid DNA (Fig. 2E). Assuming that protein expression is independent of the mutation, the observation suggested that the unitary proton flux ratio RC of wild type to mutant channel was equal to 2.5" (lines 82-85).

    Macroscopic conductance (G) depends on channel number (N), microscopic or unitary conductance (), and open probability (PO) by G=NPO. The authors assume that the level of WT and D174A mutant protein expression on plasma membrane, which determines N, are equal; however, this critical assumption does not appear to have been tested. The fact that conductance density (nS/pF) is plotted in Fig. 2E does not alter this caveat because this procedure normalizes the data only for cell surface area (i.e., size).

    The authors' conclude that "The conductance density relationship (Fig. 2E) compares the maximal conduction of both constructs; this is the fully open channel (open probability ≈ 1)"(lines 87-88). However, neither raw currents nor G-V data are shown. Typically, currents measured at large, near-saturating PO are used to compare the relative conductances of WT and mutant ion channels. The currents shown in Fig. 2A and 2B exhibit prominent 'droop' at even modest depolarizing potentials (+10 mV for D174A and +30 mV for WT), indicating that the proton gradient has been substantially perturbed by the flow of ge depolarizing voltages needed to drive channels to near-maximal PO. Furthermore, there is no evidence that maximal PO itself is also not different in WT and D174A channels. Indeed, maximal PO for native Hv1 channels measured using variance analysis is reported by significantly smaller than 1.0, and assuming that PO = 1.0 for either WT or D174A is therefore not well supported. Maximal could be altered by the D174A mutation, which has a clear and strong effect on channel gating evidenced by the large (-70 mV) negative shift in threshold potential reported both here and previously in the literature. Effects of mutations on maximal PO due to altered gating behavior could be separate and distinct from any change in plasma membrane channel number (N). Lastly, because D174A channels have a much higher PO than WT at 0 mV, the mutant will necessarily conduct inward proton currents at the physiological resting membrane potential (RMP) in tsa-201 cells (perhaps -30 mV?). Inwardly directed proton currents will therefore cause intracellular acidification under resting conditions. The constitutive acid load in cells expressing D174A, but not WT, is likely to have a variety of physiological consequences, including decreased protein expression or plasma membrane targeting of D174A. There is evidence that another-constitutively open Hv1 mutant (R205H) also generates smaller currents macroscopic conductance than WT, and this phenomenon is likely to result from decreased cell surface expression. To conclude that the microscopic conductances of WT and D174A are unequal, the authors must demonstrate that N is not different The authors' conclusion that D174A "conducts protons at a lower rate" (line 89) is therefore not well supported by the experimental data.

    1. The authors indirectly measure apparent proton flux rates (D) in LUVs containing WT and D174A mutant Hv1 channels using a fluorescence-based approach, and conclude that D is 2.4 times smaller for D174A than WT. However, the method for estimating D is not performed under voltage clamp, and the driving force for proton current is neither known nor measured. The authors state that "Transmembrane voltage constituted the driving force for proton uptake into LUVs (Figure M). It resulted from facilitated K+ efflux out of the vesicles (30)", (lines 261-262), but this voltage is unknown and not likely to equal the Nernst equilibrium potential for K+ once Hv1 channels begin to open.

    Once Hv1 channels begin to open, intra-lumenal pH (pHi) will necessarily occur during the experiment. Such changes are likely exacerbated by a) the low proton buffering capacity of the system (5 mM HEPES) and b) the absence of any counter-charge pathway to balance the effect of proton charge movement on the membrane potential. Given the small volume of LUVs, even a relatively modest difference in either membrane potential or pHi could substantially alter the driving force for proton movement. Together, these factors are highly likely to result in a rapid and potentially large change in the driving force for proton flux.

    Driving force changes may also be different for WT and D174A because their relative PO may be different under the experimental conditions used here. Because D174A activates at much more negative voltages, it is likely to open more quickly and to a higher PO than WT at early times after depolarization is initiated by addition of valinomycin (Fig. 3A). This fact will likely result in a larger initial inward current being carried by D174A than WT channels. The result would be a more rapid acidification of LUVs by D174A.

    The experimental data in Fig. 3A are consistent with the expectation that the proton gradient and driving force more rapidly approach equilibrium for D174A than WT channels: the apparent rate of AMCA fluorescence change is slower in D174A. Although the authors correctly interpret the experimental data to mean that the apparent D is slower for D174A, they do not rule out the artifactual explanation for the measured differences. Indeed, the observation in Fig. 3A that AMCA fluorescence change eventually reaches a plateau and is not affected by CCCP means that the proton gradient has become exhausted during the experiment, and directly demonstrates that the proton driving force is uncontrolled under the current experimental conditions.

    In contrast to the authors' statement that "Our experiments with the purified and reconstituted channels corroborated the conclusion (Fig. 3A)", (lines 92-93) it is not clear that unitary proton flux rates/unitary conductances are actually different in WT and D174A.

    1. The presumed differences in unitary conductances (i.e., 'transport rate') between WT and D174A are used to estimate Arrhenius activation energies (Ea): ("The difference in measures transport rates allows a rough estimation of the Arrhenius 128 activation energy Ea for HV1-mediated proton flow. It amounts to 40 kJ/mol for the wild type and 23 kJ for the mutant. Thus, Ea exceeds the corresponding 15 kJ/mol barrier measured for gramicidin A (32, 33)", (lines 128-130).

    The method for determining Ea in the current work is not well-described. In Ref. 32, the authors estimate Arrhenius activation energy (Ea = 20 kJ/mol) for gramicidin D (not gramicidin A) from the slope of a line fit to measurements of currents at various temperatures. Here, the authors measure AMCA fluorescence decay rates at 4{degree sign}C and 23{degree sign}C and observe a similar temperature-dependent difference in WT and D174A (Fig. S2). Given that the data indicate that WT and D174A are similarly temperature-dependent, it is unclear how the authors arrive at different Ea values. The authors' conclusion that "The increment in Ea suggests that the transport mechanism may be different from a pure Grotthuss type, where the proton uses an uninterrupted water wire to cross the membrane", (lines 131-133) therefore does not appear to be well-supported.

    1. The authors report no difference in water permeability in WT vs. D174A (Fig. 5 and S1) and interpret the results to mean that proton currents are not associated with measurable bulk water flow. A similar conclusion was reached for native Hv1 channels using deuterium substitution (DeCoursey & Cherny, 1997). However, the absence of bulk water flow does not itself rule out the possibility that 'trapped' waters within the Hv1 pore do not themselves carry the measured proton current. If intra-pore water molecules are tethered by hydrogen bonds with protein atoms, they may not move when Hv1 channels open. Proton transfer through a hydrogen-bonded network of waters requires only that the electronic structure of the network be rearranged during proton transfer; water is not required. As in the previous study (DeCoursey & Cherny, 1997), the lack of water flux reported here demonstrates seems to reinforce the notion that H+ moves separately from its waters of hydration (i.e., hydronium, H3O+, is not the permeant species) and does not necessarily imply information about the mechanism of proton transfer (i.e., side chain ionization vs. Grotthuss-type transfer in a water-wire).

    The authors state that: 1) "every H-bond donating or receiving pore-lining residue would have contributed an increment ΔΔ𝐺‡ of 0.1 kcal/mol to the Gibbs free energy of activation Δ𝐺‡ (25)" (lines 145-147), and 2) calculating NH from this Δ𝐺‡ allows estimation of the channel's unitary water permeability (Eqn. 2). Although hydrogen bonding patterns will undoubtedly alter the free energy for channel activation, this is not the same free energy change as that for proton transfer. Hv1 gating involves conformational changes that are both voltage and pH-dependent, and the D174A mutation is known to alter the voltage dependence of gating (Fig. 2 and previous studies). The effect of D174A on Hv1 unitary conductance, however, is speculated but not unambiguous (see above). In the absence of definitive experimental data showing differences in the unitary conductance of WT vs. D174A, the authors' assumption that water permeability would be strongly temperature-dependent (lines 154-160) seems premature and their ensuing conclusion tenuous: "pore residues interrupt the HV1 spanning water wire, trapping the water molecules inside the HV1 channel. In contrast to water, protons cross the pore by hopping from one acidic residue to another through one or more bridging water molecules (Fig. 6)" (lines 161-164).

    Furthermore, the authors calculate the number of hydrogen bonds (NH) that pore waters could form with pore-lining residues based on an X-ray structure of a chimeric proton channel protein (pdb: 3WKV) that is: a) manifests discontinuous transmembrane water density and is known to represent a non-conductive conformation, b) contains residues from Ci-VSP in the critical S2-S3 linker that form part of the proton transfer pathway, and c) exhibits structural features (i.e., highly conserved ionizable residues such as D185 and R205, which like D174 are reported to dramatically alter Hv1 gating, are packed into a solvent-free crevice) that are inconsistent with physiological function. Given that all Hv1 ionizable mutant combinations tested so far (the sole exception of D112V - other non-ionizable substitutions at D112 are tolerated) remain functional (Musset, Smith et al., 2011, Ramsey, Mokrab et al., 2010), the identities of water-interacting residues speculative. Interpreting differences in the calculated NH based on pdb: 3WKV therefore seems unlikely to reveal fundamentally important insights into Hv1 function. The author's conclusion that "The observation rules out the formation of an uninterrupted water chain spanning the open channel from the aqueous solution at one side of the membrane to the other. NH would have governed water mobility if such a water wire had formed (24)", (lines 143-145) therefore does not appear to be strongly supported.

    References

    Bennett AL, Ramsey IS (2017a) CrossTalk opposing view: proton transfer in Hv1 utilizes a water wire, and does not require transient protonation of a conserved aspartate in the S1 transmembrane helix. J Physiol

    Bennett AL, Ramsey IS (2017b) Rebuttal from Ashley L. Bennett and Ian Scott Ramsey. J Physiol

    De La Rosa V, Bennett AL, Ramsey IS (2018) Coupling between an electrostatic network and the Zn(2+) binding site modulates Hv1 activation. J Gen Physiol

    De La Rosa V, Ramsey IS (2018) Gating Currents in the Hv1 Proton Channel. Biophys J 114: 2844-2854

    DeCoursey TE (2017) CrossTalk proposal: Proton permeation through HV 1 requires transient protonation of a conserved aspartate in the S1 transmembrane helix. J Physiol 595: 6793-6795

    DeCoursey TE, Cherny VV (1997) Deuterium isotope effects on permeation and gating of proton channels in rat alveolar epithelium. J Gen Physiol 109: 415-34

    Musset B, Smith SM, Rajan S, Morgan D, Cherny VV, Decoursey TE (2011) Aspartate 112 is the selectivity filter of the human voltage-gated proton channel. Nature 480: 273-7

    Ramsey IS, Mokrab Y, Carvacho I, Sands ZA, Sansom MS, Clapham DE (2010) An aqueous H+ permeation pathway in the voltage-gated proton channel Hv1. Nat Struct Mol Biol 17: 869-75

    Ramsey IS, Moran MM, Chong JA, Clapham DE (2006) A voltage-gated proton-selective channel lacking the pore domain. Nature 440: 1213-6

    Randolph AL, Mokrab Y, Bennett AL, Sansom MS, Ramsey IS (2016) Proton currents constrain structural models of voltage sensor activation. Elife 5: e18017

    Significance

    Here the authors attempt to ascertain whether water molecules may mediate proton transfer in the voltage-gated proton channel Hv1 using a combination of whole-cell voltage clamp electrophysiology, protein purification, reconstitution, and pH-dependent AMCA fluorescence measurement and estimates of water permeability, and hydrogen bond calculations based on an X-ray structure of a chimeric Hv1 proton channel model protein. The authors address an important question that is fundamental to the exquisitely proton-selective Hv1 channel and which may be applicable to other proton transporting proteins.

    Although there is high potential for significance to a wide range of experimenters studying biologically fundamental mechanisms of proton transport, the experimental data fail to strongly support most of the authors main conclusions, and it is unclear whether the work represents a technial advance for the field. Previous work in the literature has described two main hypotheses for the proton transport mechanism in Hv1:

    • A) an intra-protein transmembrane water wire that allows permeating H+ to move along a chain of hydrogen-bonded water molecules and does not require explicit ionization of any particular amino acid side chain (Bennett & Ramsey, 2017a, Bennett & Ramsey, 2017b, Ramsey et al., 2010), and
    • B) Explicit ionization of a conserved side chain in the S1 helix (D112 in human Hv1) is required for proton transfer in Hv1 channels (DeCoursey, 2017, Musset et al., 2011). The Reviewer is an expert in the field, having originally identified and functionally characterized Hv1 channels in 2006 (Ramsey, Moran et al., 2006), contributed to the identification of key side chains and structural determinants of Hv1 function (De La Rosa, Bennett et al., 2018, Ramsey et al., 2010, Randolph, Mokrab et al., 2016), measured gating currents in Hv1 (De La Rosa & Ramsey, 2018), and authored the hypothesis that Hv1 utilizes a water-wire type mechanism for proton transfer (Ramsey et al., 2010).
  6. Version published to 10.1101/2022.05.09.491141v2 on bioRxiv
  7. Version published to 10.1101/2022.05.09.491141v1 on bioRxiv