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  1. Author Response:

    Evaluation Summary:

    This manuscript explores the mechanisms of permeation and selectivity in the unusual potassium-selective ion channel TMEM175, which lacks a canonical selectivity filter. The study is led by molecular dynamics simulations and free energy calculations, complemented by a cryo-EM analysis and electrophysiological recordings. The authors propose a novel, single ion-based mechanism of permeation, together with a partial dehydration-driven selectivity mechanism. While in principle exciting and informative, most of the conclusions in the manuscript are based on small differences in calculated values for which an estimation of the uncertainty is lacking, and on the usage of a single physics-based model. This study will appeal to readers interested in the structure and function of ion channels and in molecular mechanisms of ion translocation. It would be strengthened by a thorough exploration of alternative hypotheses.

    We thank the editor and reviewers for their positive assessment of our work. In the revised manuscript we have clarified how the uncertainties of the free energies had been estimated in the original submission. We note that Metadynamics and FEP, two radically simulation different approaches, yield results that are in excellent agreement with one another. It is also worth noting that the overwhelming majority of simulation studies of biomolecular mechanisms are based on one physical model. As we argue above there are good reasons that explain why this is the case, generally speaking; this choice is particularly logical for those studies that employ advanced sampling methods and thus entail a major computational cost.

    Reviewer #1 (Public Review):

    TMEM175 is a recently (Cang et al., 2015, Sell 162) discovered new type of cation channel, strongly diverging from the 2+4TM+pore loop fold of canonical K+/Na+/Ca2+ channels. It has been found to be relevant for the development of Parkinson's disease. Oh et al. recently published a cryoEM structure of the human TMEM175 (Oh et al. 2020, Elife 9). This is a follow-up work in which they perform further structural refinement and molecular dynamics simulations to elucidate the mechanism of selectivity in this channel.

    The calculations and experiments confirm the hypothesis formulated in their own previous and other works that a hydrophobic constriction formed by a ring of leucines (isoleucines in bacterial isoforms) in the center of the hourglass-shaped pore provides the gate for the channel as well as plays a major role in selectivity. They find that selectivity of K+ over Na+ arises from the interplay of the dehydration energies and ion-protein-interaction energies of the two ions: Both the bulk water and the channel pore actually favor Na+. But the water favors Na+ more than the pore does, leading to a better permeability for K+. These data are interesting, because they show how the electrostatics of a whole pore contribute to create an alternative selectivity mechanism for K+ ions.

    The conclusions of this paper are mostly well supported by the data, however a lot of interesting aspects could be worked out better and alternative hypothesis and publications in the field are not considered adequately.

    We thank the reviewer for their positive assessment of our work and their helpful suggestions to improve the manuscript.

    Reviewer #2 (Public Review):

    TMEM175 is a unique potassium channel that lacks a canonical selectivity filter. In this work, Oh et al. elucidated the mechanisms of permeation and ion selectivity in TMEM175. Specifically, they improved the resolutions of two existing cryo-EM maps of TMEM175 - in a closed and a putatively open state. With the reprocessed structures (not published at the time of the review), the authors used an enhanced sampling molecular dynamics (MD) simulations technique (multiple-walker Metadynamics) that allowed to reveal the main features of ion permeation. First, the previously putatively open structure was found to be indeed conductive in simulations. A single-ion mechanism, distinct from the multi-ion characteristic for canonical potassium channels, was observed, enabled by a novel collective variable in Metadynamics simulations. This variable avoided preliminary assumptions about the ion permeation mechanism. Second, a crucial part of TMEM175 was found to be a hydrophobic constriction, formed in the pore by four isoleucine residues (two per monomer). The largest energetic barrier for ion permeation was shown to be located there, as ions needed to experience a large degree of dehydration in order to pass this constriction. However, the dehydration penalty was shown to be offset through favorable electrostatic interactions of potassium ions with the channel. The ability of the open structure of TMEM175 to conduct ions was further confirmed using MD simulations with applied electric field. Another important aspect of this work was the clarification of the selectivity of TMEM175 for potassium ions. Using a similar MD approach but with sodium ions instead of potassium, a higher barrier at the constriction was detected. Together with free energy perturbation simulations, this suggested the driver for the selectivity to be the difference in dehydration energy between sodium and potassium ions in the constriction. The role of the constriction for selectivity was further underlined by simulations and electrophysiological recordings of TMEM175 mutants: an enlarged constriction lead to a lower selectivity for potassium ions. This study provides a mechanism for ion permeation and selectivity in a potassium channel that differs greatly from other ion channels. Given the previously shown association of TMEM175 mutants with Parkinson's disease, this mechanistic insight from this work may lead to a better understanding of this association. The conclusions in the manuscript are certainly exciting and informative, however not fully supported by the data.

    We thank the reviewer for their positive assessment of our work and for their helpful suggestions in improving the manuscript.

    Reviewer #3 (Public Review):

    Oh et al. examine the structure and function of a non-canonical K+ channel, TMEM175, using a combination of techniques (cryo-electron microscopy, computer simulations, and electrophysiology). They show that a surprisingly localized segment of the pore interior controls not only gating but also ionic selectivity of the channel. Improved re-processing of published EM data leads to a refined structural model of the open state of the channel that is subjected to detailed analysis using molecular dynamics simulations. Biased-sampling simulations using metadynamics confirm that a thin and narrow hydrophobic constriction consisting of 4 amino acid side chains, which is too narrow to allow water or ion permeation in the closed state of the channel, constitutes a free energy barrier to permeating K+ ions in the open state of the channel. Free energy perturbations confirm the moderate preference for K+ over Na+, which is attributed to a smaller desolvation penalty for K+ at the constriction. The role of the constriction in ionic selectivity is tested by electrophysiology measurements.

    Strengths: This is a well designed and executed study that adds to the field of K+ channels and ion channels in general. The overall complementarity between the techniques used in the study is excellent and helps support the conclusions of the paper. The inference from the structural model that the channel is open is confirmed by the simulations, and the electrophysiology confirms the role of the constriction predicted by the simulations.

    In addition, the excellent agreement between the free energy profiles or potentials of mean-force (PMF) for K+ and Na+ permeation across the length of the pore determined by metadynamics and the free energy perturbation results for the reversible replacement of K+ by Na+ at the barrier top and in bulk water validates the computational methodology, suggesting that both calculations are converged. The agreement between the relative barrier heights in the PMF and the relative free energy of the two cation types in water and at the barrier top is not trivial and offer independent validation of the relative "solvation free energy" at the constriction by exploiting two distinct pathways in a thermodynamic cycle (DeltaDeltaG calculation).

    We thank the reviewer for their positive assessment of our work and for their insightful comments on how to improve the manuscript.

    Was this evaluation helpful?
  2. Evaluation Summary:

    This manuscript explores the mechanisms of permeation and selectivity in the unusual potassium-selective ion channel TMEM175, which lacks a canonical selectivity filter. The study is led by molecular dynamics simulations and free energy calculations, complemented by a cryoEM analysis and electrophysiological recordings. The authors propose a novel, single ion-based mechanism of permeation, together with a partial dehydration-driven selectivity mechanism. While in principle exciting and informative, most of the conclusions in the manuscript are based on small differences in calculated values for which an estimation of the uncertainty is lacking, and on the usage of a single physics-based model. This study will appeal to readers interested in the structure and function of ion channels and in molecular mechanisms of ion translocation. It would be strengthened by a thorough exploration of alternative hypotheses.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    Was this evaluation helpful?
  3. Reviewer #1 (Public Review):

    TMEM175 is a recently (Cang et al., 2015, Sell 162) discovered new type of cation channel, strongly diverging from the 2+4TM+pore loop fold of canonical K+/Na+/Ca2+ channels. It has been found to be relevant for the development of Parkinson's disease. Oh et al. recently published a cryoEM structure of the human TMEM175 (Oh et al. 2020, Elife 9). This is a follow-up work in which they perform further structural refinement and molecular dynamics simulations to elucidate the mechanism of selectivity in this channel.

    The calculations and experiments confirm the hypothesis formulated in their own previous and other works that a hydrophobic constriction formed by a ring of leucines (isoleucines in bacterial isoforms) in the center of the hourglass-shaped pore provides the gate for the channel as well as plays a major role in selectivity. They find that selectivity of K+ over Na+ arises from the interplay of the dehydration energies and ion-protein-interaction energies of the two ions: Both the bulk water and the channel pore actually favor Na+. But the water favors Na+ more than the pore does, leading to a better permeability for K+. These data are interesting, because they show how the electrostatics of a whole pore contribute to create an alternative selectivity mechanism for K+ ions.

    The conclusions of this paper are mostly well supported by the data, however a lot of interesting aspects could be worked out better and alternative hypothesis and publications in the field are not considered adequately.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    TMEM175 is a unique potassium channel that lacks a canonical selectivity filter. In this work, Oh et al. elucidated the mechanisms of permeation and ion selectivity in TMEM175. Specifically, they improved the resolutions of two existing cryo-EM maps of TMEM175 - in a closed and a putatively open state.
    With the reprocessed structures (not published at the time of the review), the authors used an enhanced sampling molecular dynamics (MD) simulations technique (multiple-walker Metadynamics) that allowed to reveal the main features of ion permeation. First, the previously putatively open structure was found to be indeed conductive in simulations. A single-ion mechanism, distinct from the multi-ion characteristic for canonical potassium channels, was observed, enabled by a novel collective variable in Metadynamics simulations. This variable avoided preliminary assumptions about the ion permeation mechanism. Second, a crucial part of TMEM175 was found to be a hydrophobic constriction, formed in the pore by four isoleucine residues (two per monomer). The largest energetic barrier for ion permeation was shown to be located there, as ions needed to experience a large degree of dehydration in order to pass this constriction. However, the dehydration penalty was shown to be offset through favorable electrostatic interactions of potassium ions with the channel. The ability of the open structure of TMEM175 to conduct ions was further confirmed using MD simulations with applied electric field.
    Another important aspect of this work was the clarification of the selectivity of TMEM175 for potassium ions. Using a similar MD approach but with sodium ions instead of potassium, a higher barrier at the constriction was detected. Together with free energy perturbation simulations, this suggested the driver for the selectivity to be the difference in dehydration energy between sodium and potassium ions in the constriction. The role of the constriction for selectivity was further underlined by simulations and electrophysiological recordings of TMEM175 mutants: an enlarged constriction lead to a lower selectivity for potassium ions.
    This study provides a mechanism for ion permeation and selectivity in a potassium channel that differs greatly from other ion channels. Given the previously shown association of TMEM175 mutants with Parkinson's disease, this mechanistic insight from this work may lead to a better understanding of this association. The conclusions in the manuscript are certainly exciting and informative, however not fully supported by the data.

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    Oh et al. examine the structure and function of a non-canonical K+ channel, TMEM175, using a combination of techniques (cryo-electron microscopy, computer simulations, and electrophysiology). They show that a surprisingly localized segment of the pore interior controls not only gating but also ionic selectivity of the channel. Improved re-processing of published EM data leads to a refined structural model of the open state of the channel that is subjected to detailed analysis using molecular dynamics simulations. Biased-sampling simulations using metadynamics confirm that a thin and narrow hydrophobic constriction consisting of 4 amino acid side chains, which is too narrow to allow water or ion permeation in the closed state of the channel, constitutes a free energy barrier to permeating K+ ions in the open state of the channel. Free energy perturbations confirm the moderate preference for K+ over Na+, which is attributed to a smaller desolvation penalty for K+ at the constriction. The role of the constriction in ionic selectivity is tested by electrophysiology measurements.

    Strengths: This is a well designed and executed study that adds to the field of K+ channels and ion channels in general. The overall complementarity between the techniques used in the study is excellent and helps support the conclusions of the paper. The inference from the structural model that the channel is open is confirmed by the simulations, and the electrophysiology confirms the role of the constriction predicted by the simulations.

    In addition, the excellent agreement between the free energy profiles or potentials of mean-force (PMF) for K+ and Na+ permeation across the length of the pore determined by metadynamics and the free energy perturbation results for the reversible replacement of K+ by Na+ at the barrier top and in bulk water validates the computational methodology, suggesting that both calculations are converged. The agreement between the relative barrier heights in the PMF and the relative free energy of the two cation types in water and at the barrier top is not trivial and offer independent validation of the relative "solvation free energy" at the constriction by exploiting two distinct pathways in a thermodynamic cycle (DeltaDeltaG calculation).

    Was this evaluation helpful?