Intracellular helix-loop-helix domain modulates inactivation kinetics of mammalian TRPV5 and TRPV6 channels

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1. 

   Biophysics Colab

   Oct 2, 2023

   Evaluation statement (1 September 2023)

   Flores-Aldama and colleagues set out to identify molecular determinants of fast inactivation in the TRPV6 ion channel, a mechanism not observed in the closely related TRPV5 channel. The work focuses on a helix-loop-helix (HLH) motif, located at the interface between several important regions for channel gating. Using molecular dynamics simulations and analysis of mutations, the authors identify pairs of amino acid residues in a structural triad formed by the HLH, S2-S3 linker, and transmembrane domains, which show different conformations in the available TRPV5 and TRPV6 cryo-EM structures. An important aspect of the study is that some of the structural hypotheses were derived from an evolutionary analysis of sequences from orthologues of both channels, demonstrating the value of this type of …

   Evaluation statement (1 September 2023)

   Flores-Aldama and colleagues set out to identify molecular determinants of fast inactivation in the TRPV6 ion channel, a mechanism not observed in the closely related TRPV5 channel. The work focuses on a helix-loop-helix (HLH) motif, located at the interface between several important regions for channel gating. Using molecular dynamics simulations and analysis of mutations, the authors identify pairs of amino acid residues in a structural triad formed by the HLH, S2-S3 linker, and transmembrane domains, which show different conformations in the available TRPV5 and TRPV6 cryo-EM structures. An important aspect of the study is that some of the structural hypotheses were derived from an evolutionary analysis of sequences from orthologues of both channels, demonstrating the value of this type of analysis.

   Biophysics Colab considers this to be a convincing study and recommends it to scientists interested in the molecular determinants of ion channel gating.

   (This evaluation by Biophysics Colab refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

   Read the original source
2. 

   Biophysics Colab

   Mar 7, 2023

   Authors’ response (22 February 2023)

   GENERAL ASSESSMENT

   In this preprint, Flores-Aldama and colleagues set out to identify molecular determinants of fast inactivation in the TRPV6 ion channel, a mechanism not observed in the closely related TRPV5 channel. The work focuses on the helix-loop-helix (HLH) motif, which is a region of the channel at the interface between the intracellular and transmembrane domains, the S2-S3 linker and the TRP Domain helix (TDh). Through MD simulations, the authors identify pairs of amino acid residues in the HLH/S2-S3 linker/TDh structural triad that move differently in TRPV5 and TRPV6 based on available cryo-EM structures. They mutate the E288 residue in TRPV6 to D, which is its counterpart in TRPV5, and make the reverse mutation in TRPV5, and show that swapping this single residue is sufficient to …

   Authors’ response (22 February 2023)

   GENERAL ASSESSMENT

   In this preprint, Flores-Aldama and colleagues set out to identify molecular determinants of fast inactivation in the TRPV6 ion channel, a mechanism not observed in the closely related TRPV5 channel. The work focuses on the helix-loop-helix (HLH) motif, which is a region of the channel at the interface between the intracellular and transmembrane domains, the S2-S3 linker and the TRP Domain helix (TDh). Through MD simulations, the authors identify pairs of amino acid residues in the HLH/S2-S3 linker/TDh structural triad that move differently in TRPV5 and TRPV6 based on available cryo-EM structures. They mutate the E288 residue in TRPV6 to D, which is its counterpart in TRPV5, and make the reverse mutation in TRPV5, and show that swapping this single residue is sufficient to transfer the inactivation kinetics between the two channels. They also show that the E294A mutation in the HLH partially reduces fast inactivation in TRPV6 and that the K245A mutation in ARD6 in TRPV5 confers some fast inactivation to that channel, albeit less than that observed in TRPV6. A very rewarding aspect of the manuscript is that some of the structural hypotheses were arrived at through an evolutionary analysis of sequences from many orthologues of both channels. This work is a follow up to the authors’ previous publication (https://doi.org/10.1038/s41598-020-65679-6), where they identified the HLH as a region important for fast inactivation in TRPV6. The manuscript includes new data that provides insight into the different inactivation mechanisms in these channels and strengthens the notion that the HLH linker region plays an important role in channel gating.

   RECOMMENDATIONS

   Revisions essential for endorsement:

   1. Structural comparisons are made between TRPV5 and TRPV6 structures that were determined in different labs, under different conditions, expression systems, etc., so whether the differences are due to Ca2+ or other experimental conditions is unclear. Also, the inactivated structures are all obtained in the presence of calmodulin, so whether the changes are due to calcium or calmodulin is also unclear. Finally, some of the noninactivated structures are partially open, while some are closed. The authors should clearly state these caveats and fully discuss the limitations of the inferences obtained from this analysis.

   The critique is reasonable, and we were well aware of the diversity of structures we were dealing with. The only way we devised to partially surmount the caveat was to group them and study the average distances. By doing this, we observed that the two groups (i.e. presumably inactivated/versus presumable non-inactivated) consistently showed similar distances between the critical amino acids described in the text. We performed additional molecular dynamics simulations for the mutant channels and overall the trend in the change in distances was conserved. We edited the text accordingly and tone down the discussion regarding this point.

   1. Molecular dynamics (MD) simulations were carried out in the presence of a non-physiological (50 mM) Ca2+ concentration, presumably to increase the chance of observing a conformational change. However, throughout the duration of the simulation, no Ca2+ binding events were observed, thus leading the authors to conclude that Ca2+ induced an inactivating conformational change through global effects. It is not clear to the reviewers what the authors intend to convey with this conclusion. The high calcium concentration will increase ionic strength by a very large amount, thus affecting any electrostatic interactions through charge screening. If this is the case, the same effects on inactivation should be expected from recordings (and simulations) in high ionic strength, thus weakening the finding that this is a specific effect of calcium. New experiments to probe these effects should be carried out, but if this is not possible, the authors should tone down their conclusion of a specific effect of calcium on inactivation.

   This is correct we used 50mM in the simulations hoping to accelerate the process. We actually observed calcium binding, however, not in the interphase described here but rather at the ankyrin repeats (also associated to the mechanism described here). The revised version contains this information describing the location of the binding sites. The binding was concentration dependent and we observed even at 10mM calcium. Following the critique, we calculated the effects induced by charge in the simulations and was not significant. Additional experiments were performed with barium in the external solution showing a high specificity for calcium.

   1. The physiological importance of the fast inactivation in TRPV6 is unclear. While there is a clear, evolutionarily conserved difference between TRPV5 and TRPV6, these channels are unlikely to experience fast 50 ms voltage jumps where this fast inactivation difference would be observable. Rather they are expressed in epithelial cells with stable, or slowly changing membrane potential. At longer time scales, the two channels show similar levels of inactivation, because they both undergo slow inactivation mediated mostly by calmodulin, which is an abundant protein expressed in essentially every cell type. The authors should discuss whether they think fast inactivation is physiologically relevant in these channels or under what conditions they expect it to be relevant.

   The physiological relevance of the fast inactivation in TRPV6 channels is somewhat important, but not critical, as, independently of our physiological understanding of the phenomena, it does exist, making it intrinsically interesting from a biophysical/evolutionary perspective. Nevertheless, we think TRPV6's fast inactivation might be relevant in intestinal epithelia, so we added a short note about it in the revised text:

   In a previous study, we reported an expansion of the expression profile in mammalian TRPV6 compared to TRPV5. TRPV6, besides being expressed in the kidneys -where the [Ca2+]ext is regulated under physiological conditions-, is also expressed in the intestine (1). At this organ, TRPV6 is exposed to quick changes in the [Ca2+]ext after every meal. We reasoned that the fast inactivation phenotype of mammalian TRPV6 plays a critical role in protecting the intestinal epithelial cells from a Ca2+ overload and consequent apoptosis. In agreement with our rationale, TRPV6 expression is regulated by dietary Ca2+ levels (2-4) and its relevance in intestinal epithelial barrier dysfunction was recently discussed by Mori et al. (5).

   1. Flores-Aldama L, Vandewege MW, Zavala K, Colenso CK, Gonzalez W, Brauchi SE, et al. Evolutionary analyses reveal independent origins of gene repertoires and structural motifs associated to fast inactivation in calcium-selective TRPV channels. Sci Rep. 2020;10(1):1–13.

   2. Hoenderop JGJ, Dardenne O, Van Abel M, Van Der Kemp AWCM, Van Os CH, St -Arnaud R, et al. Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice. FASEB J. 2002;16:1398–406.

   3. Song Y, Kato S, Fleet JC. Vitamin D receptor (VDR) knockout mice reveal VDR-independent regulation of intestinal calcium absorption and ECaC2 and calbindin D9k mRNA. J Nutr. 2003;133(2):374–80.

   4. Van Cromphaut SJ, Dewerchin M, Hoenderop JG, Stockmans I, Van Herck E, Kato S, et al. Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects. Proc Natl Acad Sci U S A. 2001;98(23):13324–9.

   5. Mori Y, Omori M, Nakao A. Vital but vulnerable: Human TRPV6 is a trade-off of powerful Ca2+ uptake and susceptibility to epithelial barrier dysfunction. Cell Calcium [Internet]. 2022;107(September):102652.

   1. The charge reversal mutation experiments, though interesting, need to include the opposite mutation in the “interacting” partner in order to convincingly conclude that interacting pairs of charged residues are determinants of inactivation. In its current form, the manuscript does not provide conclusive evidence of interactions, rather, it only raises the possibility. The authors should tone down this conclusion.

   The reviewers are correct. The result might not be definitive and just rises a possibility. By additional simulations and analysis, we tried to mitigate the point raised and following reviewer’s advice we toned down the discussion of the issue.

   Additional suggestions for the authors to consider:

   1. The study will be easier to comprehend if the authors expand the introduction to provide a better description of the structure of these two channels, and specifically, the regions interrogated in this study.

   Both the introduction and first section were edited accordingly.

   1. The communication of the main findings in the study would be enhanced if the authors provide a better description of previous work on these channels and clearly state the gaps in our current knowledge that their study addresses.

   We did the best effort to address this critique in the revised version.

   1. TRPV1-4 are so different from TRPV5 and TRPV6 in many aspects that discussion about them in the context of fast inactivation is probably not relevant.

   Although we tone down the text to address the critique, we kept the data in supplementary figures and the idea at the discussion section, just because we think it is relevant and because they are not as dissimilar as the reviewers want to think. From our perspective, the evolution of temperature dependent phenotypes and the evolution of calcium dependent inactivation in TRPV5/6 points to the same region where multiple signals are integrated. In fact, we envision that the molecular rearrangements that underly both phenotypes will end up being very similar.

   1. For all figures, include the n for each experiment and increase the size of symbols for individual experiments in scatter plots.

   We address the point together with providing averaged current densities (not only the normalized response) for the individual conditions.

   Read the original source
3. 

   Biophysics Colab

   Mar 7, 2023

   Consolidated peer review report (15 December 2022)

   GENERAL ASSESSMENT

   In this preprint, Flores-Aldama and colleagues set out to identify molecular determinants of fast inactivation in the TRPV6 ion channel, a mechanism not observed in the closely related TRPV5 channel. The work focuses on the helix-loop-helix (HLH) motif, which is a region of the channel at the interface between the intracellular and transmembrane domains, the S2-S3 linker and the TRP Domain helix (TDh). Through MD simulations, the authors identify pairs of amino acid residues in the HLH/S2-S3 linker/TDh structural triad that move differently in TRPV5 and TRPV6 based on available cryo-EM structures. They mutate the E288 residue in TRPV6 to D, which is its counterpart in TRPV5, and make the reverse mutation in TRPV5, and show that swapping this single residue is …

   Consolidated peer review report (15 December 2022)

   GENERAL ASSESSMENT

   In this preprint, Flores-Aldama and colleagues set out to identify molecular determinants of fast inactivation in the TRPV6 ion channel, a mechanism not observed in the closely related TRPV5 channel. The work focuses on the helix-loop-helix (HLH) motif, which is a region of the channel at the interface between the intracellular and transmembrane domains, the S2-S3 linker and the TRP Domain helix (TDh). Through MD simulations, the authors identify pairs of amino acid residues in the HLH/S2-S3 linker/TDh structural triad that move differently in TRPV5 and TRPV6 based on available cryo-EM structures. They mutate the E288 residue in TRPV6 to D, which is its counterpart in TRPV5, and make the reverse mutation in TRPV5, and show that swapping this single residue is sufficient to transfer the inactivation kinetics between the two channels. They also show that the E294A mutation in the HLH partially reduces fast inactivation in TRPV6 and that the K245A mutation in ARD6 in TRPV5 confers some fast inactivation to that channel, albeit less than that observed in TRPV6. A very rewarding aspect of the manuscript is that some of the structural hypotheses were arrived at through an evolutionary analysis of sequences from many orthologues of both channels. This work is a follow up to the authors’ previous publication (https://doi.org/10.1038/s41598-020-65679-6), where they identified the HLH as a region important for fast inactivation in TRPV6. The manuscript includes new data that provides insight into the different inactivation mechanisms in these channels and strengthens the notion that the HLH linker region plays an important role in channel gating.

   RECOMMENDATIONS

   Revisions essential for endorsement:

   1. Structural comparisons are made between TRPV5 and TRPV6 structures that were determined in different labs, under different conditions, expression systems, etc., so whether the differences are due to Ca2+ or other experimental conditions is unclear. Also, the inactivated structures are all obtained in the presence of calmodulin, so whether the changes are due to calcium or calmodulin is also unclear. Finally, some of the non-inactivated structures are partially open, while some are closed. The authors should clearly state these caveats and fully discuss the limitations of the inferences obtained from this analysis.

   2. Molecular dynamics (MD) simulations were carried out in the presence of a non-physiological (50 mM) Ca2+ concentration, presumably to increase the chance of observing a conformational change. However, throughout the duration of the simulation, no Ca2+ binding events were observed, thus leading the authors to conclude that Ca2+ induced an inactivating conformational change through global effects. It is not clear to the reviewers what the authors intend to convey with this conclusion. The high calcium concentration will increase ionic strength by a very large amount, thus affecting any electrostatic interactions through charge screening. If this is the case, the same effects on inactivation should be expected from recordings (and simulations) in high ionic strength, thus weakening the finding that this is a specific effect of calcium. New experiments to probe these effects should be carried out, but if this is not possible, the authors should tone down their conclusion of a specific effect of calcium on inactivation.

   3. The physiological importance of the fast inactivation in TRPV6 is unclear. While there is a clear, evolutionarily conserved difference between TRPV5 and TRPV6, these channels are unlikely to experience fast 50 ms voltage jumps where this fast inactivation difference would be observable. Rather they are expressed in epithelial cells with stable, or slowly changing membrane potential. At longer time scales, the two channels show similar levels of inactivation, because they both undergo slow inactivation mediated mostly by calmodulin, which is an abundant protein expressed in essentially every cell type. The authors should discuss whether they think fast inactivation is physiologically relevant in these channels or under what conditions they expect it to be relevant.

   4. The charge reversal mutation experiments, though interesting, need to include the opposite mutation in the “interacting” partner in order to convincingly conclude that interacting pairs of charged residues are determinants of inactivation. In its current form, the manuscript does not provide conclusive evidence of interactions, rather, it only raises the possibility. The authors should tone down this conclusion.

   Additional suggestions for the authors to consider:

   1. The study will be easier to comprehend if the authors expand the introduction to provide a better description of the structure of these two channels, and specifically, the regions interrogated in this study.

   2. The communication of the main findings in the study would be enhanced if the authors provide a better description of previous work on these channels and clearly state the gaps in our current knowledge that their study addresses.

   3. TRPV1-4 are so different from TRPV5 and TRPV6 in many aspects that discussion about them in the context of fast inactivation is probably not relevant.

   4. For all figures, include the n for each experiment and increase the size of symbols for individual experiments in scatter plots.

   REVIEWING TEAM

   Reviewed by:

   Reviewer #1: TRP channel structure-function relationships and ion channel biophysics

   Reviewer #2: TRP channel physiology and biophysics

   León D Islas, Professor, National Autonomous University of Mexico, Mexico: ion channel biophysics, TRP channels

   Curated by:

   León D Islas, Professor, National Autonomous University of Mexico, Mexico

   Read the original source
4. 

   Version published to 10.1101/2022.10.04.510840v1 on bioRxiv

   Oct 5, 2022