Redox regulation of KV7 channels through EF3 hand of calmodulin
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Oxidation regulation of neuronal Kv7 channels contributes to the regulation of brain excitability. The manuscript concludes that this regulation is due to a disruption of the interaction between the S2S3 linker of Kv7 with the CaM EF3 site. The proposed mechanism is potentially important, but there are several weaknesses with the presentation and interpretation of the data that need to be addressed.
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Abstract
Neuronal K V 7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca 2+ -binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca 2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of K V 7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca 2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca 2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca 2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca 2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves K V 7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling.
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Author Response
Reviewer #1 (Public Review):
Oxidation of some KCNQ7 channels enhances channel activity. The manuscript by Nuñez and coauthors concluded that oxidation in the S2S3 linker of these channels disrupted the interaction between S2S3 and CaM EF-hand 3 (EF3). This mechanism is Ca2+-dependent. The apo EF3 no longer interacted with S2S3, and H2O2 no longer activated the channel. Electrophysiological recordings and fluorescence and NMR measurements of CaM with isolated helices A and B (CRD) and S2S3 of the channel were performed. While the results were in general clear with good quality, how the results support the conclusion was not clearly described. The approach using isolated molecular components in the study needs further validation since some of the results seem to show major conflicts with the results and mechanisms …
Author Response
Reviewer #1 (Public Review):
Oxidation of some KCNQ7 channels enhances channel activity. The manuscript by Nuñez and coauthors concluded that oxidation in the S2S3 linker of these channels disrupted the interaction between S2S3 and CaM EF-hand 3 (EF3). This mechanism is Ca2+-dependent. The apo EF3 no longer interacted with S2S3, and H2O2 no longer activated the channel. Electrophysiological recordings and fluorescence and NMR measurements of CaM with isolated helices A and B (CRD) and S2S3 of the channel were performed. While the results were in general clear with good quality, how the results support the conclusion was not clearly described. The approach using isolated molecular components in the study needs further validation since some of the results seem to show major conflicts with the results and mechanisms proposed in previous studies.
- Previous studies showed differential responses of Kv7 channels to oxidation; Kv7.2, 4, and 5 are sensitive to oxidation regulation but Kv7.1 and 3 do not change upon H2O2 treatment. These differences were attributed at least partially to the sequence differences in S2S3 among Kv7 channels (ref 10 of this manuscript). The results in this manuscript show some major differences from the previous study. First, in all experiments, no difference was observed among Kv7 channels. Second, in Fig 3-6, S2S3 from KCNQ1 was used. The rationale for using KCNQ1 S2S3 and the interpretation of results is not justified considering that KCNQ1 S2S3 has fewer Cys residues and was least affected by oxidation in the previous study.
We addressed the issue of differential sensitivity of Kv7 channels to H2O2 in the section 3.2 above (and in the discussion, lines 364-380). In brief, Kv7.3 is likely to display diminished redox-sensitivity due to its high tonic Po (as discussed in ref 10). Kv7.1 does have reduced number of Cys residues in the S2S3 linker and is also insensitive to H2O2 but introducing additional cysteine residues into Kv7.1 S2S3 confers only a fairly weak redox sensitivity. Hence, we think that on the structural level, all Kv7 channels have a redoxresponsive element (S2S3 linker) but Kv7.1 and Kv7.3 have other constrains that prevent their activity to be modulated by their redox-responsive domains.
We have performed new experiments with Kv7.2 and Kv7.4 peptides (3 cysteine residues). These new data confirm our conclusions, and are now included in Figure 6.
- In Fig 6, oxidation of S2S3 leads to a reduction of S2S3-CaM interaction, which leads to an increase of currents (Fig 1C). In Fig 4, Ca2+ loading leads to a reduced S2S3-CaM (EF3) interaction, which should also lead to an increase of currents based on Fig 6 conclusions. However, it is the EF3 mutation (destroying Ca2+ binding) that leads to the current increase (Fig 1B), contradictory to what Fig 6 data suggested.
Figure 6 and supplemental Figure 12 suggest that the effect of the peptides on the CRD is lost or reduced after oxidation. These data suggest that the oxidized S2S3 can no longer affect the CRD-CaM interaction. We propose that when EF3 is able to bind Ca2+ there is a tonic inhibition, and that oxidation relieves this inhibition leading to current increase.
As we explain above (see response 2.1), the effect is complicated due to CaMdependent promotion of surface expression.
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eLife assessment
Oxidation regulation of neuronal Kv7 channels contributes to the regulation of brain excitability. The manuscript concludes that this regulation is due to a disruption of the interaction between the S2S3 linker of Kv7 with the CaM EF3 site. The proposed mechanism is potentially important, but there are several weaknesses with the presentation and interpretation of the data that need to be addressed.
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Reviewer #1 (Public Review):
Oxidation of some KCNQ7 channels enhances channel activity. The manuscript by Nuñez and coauthors concluded that oxidation in the S2S3 linker of these channels disrupted the interaction between S2S3 and CaM EF-hand 3 (EF3). This mechanism is Ca2+-dependent. The apo EF3 no longer interacted with S2S3, and H2O2 no longer activated the channel. Electrophysiological recordings and fluorescence and NMR measurements of CaM with isolated helices A and B (CRD) and S2S3 of the channel were performed. While the results were in general clear with good quality, how the results support the conclusion was not clearly described. The approach using isolated molecular components in the study needs further validation since some of the results seem to show major conflicts with the results and mechanisms proposed in previous …
Reviewer #1 (Public Review):
Oxidation of some KCNQ7 channels enhances channel activity. The manuscript by Nuñez and coauthors concluded that oxidation in the S2S3 linker of these channels disrupted the interaction between S2S3 and CaM EF-hand 3 (EF3). This mechanism is Ca2+-dependent. The apo EF3 no longer interacted with S2S3, and H2O2 no longer activated the channel. Electrophysiological recordings and fluorescence and NMR measurements of CaM with isolated helices A and B (CRD) and S2S3 of the channel were performed. While the results were in general clear with good quality, how the results support the conclusion was not clearly described. The approach using isolated molecular components in the study needs further validation since some of the results seem to show major conflicts with the results and mechanisms proposed in previous studies.
Previous studies showed differential responses of Kv7 channels to oxidation; Kv7.2, 4, and 5 are sensitive to oxidation regulation but Kv7.1 and 3 do not change upon H2O2 treatment. These differences were attributed at least partially to the sequence differences in S2S3 among Kv7 channels (ref 10 of this manuscript). The results in this manuscript show some major differences from the previous study. First, in all experiments, no difference was observed among Kv7 channels. Second, in Fig 3-6, S2S3 from KCNQ1 was used. The rationale for using KCNQ1 S2S3 and the interpretation of results is not justified considering that KCNQ1 S2S3 has fewer Cys residues and was least affected by oxidation in the previous study.
In Fig 6, oxidation of S2S3 leads to a reduction of S2S3-CaM interaction, which leads to an increase of currents (Fig 1C). In Fig 4, Ca2+ loading leads to a reduced S2S3-CaM (EF3) interaction, which should also lead to an increase of currents based on Fig 6 conclusions. However, it is the EF3 mutation (destroying Ca2+ binding) that leads to the current increase (Fig 1B), contradictory to what Fig 6 data suggested.
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Reviewer #2 (Public Review):
The study by Nunez et al. builds upon structural work from the MacKinnon lab and the authors' labs to characterize how Ca2+, via calmodulin, interacts with Kv7 channels to mediate redox sensitivity. Using FRET experiments to support electrophysiology, the authors demonstrate an interaction defined by calmodulin, the helixA-helixB fork, and the S2-S3 linker. The experiments are well performed and the conclusions drawn are appropriate. These experiments help further define the redox signaling for Kv7 channels. A weakness is that the model in Figure 7 seems speculative, as the data provided do not appear to explain how the VSD is engaged/disengaged from the pore. Rather, most of the data concentrate on biochemical interactions and structural interpretations (via FRET signals, etc.) of conformational changes in …
Reviewer #2 (Public Review):
The study by Nunez et al. builds upon structural work from the MacKinnon lab and the authors' labs to characterize how Ca2+, via calmodulin, interacts with Kv7 channels to mediate redox sensitivity. Using FRET experiments to support electrophysiology, the authors demonstrate an interaction defined by calmodulin, the helixA-helixB fork, and the S2-S3 linker. The experiments are well performed and the conclusions drawn are appropriate. These experiments help further define the redox signaling for Kv7 channels. A weakness is that the model in Figure 7 seems speculative, as the data provided do not appear to explain how the VSD is engaged/disengaged from the pore. Rather, most of the data concentrate on biochemical interactions and structural interpretations (via FRET signals, etc.) of conformational changes in the presence of calcium. Further, the model as presented is not informative. The illustrations do not demonstrate successfully what the authors wish to claim, and the illustrations/models are not sufficiently supported by the data presented.
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Reviewer #3 (Public Review):
KV7 channels play an important role in setting the resting membrane potential of neurons. As such, modulation by reactive oxygen species is an important and physiologically relevant form of channel regulation. Here, the authors propose a mechanism for this modulation in which ROS disrupts the interaction between the S2S3 loop of the channel and CaM, resulting in an overall enhancement of channel activity. The authors propose that this S2S3/CaM interaction is selectively mediated through CaM EF3, and is dependent on Ca2+. The results are supported by patch-clamp data, as well as NMR measurements and a FRET-based binding assay. The paper contains a considerable amount of data that point towards the conclusion.
The authors conclude that the EF3 of CaM is 'by itself sufficient and necessary for the oxidative …
Reviewer #3 (Public Review):
KV7 channels play an important role in setting the resting membrane potential of neurons. As such, modulation by reactive oxygen species is an important and physiologically relevant form of channel regulation. Here, the authors propose a mechanism for this modulation in which ROS disrupts the interaction between the S2S3 loop of the channel and CaM, resulting in an overall enhancement of channel activity. The authors propose that this S2S3/CaM interaction is selectively mediated through CaM EF3, and is dependent on Ca2+. The results are supported by patch-clamp data, as well as NMR measurements and a FRET-based binding assay. The paper contains a considerable amount of data that point towards the conclusion.
The authors conclude that the EF3 of CaM is 'by itself sufficient and necessary for the oxidative response of KV7 channel complex and for gating the calcium responsive domain of KV7 channels." This is a very strong conclusion, and while much of the data points towards an important role for EF3, it is difficult to conclude that it is sufficient and necessary. The sparse description of the experiments makes the interpretation of the results a bit challenging. Based on the description provided, some of the results appear contradictory, limiting the conclusions drawn by the authors.
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