Redox regulation of KV7 channels through EF3 hand of calmodulin
- Instituto Biofisika, CSIC-UPV/EHU, Spain
- School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, United Kingdom
- Atlas Molecular Pharma S.L, Spain
- Department of Chemistry, University of Bath, United Kingdom
- Department of Chemistry, University of Oxford, United Kingdom
- Protein Stability and Inherited Disease Laboratory, CIC bioGUNE, Spain
Figures
Figure 1 with 2 supplements
EF3 hand Ca2+ binding capacity of CaM is required for H2O2-mediated potentiation of KV7.4.
(A) Response of KV7.4 transfected HEK293 cells to 150 µM H2O2 (normalized steady-state current at –60 mV, I/I0) when transfected with wild-type CaM (CaMWT n=12), mutant CaMs lacking Ca2+ binding to …
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Figure 1—source data 1
The current voltage relationship of cells transfected with mutant CaM does not differ significantly from CaMWT.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
The current voltage relationship of cells transfected with mutant CaM does not differ significantly from CaMWT.
(A) Current voltage relationship of KV4 and CaM transfected cells prior to treatment with H2O2. IV calculated through measuring tail current normalized to the maximal current in each condition …
Figure 1—figure supplement 2
Further support for the link between H2O2, CaM, and the S2S3 linker in KV7 channels.
(A) Response of KV7.4 or KV7.4 triple cysteine to alanine mutant (KV7.4CCC/AAA) to 150 µM H2O2 when co-transfected with CaMWT or CaM1234. (B) Representative current trains at –60 mV in response to …
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Figure 1—figure supplement 2—source data 1
Link between H2O2, CaM and the S2S3 linker in KV7 channels.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig1-figsupp2-data1-v2.xlsx
Figure 2 with 2 supplements
Influence of Ca2+-binding abolishing mutations in EF hands on Ca2+-dependent FRET changes.
Top: cartoon representation of CaM mutants. The EF-hands carrying a mutation that preclude Ca2+ binding are colored in red. Bottom: box-plot of the relative FRET index change produced by Ca2+ for …
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Figure 2—source data 1
Tabulated FRET values for each condition.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Residence of CaM in the calcium responsive domain (CRD) complex.
Top: cartoon representation of the assays. A blue fluorescent protein was attached to the AB fork. In one experiment (A), the tagged AB fork was complexed with CaM tagged with a yellow fluorescent …
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Figure 2—figure supplement 1—source data 1
Residence of CaM in the CRD complex.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Box plot of the influence of Ca2+-binding abolishing mutations in EF hands on Ca2+-dependent FRET change on the indicated KV7 isoforms.
Each plot represents the average of at least six independent experiments.
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Figure 2—figure supplement 2—source data 1
Tabulated FRET values for each condition.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig2-figsupp2-data1-v2.xlsx
Figure 3 with 2 supplements
Effects of a 24 residues KV7 S2S3 peptide on fluorescence emission of dansylated calmodulin (D-CAM).
(A) Emission spectra of D-CaM (50 nM) in Ca2+-free conditions (cyan), and after subsequent sequential addition of the S2S3 peptide (16 µM, green), and Ca2+ (10 µM free concentration, red). The order …
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Figure 3—source data 1
Spectra data for the indicated conditions, and tabulated peak values.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Relative disposition of calmodulin EF3 and S2S3 loop from different KV7 channel/CaM complexes solved by cryo-EM.
The PDB of each structure is indicated at the bottom of each panel. The 5VMS structure of KV7.1 suggests a potential interaction between the S2S3 loop and the EF3 hand, whereas this interaction is …
Figure 3—figure supplement 2
Effect of KV7 S2S3 peptides on fluorescence emission of dansylated calmodulin (D-CaM).
The residues forming the loop are highlighted in red in each peptide sequence. Emission spectra of D-CaM (50 nM) in Ca2+-free conditions (cyan), and after subsequent sequential addition of the S2S3 …
Figure 4 with 3 supplements
Interaction between the KV7 S2S3 peptide and CaM in complex with KV7.2 CDR.
(A) The chemical shift perturbation (CSP) analysis shows that the magnitude of local residue environmental alterations detected by NMR is larger in the C-lobe, both in the presence and in the …
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Figure 4—source data 1
Tabulated data values for NMR chemical shift perturbations.
Tabulated contact maps between calmodulin and Kv7.1 S2S3, and tabulated distance between S2S3 and EF3 mass centers.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data1-v2.xlsx
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Figure 4—source data 2
NMR raw spectra of KV7.2/Calmodulin complex with and without calcium in presence of S2S3 peptide.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data2-v2.zip
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Figure 4—source data 3
Replica 1: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data3-v2.zip
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Figure 4—source data 4
Replica 2: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data4-v2.zip
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Figure 4—source data 5
Replica 3: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data5-v2.zip
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Figure 4—source data 6
Replica 4: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data6-v2.zip
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Figure 4—source data 7
Replica 5: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data7-v2.zip
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Figure 4—source data 8
Replica 6: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data8-v2.zip
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Figure 4—source data 9
Replica 6: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data9-v2.zip
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Figure 4—source data 10
Replica 1: Molecular dynamic trajectory of int system, calcified N-lobe (no calcium C-lobe) of calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data10-v2.zip
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Figure 4—source data 11
Replica 2: Molecular dynamic trajectory of int system, calcified N-lobe (no calcium C-lobe) of calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data11-v2.zip
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Figure 4—source data 12
Replica 3: Molecular dynamic trajectory of int system, calcified N-lobe (no calcium C-lobe) of calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data12-v2.zip
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Figure 4—source data 13
Replica 4: Molecular dynamic trajectory of int system, calcified N-lobe (no calcium C-lobe) of calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data13-v2.zip
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Figure 4—source data 14
Replica 5: Molecular dynamic trajectory of int system, calcified N-lobe (no calcium C-lobe) of calmodulin in presence of S2S3.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data14-v2.zip
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Figure 4—source data 15
Molecular dynamic trajectory of S2S3 peptide y solution.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-data15-v2.zip
Figure 4—figure supplement 1
15N-HSQC of KV7.2 CDR:CaM titrated with unlabeled KV7.1-S2S3 in absence (left) and in presence of 1 mM Ca2+.
(A) 15N-HSQC of KV7.2 CDR:CaM complex at 75 µM (blue) and with KV7.1-S2S3 peptide at 1 mM (red). Signal overlap demonstrates that the structure of the complex was not altered in the presence of the …
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Figure 4—figure supplement 1—source data 1
HSQC of KV7.2 CDR:CaM titrated with unlabeled KV7.1-S2S3 in absence of 1 mM Ca2+.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Contact maps derived from molecular dynamic (MD) simulations of the S2S3/CaM complex.
(A) CaM sequence highlighting the most relevant residues for the contacts with S2S3 peptide with a cut-off at 10 Å (in gray) and at 4 Å (in green). The EF loops are underlined. (B) Normalized CaM …
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Figure 4—figure supplement 2—source data 1
HSQC of KV7.2 CDR:CaM titrated with unlabeled KV7.1-S2S3 in presence of 1 mM Ca2+.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
CD spectra of S2S3 peptide before and after treatment with H2O2.
(A) Circular Dichroism of the KV7.1 S2S3 peptide. The residues forming the loop are highlighted in green in the peptide sequence. CD (Circular Dichroism) spectra recorded at a fixed incubation time …
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Figure 4—figure supplement 3—source data 1
Raw data for CD spectra of S2S3 peptide before and after treatment with H2O2, and tabulated values of percentage helicity vs position.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig4-figsupp3-data1-v2.xlsx
Figure 5 with 1 supplement
Relative FRET changes of the human KV7.2 in complex with mutant CaM.
(A) FRET change after Ca2+ addition (10 µM) in the presence of the indicated concentrations of the S2S3 peptide: control (black), 0 µM S2S3, 5 µM S2S3 (red), and 10 µM S2S3 (green) (n=4). (B) …
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Figure 5—source data 1
Tabulated FRET values for each condition.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Relative FRET changes of KV7 CRD in complex with CaM.
(A) Relative FRET changes of KV7 calcium responsive domain (CRD) in complex with CaM. The concentration of peptide employed is indicated by the color of the symbol. Ca2+ dose-response in the …
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Figure 5—figure supplement 1—source data 1
Tabulated FRET values for each condition.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 2 supplements
FRET efficiency changes prompted by oxidized and reduced S2S3 peptides.
Difference in FRET efficiency in the absence and the presence of Ca2+. Left, KV7.4-S2S3 peptide and KV7.4 CRD. Right, KV7.2-S2S3 peptide and KV7.2 CRD. Similar results were obtained with KV7.1-S2S3 …
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Figure 6—source data 1
Tabulated FRET values for each condition.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Fluorescent emission D-CaM enhancement caused by KV7.1 S2S3 and KV7.2 S2S3 peptides before and after oxidation.
(A) Fluorescent emission dansylated calmodulin (D-CaM) enhancement caused by KV7.1 S2S3 and KV7.2 S2S3 peptides before and after oxidation. (B). Normalized FRET changes produced by 8 µM Ca2+, in the …
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Figure 6—figure supplement 1—source data 1
Tabulated fluoresce emission values for each condition.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Percentage of FRET index changes prompted by oxidized and reduced S2S3 peptides.
(A) Percentage of FRET index changes prompted by oxidized and reduced S2S3 peptides. Difference in % FRET index in the absence and the presence of Ca2+ with sensors derived from the KV7.4 and KV7.2 …
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Figure 6—figure supplement 2—source data 1
Tabulated FRET values for each condition.
- https://cdn.elifesciences.org/articles/81961/elife-81961-fig6-figsupp2-data1-v2.xlsx
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Peptide, recombinant protein | KV7.1 S2S3 | Proteogenix | RLWSAGCRSKYVGVWGRLRFARKP | |
| Peptide, recombinant protein | KV7.2 S2S3 | Proteogenix | RIWAAGCCCRYRGWRGRLKFARKP | |
| Peptide, recombinant protein | KV7.3 S2S3 | Proteogenix | RIWAAGCCCRYRKGWRLFKFARKP | |
| Peptide, recombinant protein | KV7.4 S2S3 | Proteogenix | RVWSAGCCCRYRGWQGRFRFARKP | |
| Peptide, recombinant protein | KV7.5 S2S3 | Proteogenix | RIWSAGCCCRYRGWQGRLRFARKP | |
| Recombinant DNA reagent | KV7.1 mtfp-hAB-Venus (residues I247-D456) in pPROEX HTc | This paper | NM_000218.2 | Plasmid, Fluorescence sensor |
| Recombinant DNA reagent | KV7.2 mtfp-hAB-Venus (residues I310-D549), in pPROEX HTc vector | This paper | NM_172107.3 | Plasmid, Fluorescence sensor |
| Recombinant DNA reagent | KV7.3 mtfp-hAB-Venus (residues I349-D556) in pPROEX HTc | This paper | NM_004519.3 | Plasmid, Fluorescence sensor |
| Recombinant DNA reagent | KV7.4 mtfp-hAB-Venus (residues I315-D539) in pPROEX HTc | This paper | NC_060925.1 | Plasmid, Fluorescence sensor |
| Recombinant DNA reagent | KV7.5 mtfp-hAB-Venus (residues I308-D527) in pPROEX HTc | This paper | NC_060930.1 | Plasmid, Fluorescence sensor |
| Recombinant DNA reagent | CaM in pOKD4 | Recombinant Human CALM2 in pOKD4 vector, GenScript | Genbank, NP_001292553.1 | Plasmid, Calmodulin, human CALM2 |
| Recombinant DNA reagent | hKCNQ4-eYFPc | Gamper et al., 2006 | AF105202 | Plasmid,KCNQ4 bound to YFP |
| Recombinant DNA reagent | hKCNQ4CCC/AAA -eYFPc | Mutant AF105202; Gamper et al., 2006 | Plasmid, Mutant KCNQ4 bound to YFP | |
| Recombinant DNA reagent | CaM 3 in pOKD4 | GenScript | Genbank, NP_001292553.2 | Plasmid, human CALM2, D93A mutation |
| Recombinant DNA reagent | CaM124 in pOKD4 | GenScript | Genbank, NP_001292553.3 | Plasmid, mutant human CALM2, D20A/D56A/D129A |
| Recombinant DNA reagent | CaM123 in pOKD4 | GenScript | Genbank, NP_001292553.4 | Plasmid, mutant human CALM2, D20A/D56A/D93A |
| Recombinant DNA reagent | CaM12 in pOKD4 | GenScript | Genbank, NP_001292553.5 | Plasmid, mutant human CALM2, D20A/D56A |
| Recombinant DNA reagent | CaM34 in pOKD4 | GenScript | Genbank, NP_001292553.6 | Plasmid, mutant human CALM2, D93A/D129A |
| Recombinant DNA reagent | CaM1234 in pOKD4 | GenScript | Genbank, NP_001292553.7 | Plasmid, mutant human CALM2, D20A/D56A/D93A/D129A |
| Recombinant DNA reagent | CaM WT in pCDNA3 | GenScript | Genbank, NP_001292553.8 | Plasmid, Calmodulin, human CALM2 |
| Recombinant DNA reagent | CaM3 in pCDNA3 | GenScript | Genbank, NP_001292553.9 | Plasmid, human CALM2, D93A mutations |
| Recombinant DNA reagent | CaM34 in pCDNA3 | GenScript | Genbank, NP_001292553.10 | Plasmid, human CALM2, D93A/D129A mutations |
| Recombinant DNA reagent | CaM12 in pCDNA3 | GenScript | Genbank, NP_001292553.11 | Plasmid, human CALM2, D20A/D56A mutations |
| Recombinant DNA reagent | CaM124 in pCDNA3 | GenScript | Genbank, NP_001292553.12 | Plasmid, human CALM2, D20A/D56A/D129A mutations |
| Recombinant DNA reagent | CaM1234 in pCDNA3.1 | GenScript | Genbank, NP_001292553.13 | Plasmid, human CALM2, D20A/D56A/D93A/D129A mutations |
| Recombinant DNA reagent | KV7.4 in pCDNA3.1 | GenScript | Genbank, NP_001292553.14 | Plasmid, human KV7.4 channel |
| Recombinant DNA reagent | KV7.4CCC/AAA in pCDNA3.1 | Gamper et al., 2006 | Genbank, NP_001292553.15 | Plasmid, human KV7.4 channel, C156A, C157A, C158V |
| Cell line (Homo sapiens) | Kidney (normal epithelial, embryo) | ATCC | HEK293 | |
| Chemical compound and drug | 5-(Dimethylamino)naphthalene-1-sulfonyl chloride, DNSCl | SIGMA-ALDRICH | CAS Number: 605-65-2 | Dansyl chloride |
| Chemical compound and drug | Pierce DTT (ditiotreitol) | Thermo Scientific | CAT# 20290 | DTT |
| Chemical compound and drug | Hydrogen peroxide solution | SIGMA-ALDRICH | CAS Number = 7722-84-1 / Pubchem ID = 57654227 | H2O2 30% (w/w) in H2O, contains stabilizer |
| Chemical compound and drug | X2254 | SIGMA-ALDRICH | XE-991 | |
| Chemical compound and drug | ab145545 | Abcam | Retigabine | |
| Chemical compound and drug | E2311 | Promega | Fugene | |
| Software and algorithm | PyMOL | The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC. | Use for molecular dynamics and figure preparation | |
| Software and algorithm | Patchmaster V2 | HEKA Instruments | ||
| Software and algorithm | VMD | Humphrey et al., 1996 | Use for molecular dynamics and figure preparation | |
| Software and algorithm | NAMD | Phillips et al., 2020 | Use for molecular dynamics | |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/81961/elife-81961-mdarchecklist1-v2.docx
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Supplementary file 1
S2S3 petides information.
- https://cdn.elifesciences.org/articles/81961/elife-81961-supp1-v2.docx
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Supplementary file 2
Details of the molecular dynamics simulations.
- https://cdn.elifesciences.org/articles/81961/elife-81961-supp2-v2.docx
