Redox regulation of KV7 channels through EF3 hand of calmodulin

Abstract

Neuronal K_V7 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 Ca²⁺-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 Ca²⁺ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of K_V7.4 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 Ca²⁺ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca²⁺ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca²⁺ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca²⁺ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves K_V7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling.

Keywords: E. coli; EF-hand; KCNQ; calcium; calmodulin; cell biology; neuroscience; redox; signal transduction.

Figure 1.. EF3 hand Ca²⁺ binding capacity of CaM is required for H₂O₂-mediated potentiation of K_V7.4.

(A) Response of K_V7.4 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 one or more EF hands. The number in X-axis of panel B applies and pertains to the EF hand unable to bind Ca²⁺ (CaM12 n=7, CaM124 n=7, CaM3 n=7, CaM34 n=7, and CaM1234 n=13) or with no additional CaM transfected (No CaM, n=6). (B) Current density (pA/pF; –60 mV) of K_V7.4 transfected cells prior to treatment with H₂O₂. (C) Representative currents at –60 mV in response to 150 µM H₂O₂ followed by 10 µM XE-991. Inset: representative current traces from each condition. (D) Comparative response of cells transfected with K_V7.4 and CaM3 to 300 µM H₂O₂ and 10 µM retigabine (n=8). (E) Ca²⁺ dependence of H₂O₂ response in cells transfected with K_V7.4 and CaMWT. Comparison of 300 µM H₂O₂ response in normal or low Ca²⁺ conditions induced by pre-incubation of cells in 10 µM BAPTA-AM for 30 min to chelate intracellular Ca²⁺. Control n=4, BAPTA-AM n=9. Data presented are mean ± SEM, statistical evaluation by independent measures ANOVA with Dunnett’s post hoc, **p<0.01, ***p<0.001, and ****p<0.0001 (A and B). A paired (D) or unpaired (E) two-tailed T test ***p<0.001 and ****p<0.0001.

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 K_V4 and CaM transfected cells prior to treatment with H₂O₂. IV calculated through measuring tail current normalized to the maximal current in each condition (I/Imax). (B) Change in voltage at which half maximal current is produced (ΔV_1/2) compared to CaMWT. (C) Representative IV traces for each CaM condition. Data presented is mean ± SEM, statistical evaluation by independent measures ANOVA with Dunnett’s post hoc *p<0.05. CaMWT n=6, CaM12 n=9, CaM124 n=8, CaM3 n=9, CaM34 n=6, and CaM1234 n=6.

Figure 1—figure supplement 2.. Further support for the link between H₂O₂, CaM, and the S2S3 linker in K_V7 channels.

(A) Response of K_V7.4 or K_V7.4 triple cysteine to alanine mutant (K_V7.4CCC/AAA) to 150 µM H₂O₂ when co-transfected with CaMWT or CaM1234. (B) Representative current trains at –60 mV in response to 150 µM H₂O₂ followed by 10 µM XE-991. Inset: representative current traces from each condition. (C) Current voltage relationship of K_V7.4 or K_V7.4CCC/AAA and CaMWT or CaM1234 transfected cells prior to treatment with H₂O₂. IV calculated through measuring tail current normalized to the maximal current in each condition (I/Imax). (D) Representative IV traces for each condition. Data presented are mean ± SEM, statistical evaluation by independent measures ANOVA with Dunnett’s post hoc ****p<0.0001.

Figure 2.. 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 the AB fork in complex with the indicated mutated CaM. Note that in the complex with CaM3 and CaM123, the changes prompted by 16 µM Ca²⁺ were almost obliterated, whereas in the complex with CaM124 the response was preserved. Each plot represents the average of six independent experiments. FRET index was defined as the ratio of the fluorescence peak between mcpVenus (yellow acceptor) and mTFP1 (blue donor). The index was normalized to the value obtained with WT CaM. Experiments were performed at 500 nM of hAB:CaM purified complex, in a 1:1 ratio. Inset: cartoon representing the FRET sensor in complex with CaM (mTFP1-hA-hB-Venus/CaM).

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 protein. Subsequently, the complex was incubated with excess CaM, devoid of any tag (n=4). A reduction in FRET over time is expected if there was CaM exchange. In another experiment (B), the fork was complexed with CaM and mixed with excess CaM tagged with a yellow fluorescence protein (n=4). Increase in FRET is expected if CaM exchange takes place. Bottom: FRET values over time in the presence and absence of Ca²⁺.

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 K_V7 isoforms.

Each plot represents the average of at least six independent experiments.

Figure 3.. Effects of a 24 residues K_V7 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 of additions is indicated at the left of each trace. (B) Dose-dependent relative fluorescent emission increase as a function of S2S3 peptide concentration, in the absence (green) and the presence of Ca²⁺ (10 µM, red). For this purpose, the maximum fluorescence D-CaM emission was measured between 490 and 500 nm and normalized with respect to the reference value (D-CaM with no added Ca²⁺ [green] and D-CaM with 10 µM free Ca²⁺ [red]). A Hill equation was fitted to the data (continuous line) with EC₅₀=0.88 ± 0.12 and 1.63±0.07 µM, in the absence and the presence of Ca²⁺, respectively. The K_V7.1 S2S3 peptide sequence was Ac-RLWSAGCRSKYVGVWGRLRFARK-NH₂.

Figure 3—figure supplement 1.. Relative disposition of calmodulin EF3 and S2S3 loop from different K_V7 channel/CaM complexes solved by cryo-EM.

The PDB of each structure is indicated at the bottom of each panel. The 5VMS structure of K_V7.1 suggests a potential interaction between the S2S3 loop and the EF3 hand, whereas this interaction is not apparent in cryo-EM images from other K_V7 channels, or K_V7.1 channels solved in the presence of PIP₂ (PDB 6V01). The structure S2S3 of K_V7.1 PDB 5VMS in green is superimposed in each figure. The main differences appear located at the distal region of the proximal helix of the S2S3 hairpin and the loop in the K_V7.4 structure (PDB 7VNP), which was trapped in an open channel configuration thanks to the presence of PIP₂, whereas it is remarkably similar in the structure of K_V7.4 (PDB 5BY1) trapped in a close conformation. Inset: alignment of the S2S3 sequences.

Figure 3—figure supplement 2.. Effect of K_V7 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 peptide (16 µM, green), and Ca²⁺ (10 µM free concentration, red). The order of additions is indicated at the left of each trace in the panel at the top left. A Hill equation was fitted to the emission increase data (continuous line) with EC₅₀=0.89 ± 0.04 and 1.37±0.23 µM, (n=3) in the absence and presence of Ca²⁺, respectively for K_V7.2 S2S3, 1.04±0.12 (n=3) and 1.78±0.34 µM for K_V7.3 (n=3), 0.56±0.06 and 0.89±0.07 µM for K_V7.4 (n=3), and 0.88±0.12 and 1.63±0.07 µM for K_V7.1 (n=3).

Figure 4.. Interaction between the K_V7 S2S3 peptide and CaM in complex with K_V7.2 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 absence of Ca²⁺. (B) Structural mapping of the main CSPs in the presence of Ca²⁺ over Ca²⁺-loaded K_V7.2 CaM/CDR complex. The two resides with the larger displacements are represented as balls, whereas the remaining above three times the mean are represented as sticks. The structure of the S2S3 loop was derived from the Cryo-EM PDB 5VMS (Sun and MacKinnon, 2017) and placed according to structural alignment of the C-lobe of PDB 6FEH (Bernardo-Seisdedos et al., 2018). (C) Contact map derived from molecular dynamic (MD) simulations of the S2S3/CaM complex. Normalized CaM contacts with the S2S3 peptide residues (10 Å cut-off) for int- (green) and holo-systems (red; see Figure 5—figure supplement 1). Vertical calibration bar is in arbitrary units (a.u.). (D) S2S3 contact map with CaM residues (4 Å cut-off; see Figure 5—figure supplement 1). (E) Distance as a function of time between the mass centers of the EF3 loop (residues D93-G98) and (i) the S2S3 loop (residues R164-L173; left) or (ii) the linker connecting CaM lobes (residues R74-E84; right). Bars indicate SEM (n=6).

Figure 4—figure supplement 1.. ¹⁵N-HSQC of K_V7.2 CDR:CaM titrated with unlabeled K_V7.1-S2S3 in absence (left) and in presence of 1 mM Ca²⁺.

(A) ¹⁵N-HSQC of K_V7.2 CDR:CaM complex at 75 µM (blue) and with K_V7.1-S2S3 peptide at 1 mM (red). Signal overlap demonstrates that the structure of the complex was not altered in the presence of the peptide. (B) Green spectra corresponds to the complex K_V7.2 CDR:CaM with 1 mM Ca²⁺, and orange to K_V7.2 CDR:CaM 1 mM Ca²⁺ and 1 mM K_V7.1-S2S3 peptide.

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 contacts between CaM and S2S3 peptide residues with cut-off at 4 Å, (C) 6 Å, and (D) 8 Å, for int- (green) and holo-systems (red). (E) S2S3 peptide sequence highlighting in green the most relevant residues for the contact with CaM with a cut-off at 4 Å. (F) Normalized S2S3 peptide contacts between CaM and S2S3 peptide residues with cut-off at 6 Å, (G) 8 Å, and (H) 10 Å.

Figure 4—figure supplement 3.. CD spectra of S2S3 peptide before and after treatment with H2O2.

(A) Circular Dichroism of the K_V7.1 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 of the peptide (50 nM) in control (blue) and in presence of 1 m H₂O₂ (orange). Helicity (H) was 63.4%, S+T antiparallel β-sheet conformations was 16.2%, and U for disordered structures were estimated to be 21.1%. In the presence of 1 mM H₂O₂, H=63.6%, S+T = 15.3%, and U=21.8%. Spectrum is the average of 40 runs. (B). Percentage of the S2S3 helical character relative to the whole trajectory. Bars indicate SEM (n=6). Helical character of the initial S2S3 structure is shown in black. The peptide in solution (gray line) adopts a similar structure regardless of the presence or the absence of the CaM/K_V7.2 complex. Both 3₁₀ and α-helices were combined to compute the degree of helicity. (C) Representative structures of the S2S3 peptide at initial (left) and final (right) times. Loop residues are shown in green.

Figure 5.. Relative FRET changes of the human K_V7.2 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) Superposition of helices A and B solved in the absence of Ca²⁺ (gold, PDB 6FEH, K_V7.2), in the presence of Ca²⁺ (green, PDB 6FEG, K_V7.2), and interacting with the S2S3 loop in the presence of Ca²⁺ (blue, PDB 5VMS, K_V7.1).

Figure 5—figure supplement 1.. Relative FRET changes of K_V7 CRD in complex with CaM.

(A) Relative FRET changes of K_V7 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 presence of the indicated concentrations of the S2S3 peptide, normalized to the maximal response. FRET changes expanded from 100% reduction to 70% increase. Dotted lines mark no changes in FRET. The continuous lines are the result of the fit of a Hill equation, with EC₅₀ values of 0.70±0.04 µM, 0.93±0.03 µM, 0.86±0.1, and 0.85±0.33 µM for K_V7.1, K_V7.3, K_V7.4, and K_V7.5, respectively, in the absence of S2S3. In the presence of 10 µM S2S3, the EC₅₀ values were 1.06±0.2 µM, 1.17±0.6 µM, 0.94±0.6 µM, and 0.89±0.33 µM for K_V7.1, K_V7.3, K_V7.4, and K_V7.5, respectively. Each plot represents the average of four independent experiments. (B) Ca²⁺ dose-response in the presence of the indicated concentrations of S2S3 peptide, normalized to the maximal response. FRET changes expanded from 100% reduction to 70% increase. Dotted lines mark no changes in FRET. The CaM mutant forming part of each complex is indicated on top of the corresponding dose-response curve. The continuous lines are the result of the fit of a Hill equation, with EC₅₀ values of 0.84±0.04 and 0.83±0.07 µM in the absence of S2S3 for WT and CaM124 (n=4), respectively. In the presence of 10 µM S2S3, the EC₅₀ values were 0.62±0.91, 0.62±0.16, and 1.48±0.22 µM for WT, CaM124, and CaM3, respectively (n=4).

Figure 6.. FRET efficiency changes prompted by oxidized and reduced S2S3 peptides.

Difference in FRET efficiency in the absence and the presence of Ca²⁺. Left, K_V7.4-S2S3 peptide and K_V7.4 CRD. Right, K_V7.2-S2S3 peptide and K_V7.2 CRD. Similar results were obtained with K_V7.1-S2S3 peptide (Figure 6—figure supplement 2). Red, sensor alone. Green, in the presence of 10 µM peptide. Yellow, with 10 µM oxidized peptide. Orange, oxidized peptide treated with 1 mM DTT. Bars represent mean ± SEM FRET-efficiency. *p<0.05; ***p<0.001. Each plot represents the average of at least six independent experiments.

Figure 6—figure supplement 1.. Fluorescent emission D-CaM enhancement caused by K_V7.1 S2S3 and K_V7.2 S2S3 peptides before and after oxidation.

(A) Fluorescent emission dansylated calmodulin (D-CaM) enhancement caused by K_V7.1 S2S3 and K_V7.2 S2S3 peptides before and after oxidation. (B). Normalized FRET changes produced by 8 µM Ca²⁺, in the presence of 5 µM K_V7.1 S2S3 and K_V7.2 S2S3 peptides in mTFP-Q1hAB-Venus/CaM and mTFP-Q2hAB-Venus complexes, respectively, before and after oxidation. Bars represent the means ± SEM from at least six independent experiments.

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 K_V7.4 and K_V7.2 CRD sequences. Red, sensor alone. Green, in the presence of 10 µM K_V7.1-S2S3 peptide. Yellow, with 10 µM oxidized peptide. Orange, oxidized peptide treated with 1 mM DTT. Blue, K_V7.x/CaM complex treated with 1 mM H₂O_2. Purple, K_V7.x/CaM incubated with 1 mM DTT. Bars represent mean ± SEM FRET-efficiency. *p<0.05; ***p<0.001. Each plot represents the average of at least six independent experiments. Bottom: sequence of the K_V7.1-peptide. The residues forming the loop are highlighted in red in the peptide sequence.

Author response image 1.