Journal: eLife

Article Title: Redox regulation of K V 7 channels through EF3 hand of calmodulin

doi: 10.7554/eLife.81961

Figure Lengend Snippet: ( A ) Response of K V 7.4 transfected HEK293 cells to 150 µM H 2 O 2 (normalized steady-state current at –60 mV, I/I 0 ) when transfected with wild-type CaM (CaMWT n=12), mutant CaMs lacking Ca 2+ 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 Ca 2+ (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 V 7.4 transfected cells prior to treatment with H 2 O 2 . ( C ) Representative currents at –60 mV in response to 150 µM H 2 O 2 followed by 10 µM XE-991. Inset: representative current traces from each condition. ( D ) Comparative response of cells transfected with K V 7.4 and CaM3 to 300 µM H 2 O 2 and 10 µM retigabine (n=8). ( E ) Ca 2+ dependence of H 2 O 2 response in cells transfected with K V 7.4 and CaMWT. Comparison of 300 µM H 2 O 2 response in normal or low Ca 2+ conditions induced by pre-incubation of cells in 10 µM BAPTA-AM for 30 min to chelate intracellular Ca 2+ . 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—source data 1. The current voltage relationship of cells transfected with mutant CaM does not differ significantly from CaMWT.

Article Snippet: Peptide, recombinant protein , K V 7.4 S2S3 , Proteogenix , , RVWSAGCCCRYRGWQGRFRFARKP.

Techniques: Transfection, Mutagenesis, Binding Assay, Incubation, Two Tailed Test

Journal: eLife

Article Title: Redox regulation of K V 7 channels through EF3 hand of calmodulin

doi: 10.7554/eLife.81961

Figure Lengend Snippet: The PDB of each structure is indicated at the bottom of each panel. The 5VMS structure of K V 7.1 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 V 7 channels, or K V 7.1 channels solved in the presence of PIP 2 (PDB 6V01). The structure S2S3 of K V 7.1 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 V 7.4 structure (PDB 7VNP), which was trapped in an open channel configuration thanks to the presence of PIP 2 , whereas it is remarkably similar in the structure of K V 7.4 (PDB 5BY1) trapped in a close conformation. Inset: alignment of the S2S3 sequences.

Article Snippet: Peptide, recombinant protein , K V 7.4 S2S3 , Proteogenix , , RVWSAGCCCRYRGWQGRFRFARKP.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Redox regulation of K V 7 channels through EF3 hand of calmodulin

doi: 10.7554/eLife.81961

Figure Lengend Snippet: ( A ) Emission spectra of D-CaM (50 nM) in Ca 2+ -free conditions (cyan), and after subsequent sequential addition of the S2S3 peptide (16 µM, green), and Ca 2+ (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 Ca 2+ (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 Ca 2+ [green] and D-CaM with 10 µM free Ca 2+ [red]). A Hill equation was fitted to the data (continuous line) with EC 50 =0.88 ± 0.12 and 1.63±0.07 µM, in the absence and the presence of Ca 2+ , respectively. The K V 7.1 S2S3 peptide sequence was Ac-RLWSAGCRSKYVGVWGRLRFARK-NH 2 . Figure 3—source data 1. Spectra data for the indicated conditions, and tabulated peak values.

Article Snippet: Peptide, recombinant protein , K V 7.4 S2S3 , Proteogenix , , RVWSAGCCCRYRGWQGRFRFARKP.

Techniques: Concentration Assay, Fluorescence, Sequencing

Journal: eLife

Article Title: Redox regulation of K V 7 channels through EF3 hand of calmodulin

doi: 10.7554/eLife.81961

Figure Lengend Snippet: The residues forming the loop are highlighted in red in each peptide sequence. Emission spectra of D-CaM (50 nM) in Ca 2+ -free conditions (cyan), and after subsequent sequential addition of the S2S3 peptide (16 µM, green), and Ca 2+ (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 EC 50 =0.89 ± 0.04 and 1.37±0.23 µM, (n=3) in the absence and presence of Ca 2+ , respectively for K V 7.2 S2S3, 1.04±0.12 (n=3) and 1.78±0.34 µM for K V 7.3 (n=3), 0.56±0.06 and 0.89±0.07 µM for K V 7.4 (n=3), and 0.88±0.12 and 1.63±0.07 µM for K V 7.1 (n=3).

Article Snippet: Peptide, recombinant protein , K V 7.4 S2S3 , Proteogenix , , RVWSAGCCCRYRGWQGRFRFARKP.

Techniques: Sequencing, Concentration Assay

Journal: eLife

Article Title: Redox regulation of K V 7 channels through EF3 hand of calmodulin

doi: 10.7554/eLife.81961

Figure Lengend Snippet: ( 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 Ca 2+ . ( B ) Structural mapping of the main CSPs in the presence of Ca 2+ over Ca 2+ -loaded K V 7.2 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 and placed according to structural alignment of the C-lobe of PDB 6FEH . ( 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 ). Vertical calibration bar is in arbitrary units (a.u.). ( D ) S2S3 contact map with CaM residues (4 Å cut-off; see ). ( 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—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. Figure 4—source data 2. NMR raw spectra of KV7.2/Calmodulin complex with and without calcium in presence of S2S3 peptide. Figure 4—source data 3. Replica 1: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3. Figure 4—source data 4. Replica 2: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3. Figure 4—source data 5. Replica 3: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3. Figure 4—source data 6. Replica 4: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3. Figure 4—source data 7. Replica 5: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3. Figure 4—source data 8. Replica 6: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3. Figure 4—source data 9. Replica 6: Molecular dynamic trajectory of holo system, calcified calmodulin in presence of S2S3. 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. 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. 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. 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. 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. Figure 4—source data 15. Molecular dynamic trajectory of S2S3 peptide y solution.

Article Snippet: Peptide, recombinant protein , K V 7.4 S2S3 , Proteogenix , , RVWSAGCCCRYRGWQGRFRFARKP.

Techniques: Derivative Assay, Cryo-EM Sample Prep

Journal: eLife

Article Title: Redox regulation of K V 7 channels through EF3 hand of calmodulin

doi: 10.7554/eLife.81961

Figure Lengend Snippet: ( A ) FRET change after Ca 2+ 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 Ca 2+ (gold, PDB 6FEH, K V 7.2), in the presence of Ca 2+ (green, PDB 6FEG, K V 7.2), and interacting with the S2S3 loop in the presence of Ca 2+ (blue, PDB 5VMS, K V 7.1). Figure 5—source data 1. Tabulated FRET values for each condition.

Article Snippet: Peptide, recombinant protein , K V 7.4 S2S3 , Proteogenix , , RVWSAGCCCRYRGWQGRFRFARKP.

Techniques:

Journal: eLife

Article Title: Redox regulation of K V 7 channels through EF3 hand of calmodulin

doi: 10.7554/eLife.81961

Figure Lengend Snippet: Difference in FRET efficiency in the absence and the presence of Ca 2+ . Left, K V 7.4-S2S3 peptide and K V 7.4 CRD. Right, K V 7.2-S2S3 peptide and K V 7.2 CRD. Similar results were obtained with K V 7.1-S2S3 peptide . 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—source data 1. Tabulated FRET values for each condition.

Article Snippet: Peptide, recombinant protein , K V 7.4 S2S3 , Proteogenix , , RVWSAGCCCRYRGWQGRFRFARKP.

Techniques:

Journal: eLife

Article Title: Redox regulation of K V 7 channels through EF3 hand of calmodulin

doi: 10.7554/eLife.81961

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , K V 7.4 S2S3 , Proteogenix , , RVWSAGCCCRYRGWQGRFRFARKP.

Techniques: Recombinant, Plasmid Preparation, Fluorescence, Mutagenesis, Software