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1. C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation

Author

Burel, Sophie, Coyan, Fabien C, Lorenzini, Maxime, Meyer, Matthew R, Lichti, Cheryl F, Brown, Joan H, Loussouarn, Gildas, Charpentier, Flavien, Nerbonne, Jeanne M, Townsend, R Reid, Maier, Lars S, and Marionneau, Céline

Subjects
Heart Disease, Cardiovascular, Amino Acid Substitution, Animals, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Fibroblast Growth Factors, HEK293 Cells, Heart Failure, Humans, Ion Channel Gating, Mice, Mice, Transgenic, Mutation, Missense, NAV1.5 Voltage-Gated Sodium Channel, Phosphorylation, Ca2+/calmodulin-dependent protein kinase II, FGF13, Nav1.5, calmodulin, channel inactivation, heart, phosphoproteomics, phosphorylation, sodium channel, Chemical Sciences, Biological Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology
Abstract

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

Published
2017

3. Determinants of iFGF13-mediated regulation of myocardial voltage-gated sodium (NaV) channels in mouse

Author

Lesage, Adrien, Lorenzini, Maxime, Burel, Sophie, Sarlandie, Marine, Bibault, Floriane, Lindskog, Cecilia, Maloney, Daniel, Silva, Jonathan R., Townsend, R. Reid, Nerbonne, Jeanne M., Marionneau, Celine, Lesage, Adrien, Lorenzini, Maxime, Burel, Sophie, Sarlandie, Marine, Bibault, Floriane, Lindskog, Cecilia, Maloney, Daniel, Silva, Jonathan R., Townsend, R. Reid, Nerbonne, Jeanne M., and Marionneau, Celine

Abstract

Posttranslational regulation of cardiac Na(V)1.5 channels is critical in modulating channel expression and function, yet their regulation by phosphorylation of accessory proteins has gone largely unexplored. Using phosphoproteomic analysis of Na-V channel complexes from adult mouse left ventricles, we identified nine phosphorylation sites on intracellular fibroblast growth factor 13 (iFGF13). To explore the potential roles of these phosphosites in regulating cardiac NaV currents, we abolished expression of iFGF13 in neonatal and adult mouse ventricular myocytes and rescued it with wild-type (WT), phosphosilent, or phosphomimetic iFGF13-VY. While the increased rate of closed-state inactivation of NaV channels induced by Fgf13 knockout in adult cardiomyocytes was completely restored by adenoviral-mediated expression of WT iFGF13-VY, only partial rescue was observed in neonatal cardiomyocytes after knockdown. The knockdown of iFGF13 in neonatal ventricular myocytes also shifted the voltage dependence of channel activation toward hyperpolarized potentials, a shift that was not reversed by WT iFGF13-VY expression. Additionally, we found that iFGF13-VY is the predominant isoform in adult ventricular myocytes, whereas both iFGF13-VY and iFGF13-S are expressed comparably in neonatal ventricular myocytes. Similar to WT iFGF13-VY, each of the iFGF13-VY phosphomutants studied restored NaV channel inactivation properties in both models. Lastly, Fgf13 knockout also increased the late Na+ current in adult cardiomyocytes, and this effect was restored with expression of WT and phosphosilent iFGF13-VY. Together, our results demonstrate that iFGF13 is highly phosphorylated and displays differential isoform expression in neonatal and adult ventricular myocytes. While we found no roles for iFGF13 phosphorylation, our results demonstrate differential effects of iFGF13 on neonatal and adult mouse ventricular NaV channels.

Published
2023
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4. Supplementary Figures;Supplementary Table 1 from Mapping of a N-terminal α-helix domain required for human PINK1 stabilization, Serine228 autophosphorylation and activation in cells

Author

Kakade, Poonam, Ojha, Hina, Raimi, Olawale G., Shaw, Andrew, Waddell, Andrew D., Ault, James R., Burel, Sophie, Brockmann, Kathrin, Kumar, Atul, Ahangar, Mohd Syed, Krysztofinska, Ewelina M., Macartney, Thomas, Bayliss, Richard, Fitzgerald, Julia C., and Muqit, Miratul M. K.

Abstract

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

Published
2022
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5. Mapping of a N-terminal ���-helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells

Author

Kakade Poonam, Ojha Hina, Raimi Olawale G., Shaw Andrew, Waddell Andrew D., Ault James R, Burel Sophie, Brockmann Kathrin, Kumar Atul, Ahangar Mohd Syed, Krysztofinska Ewelina M., Macartney Thomas, Bayliss Richard, Fitzgerald Julia C., and Muqit Miratul M. K.

Abstract

Human autosomal recessive mutations in the PINK1 gene are causal for Parkinson’s disease (PD). PINK1 encodes a mitochondrial localised protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function, however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain are unknown. We have employed mutagenesis studies of human PINK1 in cells to define the minimal region of PINK1, required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Bioinformatic analysis of the region spanning Ile111 to the kinase domain and inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal a-helical domain extension (NTE domain) within this region corroborated by hydrogen/deuterium exchange mass spectrometry (HDX-MS) of recombinant insect PINK1 protein. The AlphaFold structure also predicts the NTE domain forms an intramolecular interaction with the C-terminal extension (CTE). Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE:CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228); and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE domain mutants do not affect intrinsic catalytic kinase activity but do disrupt PINK1 stabilisation at the mitochondrial Translocase of outer membrane (TOM) complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilisation and activation and show that loss of NTE:CTE interactions is a major mechanism of PINK1-associated mutations linked to PD. Description of files Figure 1B PINK1 First 16 lanes. Other lanes are from unrelated experiments Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3111hPINK1 (-CCCP), Lane 12-13: OPA3111hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60111hPINK1 (-CCCP), Lane 16-17: HSP60111hPINK1 (+CCCP 3 hour) OPA1 (High exposure) Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3111hPINK1 (-CCCP), Lane 12-13: OPA3111hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60111hPINK1 (-CCCP), Lane 16-17: HSP60111hPINK1 (+CCCP 3 hour) GAPDH (Low exposure) Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3111hPINK1 (-CCCP), Lane 12-13: OPA3111hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60111hPINK1 (-CCCP), Lane 16-17: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C Figure 1C OPA1: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C Parkin: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C PINK1: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C pS65 Parkin: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C pUb: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1C Vinculin: Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA111hPINK1 (-CCCP), Lane 16-17: OPA111hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60111hPINK1 (-CCCP), Lane 20-21: HSP60111hPINK1 (+CCCP 3 hour) Figure 1D Figure 1D OPA1, PINK1 and GAPDH: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O) (not shown in paper), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) (not shown in paper), Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Lower left blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: PINK1(1-34)-PINK1(111- 581), (-A/O), Lane 16-17: PINK1(1-34)-PINK1(111- 581), (+A/O 9 hour). Upper right blot 3: Lane 1: Marker, Lane 2-3: pOTC(1-30)-PINK1(111-581) (-A/O), Lane 4-5: pOTC(1-30)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (-A/O), Lane 8-9: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), Lane 10: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), (not shown in paper), Lane 11-12: pF1ATPase(1-30)- PINK1(111-581) (-A/O), Lane 13-14: pF1ATPase(1-30)- PINK1(111-581) (+A/O 9 hour) lane 15-16: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (-A/O), lane 17-18: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (+A/O 9 hour). Lower right: blot 4: (not shown in paper) Lane 1: Marker, Lane 2-3: pALDH(1-19)-PINK1(111-581) (-A/O), Lane 4-5: pALDH(1-19)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pALDH (1-19) -(L15A) PINK1(111-581) (-A/O), Lane 8-9: pALDH (1-19) -(L15A) PINK1(111-581) (+A/O 9 hour. Figure 1D pUb: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O) (not shown in paper), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) (not shown in paper), Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Lower left blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: PINK1(1-34)-PINK1(111- 581), (-A/O), Lane 16-17: PINK1(1-34)-PINK1(111- 581), (+A/O 9 hour). Upper right blot 3: Lane 1: Marker, Lane 2-3: pOTC(1-30)-PINK1(111-581) (-A/O), Lane 4-5: pOTC(1-30)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (-A/O), Lane 8-9: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), Lane 10: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), (not shown in paper), Lane 11-12: pF1ATPase(1-30)- PINK1(111-581) (-A/O), Lane 13-14: pF1ATPase(1-30)- PINK1(111-581) (+A/O 9 hour) lane 15-16: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (-A/O), lane 17-18: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (+A/O 9 hour). Lower right: blot 4: (not shown in paper) Lane 1: Marker, Lane 2-3: pALDH(1-19)-PINK1(111-581) (-A/O), Lane 4-5: pALDH(1-19)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pALDH (1-19) -(L15A) PINK1(111-581) (-A/O), Lane 8-9: pALDH(1-19)-(L15A) PINK1(111-581) (+A/O 9 hour). Figure 2B Figure 2B pUb: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Upper right blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour). Lower left blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour). Figure 2B PINK1: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Upper right blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour). Lower left blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour). Figure 2B OPA1: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Upper right blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour). Lower left blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour). Figure 2B GAPDH: Upper left: blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Upper right blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour). Lower left blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour). Figure 2C Figure 2C pUb: Blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour). Blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(113- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(113- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(114- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(114- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(115-581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(115- 581) (+A/O 9 hour). Figure 2C PINK1: Blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour). Blot 3: Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(113- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(113- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(114- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(114- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(115-581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(115- 581) (+A/O 9 hour). Figure 2C OPA1: Blot 1: Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour). Blot 2: Lane 1: Marker, Lane 2-3: 111PINK1(111-581) (-A/O), Lane 4-5: 111PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour). Blot 3

Published
2021
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6. Mapping of a N-terminal α-helix domain required for human PINK1 stabilization, Serine228 autophosphorylation and activation in cells

Author

Kakade, Poonam, primary, Ojha, Hina, additional, Raimi, Olawale G., additional, Shaw, Andrew, additional, Waddell, Andrew D., additional, Ault, James R., additional, Burel, Sophie, additional, Brockmann, Kathrin, additional, Kumar, Atul, additional, Ahangar, Mohd Syed, additional, Krysztofinska, Ewelina M., additional, Macartney, Thomas, additional, Bayliss, Richard, additional, Fitzgerald, Julia C., additional, and Muqit, Miratul M. K., additional

Published
2022
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7. Supplementary Figures;Supplementary Table 1 from Mapping of a N-terminal ��-helix domain required for human PINK1 stabilization, Serine228 autophosphorylation and activation in cells

Author

Kakade, Poonam, Ojha, Hina, Raimi, Olawale G., Shaw, Andrew, Waddell, Andrew D., Ault, James R., Burel, Sophie, Brockmann, Kathrin, Kumar, Atul, Ahangar, Mohd Syed, Krysztofinska, Ewelina M., Macartney, Thomas, Bayliss, Richard, Fitzgerald, Julia C., and Muqit, Miratul M. K.

Abstract

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal ��-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

Published
2021
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8. Fluorescent‐ and tagged‐protoxin II peptides: potent markers of the Na v 1.7 channel pain target

Author

Montnach, Jérôme, primary, De Waard, Stephan, additional, Nicolas, Sébastien, additional, Burel, Sophie, additional, Osorio, Nancy, additional, Zoukimian, Claude, additional, Mantegazza, Massimo, additional, Boukaiba, Rachid, additional, Béroud, Rémy, additional, Partiseti, Michel, additional, Delmas, Patrick, additional, Marionneau, Céline, additional, and De Waard, Michel, additional

Published
2021
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9. Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites

Author

Lorenzini, Maxime, primary, Burel, Sophie, additional, Lesage, Adrien, additional, Wagner, Emily, additional, Charrière, Camille, additional, Chevillard, Pierre-Marie, additional, Evrard, Bérangère, additional, Maloney, Dan, additional, Ruff, Kiersten M., additional, Pappu, Rohit V., additional, Wagner, Stefan, additional, Nerbonne, Jeanne M., additional, Silva, Jonathan R., additional, Townsend, R. Reid, additional, Maier, Lars S., additional, and Marionneau, Céline, additional

Published
2021
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10. RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome

Author

Belbachir, Nadjet, primary, Portero, Vincent, additional, Al Sayed, Zeina R, additional, Gourraud, Jean-Baptiste, additional, Dilasser, Florian, additional, Jesel, Laurence, additional, Guo, Hongchao, additional, Wu, Haodi, additional, Gaborit, Nathalie, additional, Guilluy, Christophe, additional, Girardeau, Aurore, additional, Bonnaud, Stephanie, additional, Simonet, Floriane, additional, Karakachoff, Matilde, additional, Pattier, Sabine, additional, Scott, Carol, additional, Burel, Sophie, additional, Marionneau, Céline, additional, Chariau, Caroline, additional, Gaignerie, Anne, additional, David, Laurent, additional, Genin, Emmanuelle, additional, Deleuze, Jean-François, additional, Dina, Christian, additional, Sauzeau, Vincent, additional, Loirand, Gervaise, additional, Baró, Isabelle, additional, Schott, Jean-Jacques, additional, Probst, Vincent, additional, Wu, Joseph C, additional, Redon, Richard, additional, Charpentier, Flavien, additional, and Le Scouarnec, Solena, additional

Published
2019
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11. Fluorescent- and tagged-protoxin II peptides: potent markers of the Nav 1.7 channel pain target.

Author

Montnach, Jérôme, De Waard, Stephan, Nicolas, Sébastien, Burel, Sophie, Osorio, Nancy, Zoukimian, Claude, Mantegazza, Massimo, Boukaiba, Rachid, Béroud, Rémy, Partiseti, Michel, Delmas, Patrick, Marionneau, Céline, and De Waard, Michel

Subjects
VOLTAGE-gated ion channels, PAIN management, NOCICEPTORS, PEPTIDES, SODIUM channels, SENSORIMOTOR integration, RESEARCH, PAIN, RESEARCH methodology, MEDICAL cooperation, EVALUATION research, COMPARATIVE studies, ARTHROPOD venom, MEMBRANE transport proteins, RESEARCH funding
Abstract

Background and Purpose: Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the Nav 1.7 voltage-dependent Na+ channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its Nav channel targets.Experimental Approach: We took advantage of the high affinity properties of the peptide, combined to its slow off rate, to design a number of new tagged analogues useful for imaging and biochemistry purposes. We used high-throughput automated patch-clamp to identify the analogues best matching the native properties of ProTx II and validated them on various Nav -expressing cells in pull-down and cell distribution studies.Key Results: Two of the produced ProTx II analogues, Biot-ProTx II and ATTO488-ProTx II, best emulate the pharmacological properties of unlabelled ProTx II, whereas other analogues remain high affinity blockers of Nav 1.7. The biotinylated version of ProTx II efficiently works for the pull-down of several Nav isoforms tested in a concentration-dependent manner, whereas the fluorescent ATTO488-ProTx II specifically labels the Nav 1.7 channel over other Nav isoforms tested in various experimental conditions.Conclusions and Implications: The properties of these ProTx II analogues as tools for Nav channel purification and cell distribution studies pave the way for a better understanding of ProTx II channel receptors in pain and their pathophysiological implications in sensory neuronal processing. The new fluorescent ProTx II should also be useful in the design of new drug screening strategies. [ABSTRACT FROM AUTHOR]

Published
2021
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12. Supplementary Table 4 from Phosphorylation of Parkin at serine 65 is essential for its activation in vivo

Author

McWilliams, Thomas G., Barini, Erica, Pohjolan-Pirhonen, Risto, Brooks, Simon P., Singh, François, Burel, Sophie, Balk, Kristin, Kumar, Atul, Montava-Garriga, Lambert, Prescott, Alan R., Hassoun, Sidi Mohamed, Mouton-Liger, François, Ball, Graeme, Hills, Rachel, Knebel, Axel, Ayse Ulusoy, Monte, Donato A. Di, Tamjar, Jevgenia, Antico, Odetta, Fears, Kyle, Smith, Laura, Brambilla, Riccardo, Palin, Eino, Valori, Miko, Eerola-Rautio, Johanna, Tienari, Pentti, Corti, Olga, Dunnett, Stephen B., Ganley, Ian G., Suomalainen, Anu, and Miratul M. K. Muqit

Subjects
nervous system diseases
Abstract

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Published
2018
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13. Supplementary Table 1 from Phosphorylation of Parkin at serine 65 is essential for its activation in vivo

Author

McWilliams, Thomas G., Barini, Erica, Pohjolan-Pirhonen, Risto, Brooks, Simon P., Singh, François, Burel, Sophie, Balk, Kristin, Kumar, Atul, Montava-Garriga, Lambert, Prescott, Alan R., Hassoun, Sidi Mohamed, Mouton-Liger, François, Ball, Graeme, Hills, Rachel, Knebel, Axel, Ayse Ulusoy, Monte, Donato A. Di, Tamjar, Jevgenia, Antico, Odetta, Fears, Kyle, Smith, Laura, Brambilla, Riccardo, Palin, Eino, Valori, Miko, Eerola-Rautio, Johanna, Tienari, Pentti, Corti, Olga, Dunnett, Stephen B., Ganley, Ian G., Suomalainen, Anu, and Miratul M. K. Muqit

Subjects
nervous system diseases
Abstract

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Published
2018
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14. Supplementary Table 3 from Phosphorylation of Parkin at serine 65 is essential for its activation in vivo

Author

McWilliams, Thomas G., Barini, Erica, Pohjolan-Pirhonen, Risto, Brooks, Simon P., Singh, François, Burel, Sophie, Balk, Kristin, Kumar, Atul, Montava-Garriga, Lambert, Prescott, Alan R., Hassoun, Sidi Mohamed, Mouton-Liger, François, Ball, Graeme, Hills, Rachel, Knebel, Axel, Ayse Ulusoy, Monte, Donato A. Di, Tamjar, Jevgenia, Antico, Odetta, Fears, Kyle, Smith, Laura, Brambilla, Riccardo, Palin, Eino, Valori, Miko, Eerola-Rautio, Johanna, Tienari, Pentti, Corti, Olga, Dunnett, Stephen B., Ganley, Ian G., Suomalainen, Anu, and Miratul M. K. Muqit

Subjects
nervous system diseases
Abstract

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Published
2018
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15. Phosphorylation of Parkin at serine 65 is essential for its activation in vivo

Author

McWilliams, Thomas G., primary, Barini, Erica, additional, Pohjolan-Pirhonen, Risto, additional, Brooks, Simon P., additional, Singh, François, additional, Burel, Sophie, additional, Balk, Kristin, additional, Kumar, Atul, additional, Montava-Garriga, Lambert, additional, Prescott, Alan R., additional, Hassoun, Sidi Mohamed, additional, Mouton-Liger, François, additional, Ball, Graeme, additional, Hills, Rachel, additional, Knebel, Axel, additional, Ulusoy, Ayse, additional, Di Monte, Donato A., additional, Tamjar, Jevgenia, additional, Antico, Odetta, additional, Fears, Kyle, additional, Smith, Laura, additional, Brambilla, Riccardo, additional, Palin, Eino, additional, Valori, Miko, additional, Eerola-Rautio, Johanna, additional, Tienari, Pentti, additional, Corti, Olga, additional, Dunnett, Stephen B., additional, Ganley, Ian G., additional, Suomalainen, Anu, additional, and Muqit, Miratul M. K., additional

Published
2018
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16. Dysfunction of the Voltage-Gated K + Channel b2 Subunit in a Familial Case of Brugada Syndrome

Author

Portero, Vincent, Le Scouarnec, Solena, Es-Salah-Lamoureux, Zeineb, Burel, Sophie, Gourraud, Jean-Baptiste, Bonnaud, Stephanie, Lindenbaum, Pierre, Simonet, Floriane, Violleau, Jade, Baron, Estelle, Moreau, Eleonore, Scott, Carol, Chatel, Stephanie, Loussouarn, Gildas, O 'hara, Thomas, Mabo, Philippe, Dina, Christian, Le Marec, Herve, Schott, Jean-Jacques, Probst, Vincent, Baro, Isabelle, Marionneau, Céline, Charpentier, Flavien, Redon, Richard, unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), The Wellcome Trust Sanger Institute [Cambridge], Johns Hopkins University (JHU), Service de cardiologie et maladies vasculaires, Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Hôpital Pontchaillou-CHU Pontchaillou [Rennes]

Subjects
[SDV.GEN]Life Sciences [q-bio]/Genetics, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, cardiovascular diseases
Abstract

International audience; Background - The Brugada syndrome is an inherited cardiac arrhythmia associated with high risk of sudden death. Although 20% of patients with Brugada syndrome carry mutations in SCN5A, the molecular mechanisms underlying this condition are still largely unknown.Methods and Results - We combined whole‐exome sequencing and linkage analysis to identify the genetic variant likely causing Brugada syndrome in a pedigree for which SCN5A mutations had been excluded. This approach identified 6 genetic variants cosegregating with the Brugada electrocardiographic pattern within the pedigree. In silico gene prioritization pointed to 1 variant residing in KCNAB2, which encodes the voltage‐gated K+ channel β2‐subunit (Kvβ2‐R12Q). Kvβ2 is widely expressed in the human heart and has been shown to interact with the fast transient outward K+ channel subunit Kv4.3, increasing its current density. By targeted sequencing of the KCNAB2 gene in 167 unrelated patients with Brugada syndrome, we found 2 additional rare missense variants (L13F and V114I). We then investigated the physiological effects of the 3 KCNAB2 variants by using cellular electrophysiology and biochemistry. Patch‐clamp experiments performed in COS‐7 cells expressing both Kv4.3 and Kvβ2 revealed a significant increase in the current density in presence of the R12Q and L13F Kvβ2 mutants. Although biotinylation assays showed no differences in the expression of Kv4.3, the total and submembrane expression of Kvβ2‐R12Q were significantly increased in comparison with wild‐type Kvβ2.Conclusions - Altogether, our results indicate that Kvβ2 dysfunction can contribute to the Brugada electrocardiographic pattern.

Published
2016

17. Dysfunction of the Voltage‐Gated K + Channel β2 Subunit in a Familial Case of Brugada Syndrome

Author

Portero, Vincent, primary, Le Scouarnec, Solena, additional, Es‐Salah‐Lamoureux, Zeineb, additional, Burel, Sophie, additional, Gourraud, Jean‐Baptiste, additional, Bonnaud, Stéphanie, additional, Lindenbaum, Pierre, additional, Simonet, Floriane, additional, Violleau, Jade, additional, Baron, Estelle, additional, Moreau, Eléonore, additional, Scott, Carol, additional, Chatel, Stéphanie, additional, Loussouarn, Gildas, additional, O'Hara, Thomas, additional, Mabo, Philippe, additional, Dina, Christian, additional, Le Marec, Hervé, additional, Schott, Jean‐Jacques, additional, Probst, Vincent, additional, Baró, Isabelle, additional, Marionneau, Céline, additional, Charpentier, Flavien, additional, and Redon, Richard, additional

Published
2016
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21. Dysfunction of the Voltage-Gated K+ Channel β2 Subunit in a Familial Case of Brugada Syndrome.

Author

Portero, Vincent, Le Scouarnec, Solena, Es ‐ Salah ‐ Lamoureux, Zeineb, Burel, Sophie, Gourraud, Jean ‐ Baptiste, Bonnaud, Stéphanie, Lindenbaum, Pierre, Simonet, Floriane, Violleau, Jade, Baron, Estelle, Moreau, Eléonore, Scott, Carol, Chatel, Stéphanie, Loussouarn, Gildas, O'Hara, Thomas, Mabo, Philippe, Dina, Christian, Le Marec, Hervé, Schott, Jean ‐ Jacques, and Probst, Vincent

Published
2016
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22. FHF2 phosphorylation and regulation of native myocardial Na V 1.5 channels.

Author

Lesage A, Lorenzini M, Burel S, Sarlandie M, Bibault F, Maloney D, Silva JR, Reid Townsend R, Nerbonne JM, and Marionneau C

Abstract

Phosphorylation of the cardiac Na V 1.5 channel pore-forming subunit is extensive and critical in modulating channel expression and function, yet the regulation of Na V 1.5 by phosphorylation of its accessory proteins remains elusive. Using a phosphoproteomic analysis of Na V channel complexes purified from mouse left ventricles, we identified nine phosphorylation sites on Fibroblast growth factor Homologous Factor 2 (FHF2). To determine the roles of phosphosites in regulating Na V 1.5, we developed two models from neonatal and adult mouse ventricular cardiomyocytes in which FHF2 expression is knockdown and rescued by WT, phosphosilent or phosphomimetic FHF2-VY. While the increased rates of closed-state and open-state inactivation of Na V channels induced by the FHF2 knockdown are completely restored by the FHF2-VY isoform in adult cardiomyocytes, sole a partial rescue is obtained in neonatal cardiomyocytes. The FHF2 knockdown also shifts the voltage-dependence of activation towards hyperpolarized potentials in neonatal cardiomyocytes, which is not rescued by FHF2-VY. Parallel investigations showed that the FHF2-VY isoform is predominant in adult cardiomyocytes, while expression of FHF2-VY and FHF2-A is comparable in neonatal cardiomyocytes. Similar to WT FHF2-VY, however, each FHF2-VY phosphomutant restores the Na V channel inactivation properties in both models, preventing identification of FHF2 phosphosite roles. FHF2 knockdown also increases the late Na + current in adult cardiomyocytes, which is restored similarly by WT and phosphosilent FHF2-VY. Together, our results demonstrate that ventricular FHF2 is highly phosphorylated, implicate differential roles for FHF2 in regulating neonatal and adult mouse ventricular Na V 1.5, and suggest that the regulation of Na V 1.5 by FHF2 phosphorylation is highly complex., Etoc Summary: Lesage et al . identify the phosphorylation sites of FHF2 from mouse left ventricular Na V 1.5 channel complexes. While no roles for FHF2 phosphosites could be recognized yet, the findings demonstrate differential FHF2-dependent regulation of neonatal and adult mouse ventricular Na V 1.5 channels.

Published
2023
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