Isabelle Baró - Academia.edu (original) (raw)
Papers by Isabelle Baró
ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of io... more ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the c...
EP Europace, 2017
Introduction: Kv11.1 (hERG) channels play a critical role in repolarization of cardiomyocytes dur... more Introduction: Kv11.1 (hERG) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug mediated Kv11.1 blockade results in AP prolongation which poses an increased risk of sudden cardiac death. An increasing number of drugs, like pentamidine, that lack direct channel blocking ability of Kv11.1 are now known to interfere with normal Kv11.1 trafficking and thus reduce functional Kv11.1 channel densities. Class III antiarrhythmics, e.g.dofetilide,are able to rescue congenital and acquired trafficking defects and it is found that channel inhibition potency correlates with rescue efficacy. Unfortunately, dofetilide-induced rescue is counteracted by direct channel block. Purpose: To develop a 'non-channel blocking' pharmacological corrector for congenital and acquired Kv11.1 trafficking defects. Methods: LUF7244 was custom synthesized. IKv11.1 current in HEK-hERG cells was measured by whole cell patch clamp. AP characteristics were determined in isolated SR (sinus rhythm) and cAVB (chronic complete atrioventricular block) dog ventricular cardiomyocytes by patch clamp electrophysiology. Kv11.1 channel trafficking was determined by western blot and immune-fluorescence microscopy using hKV11.1 antibody in HEK-hERG cells (Kv11.1/HERG wild type) and G601S cells (traffickingdeficient Kv11.1). Results:Application of 0.5 lM, 3 lM and 10 lM LUF7244 increased the Ikv11.1 current by 8.0-, 9.6-and 12-fold, respectively. Importantly, LUF7244 antagonizes dofetilide induced IKv11.1 inhibition. LUF7244 shortened AP duration in canine ventricular cardiomyocytes; (-0.1560.09,-0.5360.17 and-0.6360.13 fold in SR cells;-0.1360.10,-0.4360.11 and-0.4060.14 fold in cAVB dog cells for 0.5, 3 and 10 lM LUF7244, respectively). 10 lM LUF7244 suppressed dofetilide-induced early-after-depolarization in 100% of SR (n¼7) and 80% of cAVB(n¼10) cells. LUF7244 treatment at 0.1 and 1 lM for 48 h did not disturb WT or G601S Kv11.1 trafficking. Pentamidine decreased mature WT Kv11.1 levels (0.4560.14 vs. 1.00 (control)), which was rescued by 10 lM dofetilide (0.8160.16) or 10 lM dofetilide þ 5 lM LUF7244 (0.8460.18). In G601S Kv11.1 cells, dofetilide (10 lM) or dofetilideþLUF7244 (10þ5 lM) restored Kv11.1 trafficking as demonstrated by western blot (1.0960.07 and 0.7660.08 fold respectively) and immunofluorescence microscopy. Conclusions: LUF7244 increases IKv11.1 and shortens the cardiac AP. LUF7244 counteracts dofetilide-induced AP prolongation and early after depolarization. LUF7244 does not interfere in dofetilide-mediated rescue of aberrant hERG trafficking. LUF7244 uncouples dofetilide-mediated hERG trafficking rescue from its direct channel blockade.
Heart Rhythm, 2014
Background: In a recent GWAS, we identified HEY2 as a novel susceptibility gene for Brugada syndr... more Background: In a recent GWAS, we identified HEY2 as a novel susceptibility gene for Brugada syndrome. Studies in heterozygous Hey2 knockout mice (Hey2 þ/-) demonstrated its role in mediating conduction velocity in the right ventricular outflow tract (RVOT). HEY2 is a transcriptional repressor putatively involved in establishing transmural gradients of genes across the ventricular wall. We investigated the effects of HEY2 on regional and transmural depolarization and repolarization patterns. Methods: Male adult Hey2 þ/mice and wild-type (WT) littermates were studied. Action potentials and membrane currents were measured in cardiomyocytes isolated from RVOT and subepicardial (EPI) and subendocardial (ENDO) regions of the right ventricle using the patchclamp technique. Real-time RT-PCR was performed on EPI and ENDO tissues from the left ventricle. Results: In WT cardiomyocytes, action potential duration (APD 90) and upstroke velocity (V max) were significantly lower in WT-RVOT and WT-EPI compared to WT-ENDO, demonstrating regional differences in depolarization and repolarization. Preliminary voltage-clamp experiments demonstrated higher transient outward K þ current (I to) densities in WT-RVOT and WT-EPI compared to WT-ENDO, whereas steady-state K þ currents were similar among the 3 sites. In line with this, Kcnd2 mRNA expression was significantly higher in WT-EPI compared to WT-ENDO. These regional and transmural differences were abrogated in Hey2 þ/hearts due to an increase in APD 90 and V max specifically in EPI and RVOT. I to density and Kcnd2 mRNA expression was significantly decreased in Hey2 þ/-EPI but was unaffected in Hey2 þ/-ENDO; hence a flattened transmural gradient of I to and Kcnd2 was observed in Hey2 þ/-. Ongoing experiments are focusing on the regional and transmural distribution of the Na þ current in WT vs Hey2 þ/-. Conclusions: The transcriptional repressor HEY2, a novel susceptibility gene for Brugada syndrome, regulates APD 90 and upstroke velocity in the RVOT and controls the transmural depolarization and repolarization gradient across the ventricular wall. These findings indicate that HEY2 modulates Brugada syndrome susceptibility through effects on both depolarization and repolarization processes.
Ion Channels, 2006
Analysis of genetically engineered mice is crucial for our understanding of the in vivo function ... more Analysis of genetically engineered mice is crucial for our understanding of the in vivo function of genes and proteins in the whole organism. This includes inactivation of a gene or the generation of specific mutations. The development of knockout and transgenic technologies in the mouse, therefore, represents a powerful tool for elucidating gene function, for modeling of human diseases, and potentially for the evaluation of drugs. In particular, conditional gene targeting applying the Cre/loxP-mediated recombination system is increasingly used to evaluate the role of the gene of interest in a cell-type-specific or even inducible manner. The experimental steps start with the characterization of the gene locus, followed by construction of a vector, gene targeting in ES cells, and establishment of mouse lines carrying the desired mutation. These are then bred to transgenic mice expressing Cre recombinase in a tissue-specific manner, thus allowing gene inactivation in a cell type of interest.
The Journal of Physiology, 2010
KCNQ1 osmosensitivity is of physiological and pathophysiological relevance in epithelial and card... more KCNQ1 osmosensitivity is of physiological and pathophysiological relevance in epithelial and cardiac cells, but the mechanism involved remains elusive. In COS-7 cells expressing the KCNE1-KCNQ1 fusion protein, extracellular hypoosmolarity and hyperosmolarity modify the channel biophysical parameters. These changes are consistent with hypoosmolarity increasing the level of membrane phosphatidylinositol-4,5-bisphosphate (PIP 2), which in turn upregulates KCNE1-KCNQ1 channels. We showed that increasing PIP 2 levels with a water-soluble PIP 2 analogue prevented channel upregulation in hypoosmotic condition, suggesting a variation of the channel-PIP 2 interaction during channel osmoregulation. Furthermore, we showed that polyamines and Mg 2+ , already known to tonically inhibit KCNQ channels by screening PIP 2 negative charges, are involved in the osmoregulatory process. Indeed, intracellular Mg 2+ removal and polyamines chelation inhibited the channel osmoregulation. Thus, the dilution of those cations during cell swelling might decrease channel inhibition and explain the channel upregulation by hypoosmolarity. To support this idea, we quantified the role of Mg 2+ in the osmodependent channel activity. Direct measurement of intracellular [Mg 2+ ] variations during osmotic changes and characterization of the channel Mg 2+ sensitivity showed that Mg 2+ participates significantly to the osmoregulation. Using intracellular solutions that mimic the variation of Mg 2+ and polyamines, we were able to recapitulate the current amplitude variations in response to extracellular osmolarity changes. Altogether, these results support the idea of a modulation of the channel-PIP 2 interactions by Mg 2+ and polyamines during cell volume changes. It is likely that this mechanism applies to other channels that are sensitive to both osmolarity and PIP 2 .
Journal of Biological Chemistry, 2010
Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) prote... more Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5 L) and S6 C terminus (S6 T). From these data, we hypothesized that S4S5 L behaves like a ligand specifically interacting with S6 T and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5 L and S6 T of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5 L peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5 L peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6 T , consistent with S4S5 L binding to S6 T. Val 254 in S4S5 L is known to contact Leu 353 in S6 T when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5 L binding to S6 T. Our results suggest a mechanistic model in which S4S5 L acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5 L away from S6 T , allowing channel opening.
Archives of Cardiovascular Diseases, 2009
Biophysical Journal, 2010
Biophysical Journal, Sep 4, 2004
Gabriel Laurent, MD, PHD,* Samuel Saal, MD,† Mohamed Yassine Amarouch, PHD,‡§ Delphine M. Béziau,... more Gabriel Laurent, MD, PHD,* Samuel Saal, MD,† Mohamed Yassine Amarouch, PHD,‡§ Delphine M. Béziau, MSC,‡§ Roos F. J. Marsman, MSC,¶ Laurence Faivre, MD, PHD,† Julien Barc, PHD,‡ Christian Dina, PHD,‡ Geraldine Bertaux, MD,* Olivier Barthez, MD,* Christel Thauvin-Robinet, MD, PHD,† Philippe Charron, MD, PHD,# Véronique Fressart, MD, PHD,** Alice Maltret, MD,†† Elisabeth Villain, MD,†† Estelle Baron, BA,‡ Jean Mérot, PHD,‡§ Rodolphe Turpault, PHD,‡‡ Yves Coudière, PHD,‡‡ Flavien Charpentier, PHD,‡§ §§ Jean-Jacques Schott, PHD,‡ §§ Gildas Loussouarn, PHD,‡§ Arthur A. M. Wilde, MD, PHD,¶ Jean-Eric Wolf, MD, PHD,* Isabelle Baró, PHD,‡§ Florence Kyndt, PHARMD, PHD,‡ Vincent Probst, MD, PHD‡ §§
Pharmacological Research, 2020
The Journal of Physiology, 1995
![Research paper thumbnail of Changes of intracellular [Ca2+] during refilling of sarcoplasmic reticulum in rat ventricular and vascular smooth muscle](https://attachments.academia-assets.com/84469731/thumbnails/1.jpg)
](The Journal of Physiology, 1993
Journal of molecular and cellular cardiology, Jan 31, 2016
Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases a... more Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, f...
Biophysical Journal, 2016
forces that destroy molecules. Our model reproduces signature properties of gating current: (1) e... more forces that destroy molecules. Our model reproduces signature properties of gating current: (1) equality of ON and OFF charge (2) saturating voltage dependence and (3) many (but not all) details of the shape of charge movement as a function of voltage, time, and solution composition. The model computes gating current flowing in the baths produced by arginines moving in the voltage sensor. The movement of arginines induces current flow everywhere producing 'capacitive' pile ups at the ends of the channel. Such pileups at charged interfaces are well studied in measurements and theories of physical chemistry but they are not typically included in models of gating current or ion channels. The pileups of charge change local electric fields, and they store charge in series with the charge storage of the arginines of the voltage sensor. Implications are being investigated.
Basic Research in Cardiology, 2014
The Journal of Physiology, 2008
Ion Channels, 2006
KCNQ1 is a voltage-activated potassium channel alpha-subunit expressed in various cell types, inc... more KCNQ1 is a voltage-activated potassium channel alpha-subunit expressed in various cell types, including cardiac myocytes and epithelial cells. KCNQ1 associates with different beta-subunits of the KCNE protein family. In the human heart, KCNQ1 associates with KCNE1 to generate the IKs current characterized by its slow activation and deactivation kinetics. Mutations in either KCNQ1 or KCNE1 are responsible for at least four channelopathies that lead to cardiac dysfunction and one that leads to congenital deafness: the Romano-Ward syndrome, the short QT syndrome, atrial fibrillation, and the Jervell and Lange-Nielsen syndrome (cardioauditory syndrome). To date, nearly 100 different KCNQ1 mutations have been reported as responsible for the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. Patch clamp and immunofluorescence techniques are instrumental for characterization of the molecular mechanisms responsible for the altered function of KCNQ1 and its partners.
Pfl�gers Archiv European Journal of Physiology, 1989
I n troduc t ion Ca2+-dependent K + channels have been described in various cell types (for a rec... more I n troduc t ion Ca2+-dependent K + channels have been described in various cell types (for a recent review see Blatz and Magleby 1987). In cardiac cells, Ca2+-activated K + currents usually exhibit a brief transient time course and are thought to participate in the initial repolarization (Carmeliet et al. 1987). In the present study, we report the existence of a sustained Ca2+-dependent K + current in guinea-pig atrial myocytes.
Journal of the American College of Cardiology, 2012
ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of io... more ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the c...
EP Europace, 2017
Introduction: Kv11.1 (hERG) channels play a critical role in repolarization of cardiomyocytes dur... more Introduction: Kv11.1 (hERG) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug mediated Kv11.1 blockade results in AP prolongation which poses an increased risk of sudden cardiac death. An increasing number of drugs, like pentamidine, that lack direct channel blocking ability of Kv11.1 are now known to interfere with normal Kv11.1 trafficking and thus reduce functional Kv11.1 channel densities. Class III antiarrhythmics, e.g.dofetilide,are able to rescue congenital and acquired trafficking defects and it is found that channel inhibition potency correlates with rescue efficacy. Unfortunately, dofetilide-induced rescue is counteracted by direct channel block. Purpose: To develop a 'non-channel blocking' pharmacological corrector for congenital and acquired Kv11.1 trafficking defects. Methods: LUF7244 was custom synthesized. IKv11.1 current in HEK-hERG cells was measured by whole cell patch clamp. AP characteristics were determined in isolated SR (sinus rhythm) and cAVB (chronic complete atrioventricular block) dog ventricular cardiomyocytes by patch clamp electrophysiology. Kv11.1 channel trafficking was determined by western blot and immune-fluorescence microscopy using hKV11.1 antibody in HEK-hERG cells (Kv11.1/HERG wild type) and G601S cells (traffickingdeficient Kv11.1). Results:Application of 0.5 lM, 3 lM and 10 lM LUF7244 increased the Ikv11.1 current by 8.0-, 9.6-and 12-fold, respectively. Importantly, LUF7244 antagonizes dofetilide induced IKv11.1 inhibition. LUF7244 shortened AP duration in canine ventricular cardiomyocytes; (-0.1560.09,-0.5360.17 and-0.6360.13 fold in SR cells;-0.1360.10,-0.4360.11 and-0.4060.14 fold in cAVB dog cells for 0.5, 3 and 10 lM LUF7244, respectively). 10 lM LUF7244 suppressed dofetilide-induced early-after-depolarization in 100% of SR (n¼7) and 80% of cAVB(n¼10) cells. LUF7244 treatment at 0.1 and 1 lM for 48 h did not disturb WT or G601S Kv11.1 trafficking. Pentamidine decreased mature WT Kv11.1 levels (0.4560.14 vs. 1.00 (control)), which was rescued by 10 lM dofetilide (0.8160.16) or 10 lM dofetilide þ 5 lM LUF7244 (0.8460.18). In G601S Kv11.1 cells, dofetilide (10 lM) or dofetilideþLUF7244 (10þ5 lM) restored Kv11.1 trafficking as demonstrated by western blot (1.0960.07 and 0.7660.08 fold respectively) and immunofluorescence microscopy. Conclusions: LUF7244 increases IKv11.1 and shortens the cardiac AP. LUF7244 counteracts dofetilide-induced AP prolongation and early after depolarization. LUF7244 does not interfere in dofetilide-mediated rescue of aberrant hERG trafficking. LUF7244 uncouples dofetilide-mediated hERG trafficking rescue from its direct channel blockade.
Heart Rhythm, 2014
Background: In a recent GWAS, we identified HEY2 as a novel susceptibility gene for Brugada syndr... more Background: In a recent GWAS, we identified HEY2 as a novel susceptibility gene for Brugada syndrome. Studies in heterozygous Hey2 knockout mice (Hey2 þ/-) demonstrated its role in mediating conduction velocity in the right ventricular outflow tract (RVOT). HEY2 is a transcriptional repressor putatively involved in establishing transmural gradients of genes across the ventricular wall. We investigated the effects of HEY2 on regional and transmural depolarization and repolarization patterns. Methods: Male adult Hey2 þ/mice and wild-type (WT) littermates were studied. Action potentials and membrane currents were measured in cardiomyocytes isolated from RVOT and subepicardial (EPI) and subendocardial (ENDO) regions of the right ventricle using the patchclamp technique. Real-time RT-PCR was performed on EPI and ENDO tissues from the left ventricle. Results: In WT cardiomyocytes, action potential duration (APD 90) and upstroke velocity (V max) were significantly lower in WT-RVOT and WT-EPI compared to WT-ENDO, demonstrating regional differences in depolarization and repolarization. Preliminary voltage-clamp experiments demonstrated higher transient outward K þ current (I to) densities in WT-RVOT and WT-EPI compared to WT-ENDO, whereas steady-state K þ currents were similar among the 3 sites. In line with this, Kcnd2 mRNA expression was significantly higher in WT-EPI compared to WT-ENDO. These regional and transmural differences were abrogated in Hey2 þ/hearts due to an increase in APD 90 and V max specifically in EPI and RVOT. I to density and Kcnd2 mRNA expression was significantly decreased in Hey2 þ/-EPI but was unaffected in Hey2 þ/-ENDO; hence a flattened transmural gradient of I to and Kcnd2 was observed in Hey2 þ/-. Ongoing experiments are focusing on the regional and transmural distribution of the Na þ current in WT vs Hey2 þ/-. Conclusions: The transcriptional repressor HEY2, a novel susceptibility gene for Brugada syndrome, regulates APD 90 and upstroke velocity in the RVOT and controls the transmural depolarization and repolarization gradient across the ventricular wall. These findings indicate that HEY2 modulates Brugada syndrome susceptibility through effects on both depolarization and repolarization processes.
Ion Channels, 2006
Analysis of genetically engineered mice is crucial for our understanding of the in vivo function ... more Analysis of genetically engineered mice is crucial for our understanding of the in vivo function of genes and proteins in the whole organism. This includes inactivation of a gene or the generation of specific mutations. The development of knockout and transgenic technologies in the mouse, therefore, represents a powerful tool for elucidating gene function, for modeling of human diseases, and potentially for the evaluation of drugs. In particular, conditional gene targeting applying the Cre/loxP-mediated recombination system is increasingly used to evaluate the role of the gene of interest in a cell-type-specific or even inducible manner. The experimental steps start with the characterization of the gene locus, followed by construction of a vector, gene targeting in ES cells, and establishment of mouse lines carrying the desired mutation. These are then bred to transgenic mice expressing Cre recombinase in a tissue-specific manner, thus allowing gene inactivation in a cell type of interest.
The Journal of Physiology, 2010
KCNQ1 osmosensitivity is of physiological and pathophysiological relevance in epithelial and card... more KCNQ1 osmosensitivity is of physiological and pathophysiological relevance in epithelial and cardiac cells, but the mechanism involved remains elusive. In COS-7 cells expressing the KCNE1-KCNQ1 fusion protein, extracellular hypoosmolarity and hyperosmolarity modify the channel biophysical parameters. These changes are consistent with hypoosmolarity increasing the level of membrane phosphatidylinositol-4,5-bisphosphate (PIP 2), which in turn upregulates KCNE1-KCNQ1 channels. We showed that increasing PIP 2 levels with a water-soluble PIP 2 analogue prevented channel upregulation in hypoosmotic condition, suggesting a variation of the channel-PIP 2 interaction during channel osmoregulation. Furthermore, we showed that polyamines and Mg 2+ , already known to tonically inhibit KCNQ channels by screening PIP 2 negative charges, are involved in the osmoregulatory process. Indeed, intracellular Mg 2+ removal and polyamines chelation inhibited the channel osmoregulation. Thus, the dilution of those cations during cell swelling might decrease channel inhibition and explain the channel upregulation by hypoosmolarity. To support this idea, we quantified the role of Mg 2+ in the osmodependent channel activity. Direct measurement of intracellular [Mg 2+ ] variations during osmotic changes and characterization of the channel Mg 2+ sensitivity showed that Mg 2+ participates significantly to the osmoregulation. Using intracellular solutions that mimic the variation of Mg 2+ and polyamines, we were able to recapitulate the current amplitude variations in response to extracellular osmolarity changes. Altogether, these results support the idea of a modulation of the channel-PIP 2 interactions by Mg 2+ and polyamines during cell volume changes. It is likely that this mechanism applies to other channels that are sensitive to both osmolarity and PIP 2 .
Journal of Biological Chemistry, 2010
Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) prote... more Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5 L) and S6 C terminus (S6 T). From these data, we hypothesized that S4S5 L behaves like a ligand specifically interacting with S6 T and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5 L and S6 T of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5 L peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5 L peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6 T , consistent with S4S5 L binding to S6 T. Val 254 in S4S5 L is known to contact Leu 353 in S6 T when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5 L binding to S6 T. Our results suggest a mechanistic model in which S4S5 L acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5 L away from S6 T , allowing channel opening.
Archives of Cardiovascular Diseases, 2009
Biophysical Journal, 2010
Biophysical Journal, Sep 4, 2004
Gabriel Laurent, MD, PHD,* Samuel Saal, MD,† Mohamed Yassine Amarouch, PHD,‡§ Delphine M. Béziau,... more Gabriel Laurent, MD, PHD,* Samuel Saal, MD,† Mohamed Yassine Amarouch, PHD,‡§ Delphine M. Béziau, MSC,‡§ Roos F. J. Marsman, MSC,¶ Laurence Faivre, MD, PHD,† Julien Barc, PHD,‡ Christian Dina, PHD,‡ Geraldine Bertaux, MD,* Olivier Barthez, MD,* Christel Thauvin-Robinet, MD, PHD,† Philippe Charron, MD, PHD,# Véronique Fressart, MD, PHD,** Alice Maltret, MD,†† Elisabeth Villain, MD,†† Estelle Baron, BA,‡ Jean Mérot, PHD,‡§ Rodolphe Turpault, PHD,‡‡ Yves Coudière, PHD,‡‡ Flavien Charpentier, PHD,‡§ §§ Jean-Jacques Schott, PHD,‡ §§ Gildas Loussouarn, PHD,‡§ Arthur A. M. Wilde, MD, PHD,¶ Jean-Eric Wolf, MD, PHD,* Isabelle Baró, PHD,‡§ Florence Kyndt, PHARMD, PHD,‡ Vincent Probst, MD, PHD‡ §§
Pharmacological Research, 2020
The Journal of Physiology, 1995
The Journal of Physiology, 1993
Journal of molecular and cellular cardiology, Jan 31, 2016
Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases a... more Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, f...
Biophysical Journal, 2016
forces that destroy molecules. Our model reproduces signature properties of gating current: (1) e... more forces that destroy molecules. Our model reproduces signature properties of gating current: (1) equality of ON and OFF charge (2) saturating voltage dependence and (3) many (but not all) details of the shape of charge movement as a function of voltage, time, and solution composition. The model computes gating current flowing in the baths produced by arginines moving in the voltage sensor. The movement of arginines induces current flow everywhere producing 'capacitive' pile ups at the ends of the channel. Such pileups at charged interfaces are well studied in measurements and theories of physical chemistry but they are not typically included in models of gating current or ion channels. The pileups of charge change local electric fields, and they store charge in series with the charge storage of the arginines of the voltage sensor. Implications are being investigated.
Basic Research in Cardiology, 2014
The Journal of Physiology, 2008
Ion Channels, 2006
KCNQ1 is a voltage-activated potassium channel alpha-subunit expressed in various cell types, inc... more KCNQ1 is a voltage-activated potassium channel alpha-subunit expressed in various cell types, including cardiac myocytes and epithelial cells. KCNQ1 associates with different beta-subunits of the KCNE protein family. In the human heart, KCNQ1 associates with KCNE1 to generate the IKs current characterized by its slow activation and deactivation kinetics. Mutations in either KCNQ1 or KCNE1 are responsible for at least four channelopathies that lead to cardiac dysfunction and one that leads to congenital deafness: the Romano-Ward syndrome, the short QT syndrome, atrial fibrillation, and the Jervell and Lange-Nielsen syndrome (cardioauditory syndrome). To date, nearly 100 different KCNQ1 mutations have been reported as responsible for the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. Patch clamp and immunofluorescence techniques are instrumental for characterization of the molecular mechanisms responsible for the altered function of KCNQ1 and its partners.
Pfl�gers Archiv European Journal of Physiology, 1989
I n troduc t ion Ca2+-dependent K + channels have been described in various cell types (for a rec... more I n troduc t ion Ca2+-dependent K + channels have been described in various cell types (for a recent review see Blatz and Magleby 1987). In cardiac cells, Ca2+-activated K + currents usually exhibit a brief transient time course and are thought to participate in the initial repolarization (Carmeliet et al. 1987). In the present study, we report the existence of a sustained Ca2+-dependent K + current in guinea-pig atrial myocytes.
Journal of the American College of Cardiology, 2012