The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Michael F. Waters - One of the best experts on this subject based on the ideXlab platform.

  • c terminal proline deletions in KCNC3 cause delayed channel inactivation and an adult onset progressive sca13 with spasticity
    The Cerebellum, 2018
    Co-Authors: Swati Khare, Jerelyn A. Nick, Yalan Zhang, Kira Galeano, Harry S Nick, S H Subramony, Jacinda B Sampson, Leonard K Kaczmarek, Michael F. Waters
    Abstract:

    Mutations in the potassium channel gene KCNC3 (Kv3.3) cause the autosomal dominant neurological disease, spinocerebellar ataxia 13 (SCA13). In this study, we expand the genotype-phenotype repertoire of SCA13 by describing the novel KCNC3 deletion p.Pro583_Pro585del highlighting the allelic heterogeneity observed in SCA13 patients. We characterize adult-onset, progressive clinical symptoms of two afflicted kindred and introduce the symptom of profound spasticity not previously associated with the SCA13 phenotype. We also present molecular and electrophysiological characterizations of the mutant protein in mammalian cell culture. Mechanistically, the p.Pro583_Pro585del protein showed normal membrane trafficking with an altered electrophysiological profile, including slower inactivation and decreased sensitivity to the inactivation-accelerating effects of the actin depolymerizer latrunculin B. Taken together, our results highlight the clinical importance of the intracellular C-terminal portion of Kv3.3 and its association with ion channel function.

  • Frequency of KCNC3 DNA Variants as Causes of
    2013
    Co-Authors: Spinocerebellar Ataxia, Natali A Minassian, Vartan Garibyan, Christopher M. Gomez, Diane M. Papazian, Michael F. Waters, Laura P W Ranum, Thomas D. Bird, Karla P Figueroa, Stefan M Pulst
    Abstract:

    Background: Gain-of function or dominant-negative mutations in the voltage-gated potassium channel KCNC3 (Kv3.3) were recently identified as a cause of autosomal dominant spinocerebellar ataxia. Our objective was to describe the frequency of mutations associated with KCNC3 in a large cohort of index patients with sporadic or familial ataxia presenting to three US ataxia clinics at academic medical centers. Methodology: DNA sequence analysis of the coding region of the KCNC3 gene was performed in 327 index cases with ataxia. Analysis of channel function was performed by expression of DNA variants in Xenopus oocytes. Principal Findings: Sequence analysis revealed two non-synonymous substitutions in exon 2 and five intronic changes, which were not predicted to alter splicing. We identified another pedigree with the p.Arg423His mutation in the highly conserved S4 domain of this channel. This family had an early-onset of disease and associated seizures in one individual. The second coding change, p.Gly263Asp, subtly altered biophysical properties of the channel, but was unlikely to be disease-associated as it occurred in an individual with an expansion of the CAG repeat in the CACNA1A calcium channel. Conclusions: Mutations in KCNC3 are a rare cause of spinocerebellar ataxia with a frequency of less than 1%. Th

  • frequency of KCNC3 dna variants as causes of spinocerebellar ataxia 13 sca13
    PLOS ONE, 2011
    Co-Authors: Karla P Figueroa, Natali A Minassian, Vartan Garibyan, Christopher M. Gomez, Diane M. Papazian, Michael F. Waters, Laura P W Ranum, Thomas D. Bird, Stefan M Pulst
    Abstract:

    Background Gain-of function or dominant-negative mutations in the voltage-gated potassium channel KCNC3 (Kv3.3) were recently identified as a cause of autosomal dominant spinocerebellar ataxia. Our objective was to describe the frequency of mutations associated with KCNC3 in a large cohort of index patients with sporadic or familial ataxia presenting to three US ataxia clinics at academic medical centers. Methodology DNA sequence analysis of the coding region of the KCNC3 gene was performed in 327 index cases with ataxia. Analysis of channel function was performed by expression of DNA variants in Xenopus oocytes. Principal Findings Sequence analysis revealed two non-synonymous substitutions in exon 2 and five intronic changes, which were not predicted to alter splicing. We identified another pedigree with the p.Arg423His mutation in the highly conserved S4 domain of this channel. This family had an early-onset of disease and associated seizures in one individual. The second coding change, p.Gly263Asp, subtly altered biophysical properties of the channel, but was unlikely to be disease-associated as it occurred in an individual with an expansion of the CAG repeat in the CACNA1A calcium channel. Conclusions Mutations in KCNC3 are a rare cause of spinocerebellar ataxia with a frequency of less than 1%. The p.Arg423His mutation is recurrent in different populations and associated with early onset. In contrast to previous p.Arg423His mutation carriers, we now observed seizures and mild mental retardation in one individual. This study confirms the wide phenotypic spectrum in SCA13.

  • Frequency of KCNC3 DNA variants as causes of spinocerebellar ataxia 13 (SCA13).
    Public Library of Science (PLoS), 2011
    Co-Authors: Karla P Figueroa, Natali A Minassian, Vartan Garibyan, Christopher M. Gomez, Diane M. Papazian, Michael F. Waters, Laura P W Ranum, Thomas D. Bird, Stefan M Pulst
    Abstract:

    Gain-of function or dominant-negative mutations in the voltage-gated potassium channel KCNC3 (Kv3.3) were recently identified as a cause of autosomal dominant spinocerebellar ataxia. Our objective was to describe the frequency of mutations associated with KCNC3 in a large cohort of index patients with sporadic or familial ataxia presenting to three US ataxia clinics at academic medical centers.DNA sequence analysis of the coding region of the KCNC3 gene was performed in 327 index cases with ataxia. Analysis of channel function was performed by expression of DNA variants in Xenopus oocytes.Sequence analysis revealed two non-synonymous substitutions in exon 2 and five intronic changes, which were not predicted to alter splicing. We identified another pedigree with the p.Arg423His mutation in the highly conserved S4 domain of this channel. This family had an early-onset of disease and associated seizures in one individual. The second coding change, p.Gly263Asp, subtly altered biophysical properties of the channel, but was unlikely to be disease-associated as it occurred in an individual with an expansion of the CAG repeat in the CACNA1A calcium channel.Mutations in KCNC3 are a rare cause of spinocerebellar ataxia with a frequency of less than 1%. The p.Arg423His mutation is recurrent in different populations and associated with early onset. In contrast to previous p.Arg423His mutation carriers, we now observed seizures and mild mental retardation in one individual. This study confirms the wide phenotypic spectrum in SCA13

  • mutations in voltage gated potassium channel KCNC3 cause degenerative and developmental central nervous system phenotypes
    Nature Genetics, 2006
    Co-Authors: Michael F. Waters, Karla P Figueroa, Ngatali A Minassian, Giovanni Stevanin, John P Bannister, Dagmar Nolte, Allan F Mock, Virgilio Gerald H Evidente, Dominic B Fee, Ulrich Muller
    Abstract:

    Potassium channel mutations have been described in episodic neurological diseases1. We report that K+ channel mutations cause disease phenotypes with neurodevelopmental and neurodegenerative features. In a Filipino adult-onset ataxia pedigree, the causative gene maps to 19q13, overlapping the SCA13 disease locus described in a French pedigree with childhood-onset ataxia and cognitive delay2. This region contains KCNC3 (also known as Kv3.3), encoding a voltage-gated Shaw channel with enriched cerebellar expression3. Sequencing revealed two missense mutations, both of which alter KCNC3 function in Xenopus laevis expression systems. KCNC3R420H, located in the voltage-sensing domain4, had no channel activity when expressed alone and had a dominant-negative effect when co-expressed with the wild-type channel. KCNC3F448L shifted the activation curve in the negative direction and slowed channel closing. Thus, KCNC3R420H and KCNC3F448L are expected to change the output characteristics of fast-spiking cerebellar neurons, in which KCNC channels confer capacity for high-frequency firing. Our results establish a role for KCNC3 in phenotypes ranging from developmental disorders to adult-onset neurodegeneration and suggest voltage-gated K+ channels as candidates for additional neurodegenerative diseases.

Peter H Larsson - One of the best experts on this subject based on the ideXlab platform.

  • kcne1 and kcne3 modulate kcnq1 channels by affecting different gating transitions
    Proceedings of the National Academy of Sciences of the United States of America, 2017
    Co-Authors: Rene Barrosoria, Rosamary Ramentol, Sara I Liin, Marta E Perez, Robert S Kass, Peter H Larsson
    Abstract:

    Abstract KCNE β-subunits assemble with and modulate the properties of voltage-gated K+ channels. In the heart, KCNE1 associates with the α-subunit KCNQ1 to generate the slowly activating, voltage-dependent potassium current (IKs) in the heart that controls the repolarization phase of cardiac action potentials. By contrast, in epithelial cells from the colon, stomach, and kidney, KCNE3 coassembles with KCNQ1 to form K+ channels that are voltage-independent K+ channels in the physiological voltage range and important for controlling water and salt secretion and absorption. How KCNE1 and KCNE3 subunits modify KCNQ1 channel gating so differently is largely unknown. Here, we use voltage clamp fluorometry to determine how KCNE1 and KCNE3 affect the voltage sensor and the gate of KCNQ1. By separating S4 movement and gate opening by mutations or phosphatidylinositol 4,5-bisphosphate depletion, we show that KCNE1 affects both the S4 movement and the gate, whereas KCNE3 affects the S4 movement and only affects the gate in KCNQ1 if an intact S4-to-gate coupling is present. Further, we show that a triple mutation in the middle of the transmembrane (TM) segment of KCNE3 introduces KCNE1-like effects on the second S4 movement and the gate. In addition, we show that differences in two residues at the external end of the KCNE TM segments underlie differences in the effects of the different KCNEs on the first S4 movement and the voltage sensor-to-gate coupling.

  • kcne1 and kcne3 modulate kcnq1 channels by affecting different gating transitions
    Biophysical Journal, 2017
    Co-Authors: Rene Barrosoria, Rosamary Ramentol, Sara I Liin, Marta E Perez, Robert S Kass, Peter H Larsson
    Abstract:

    KCNE β subunits assemble with and modulate the properties of voltage-gated K+ channels. In the heart, KCNE1 associates with KCNQ1 to generate the slowly activating, voltage-dependent IKs current that controls the repolarization phase of cardiac action potentials. By contrast, in epithelial cells from the colon, stomach and kidney, KCNE3 coassembles with the α-subunit KCNQ1 to form apparent voltage-independent K+ channels important for controlling water and salt secretion. How KCNE1 and KCNE3 subunits modify KCNQ1 channel gating so differently is largely unknown. Different molecular mechanisms have been proposed to explain the effects of KCNE1 and KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometry to determine how KCNE1 and KCNE3 affect the voltage sensor and the gate of KCNQ1. By separating S4 movement and gate opening by a mutation, we show that KCNE1 affects both the S4 movement and the gate, whereas KCNE3 directly affects the S4 movement and only indirectly affects the gate in KCNQ1. Further, we show that a triple mutation in KCNE3 converts KCNQ1/KCNE3 channels into KCNQ1/KCNE1-like channels by introducing KCNE1-like effects on the KCNQ1 gate. Our results suggest that the difference between the effects of KCNE1 and KCNE3 on KCNQ1 is that KCNE1 affects both the voltage-sensing domain and the gate, whereas KCNE3 primarily affects the voltage-sensing domain and only indirectly affects the gate.

  • kcne3 stabilizes the voltage sensor s4 of kcnq1 channel kcne1 uncouples s4 and the gate
    Biophysical Journal, 2015
    Co-Authors: Rene Barrosoria, Robert S Kass, Kevin J Sampson, Gary Peng, Peter H Larsson
    Abstract:

    KCNEs are single-span transmembrane β-subunits that assemble with and modulate the biophysical properties of voltage-gated K+ (Kv) channels. In the heart, the pore forming α-subunit KCNQ1 associates with KCNE1 to form the slowly-activating, voltage-gated IKs channels that contribute to the repolarization of the cardiac action potential. In tissues such as the colon, stomach and kidney, KCNQ1 coassembles with the β-subunit KCNE3 to form voltage-independent K+ channels important for K+ and Cl- secretion. KCNE3 has also been shown to be expressed in the human heart, although its physiological function remains unknown. Different mechanisms have been proposed to explain how different KCNE subunits alter KCNQ1 gating and permeation. For instance, KCNE3 is assumed to lock the voltage sensor (S4) of KCNQ1 channel in the activated state, resulting in a constitutively open channel. Here, we use voltage clamp fluorometry (VCF) to understand how KCNE3 affects the voltage sensor and the gate of KCNQ1 channel. We show that KCNE3, contrary to what was previously assumed, allows S4 movement in KCNQ1/KCNE3 channels. KCNE3 shifts the closing and S4 movement of KCNQ1 to extreme hyperpolarized potentials, such that at physiological voltage range (−80 mV to +40 mV), the channel is always open. By decoupling S4 and the gate, either by mutations or PIP2 depletion, we show that KCNE3 mainly affects S4 movement in KCNQ1. Two negatively charged residues in the N-terminus of KCNE3 (D54 and D55) are, at least partly, responsible for stabilizing S4 in an outward position, therefore stabilizing KCNQ1/KCNE3 channels in the activated open state. Further, we unitized a triple mutation of KCNE3, previously shown to convert KCNQ1/KCNE1 channel to a KCNQ1/KCNE1-like current, and observe a decoupling of S4 and the gate

Thomas J. Jentsch - One of the best experts on this subject based on the ideXlab platform.

  • k2p task 2 and kcnq1 kcne3 k channels are major players contributing to intestinal anion and fluid secretion
    The Journal of Physiology, 2017
    Co-Authors: Francisca Juliokalajzic, Thomas J. Jentsch, Sandra Villanueva, Johanna Burgos, Margarita Ojeda, Francisco V Sepulveda
    Abstract:

    KEY POINTS: K+ channels are important in intestinal epithelium as they ensure the ionic homeostasis and electrical potential of epithelial cells during anion and fluid secretion. Intestinal epithelium cAMP-activated anion secretion depends on the activity of the (also cAMP dependent) KCNQ1-KCNE3 K+ channel, but the secretory process survives after genetic inactivation of the K+ channel in the mouse. Here we use double mutant mice to investigate which alternative K+ channels come into action to compensate for the absence of KCNQ1-KCNE3 K+ channels. Our data establish that whilst Ca2+ -activated KCa 3.1 channels are not involved, K2P two-pore domain TASK-2 K+ channels are major players providing an alternative conductance to sustain the intestinal secretory process. Work with double mutant mice lacking both TASK-2 and KCNQ1-KCNE3 channels nevertheless points to yet-unidentified K+ channels that contribute to the robustness of the cAMP-activated anion secretion process. ABSTRACT: Anion and fluid secretion across the intestinal epithelium, a process altered in cystic fibrosis and secretory diarrhoea, is mediated by cAMP-activated CFTR Cl- channels and requires the simultaneous activity of basolateral K+ channels to maintain cellular ionic homeostasis and membrane potential. This function is fulfilled by the cAMP-activated K+ channel formed by the association of pore-forming KCNQ1 with its obligatory KCNE3 β-subunit. Studies using mice show sizeable cAMP-activated intestinal anion secretion in the absence of either KCNQ1 or KCNE3 suggesting that an alternative K+ conductance must compensate for the loss of KCNQ1-KCNE3 activity. We used double mutant mouse and pharmacological approaches to identify such a conductance. Ca2+ -dependent anion secretion can also be supported by Ca2+ -dependent KCa 3.1 channels after independent CFTR activation, but cAMP-dependent anion secretion is not further decreased in the combined absence of KCa 3.1 and KCNQ1-KCNE3 K+ channel activity. We show that the K2P K+ channel TASK-2 is expressed in the epithelium of the small and large intestine. Tetrapentylammonium, a TASK-2 inhibitor, abolishes anion secretory current remaining in the absence of KCNQ1-KCNE3 activity. A double mutant mouse lacking both KCNQ1-KCNE3 and TASK-2 showed a much reduced cAMP-mediated anion secretion compared to that observed in the single KCNQ1-KCNE3 deficient mouse. We conclude that KCNQ1-KCNE3 and TASK-2 play major roles in the intestinal anion and fluid secretory phenotype. The persistence of an, admittedly reduced, secretory activity in the absence of these two conductances suggests that further additional K+ channel(s) as yet unidentified contribute to the robustness of the intestinal anion secretory process.

  • kcnq potassium channels modulate sensitivity of skin down hair d hair mechanoreceptors
    Journal of Biological Chemistry, 2016
    Co-Authors: Sebastian Schutze, Thomas J. Jentsch, Ian J Orozco
    Abstract:

    M-current-mediating KCNQ (Kv7) channels play an important role in regulating the excitability of neuronal cells, as highlighted by mutations in Kcnq2 and Kcnq3 that underlie certain forms of epilepsy. In addition to their expression in brain, KCNQ2 and -3 are also found in the somatosensory system. We have now detected both KCNQ2 and KCNQ3 in a subset of dorsal root ganglia neurons that correspond to D-hair Aδ-fibers and demonstrate KCNQ3 expression in peripheral nerve endings of cutaneous D-hair follicles. Electrophysiological recordings from single D-hair afferents from Kcnq3(-/-) mice showed increased firing frequencies in response to mechanical ramp-and-hold stimuli. This effect was particularly pronounced at slow indentation velocities. Additional reduction of KCNQ2 expression further increased D-hair sensitivity. Together with previous work on the specific role of KCNQ4 in rapidly adapting skin mechanoreceptors, our results show that different KCNQ isoforms are specifically expressed in particular subsets of mechanosensory neurons and modulate their sensitivity directly in sensory nerve endings.

  • disruption of erythroid k cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in sad mice
    Journal of Clinical Investigation, 2007
    Co-Authors: Marco B Rust, Thomas J. Jentsch, Ruben Vicente, York Rudhard, Boris E. Shmukler, Mathieu Trudel, Seth L Alper, Carlo Brugnara, Christian A Hubner
    Abstract:

    K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1–/– mice, decreased in Kcc3–/– mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1–/– or Kcc3–/– mice were of normal density, rbc of Kcc1–/–Kcc3–/– mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1–/–Kcc3–/– SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K+ content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

  • moderate loss of function of cyclic amp modulated kcnq2 kcnq3 k channels causes epilepsy
    Nature, 1998
    Co-Authors: Bjorn C Schroeder, Christian Kubisch, Valentin Stein, Thomas J. Jentsch
    Abstract:

    Epilepsy affects more than 0.5% of the world's population and has a large genetic component1. It is due to an electrical hyperexcitability in the central nervous system. Potassium channels are important regulators of electrical signalling, and benign familial neonatal convulsions (BFNC), an autosomal dominant epilepsy of infancy, is caused by mutations in the KCNQ2 or the KCNQ3 potassium channel genes2,3,4. Here we show that KCNQ2 and KCNQ3 are distributed broadly in brain with expression patterns that largely overlap. Expression in Xenopus oocytes indicates the formation of heteromeric KCNQ2/KCNQ3 potassium channels with currents that are at least tenfold larger than those of the respective homomeric channels. KCNQ2/KCNQ3 currents can be increased by intracellular cyclic AMP, an effect that depends on an intact phosphorylation site in the KCNQ2 amino terminus. KCNQ2 and KCNQ3 mutations identified in BFNC pedigrees compromised the function of the respective subunits, but exerted no dominant-negative effect on KCNQ2/KCNQ3 heteromeric channels. We predict that a 25% loss of heteromeric KCNQ2/KCNQ3-channel function is sufficient to cause the electrical hyperexcitability in BFNC. Drugs raising intracellular cAMP may prove beneficial in this form of epilepsy.

Mélanie Lambert - One of the best experts on this subject based on the ideXlab platform.

  • etude de l implication de la dysfonction de kcnk3 dans le developpement de l hypertension arterielle pulmonaire
    2019
    Co-Authors: Mélanie Lambert
    Abstract:

    L’hypertension arterielle pulmonaire (HTAP) est une maladie rare resultant de l’obstruction progressive des petites arteres pulmonaires, via un remodelage de la paroi vasculaire, associee a une vasoconstriction entrainant une hypertrophie puis une defaillance cardiaque droite qui aboutit au deces du patient. Depuis 2013, 10 mutations, dans le gene KCNK3 (codant pour un canal potassique), ont ete identifiees chez des patients atteints d’HTAP. Toutes les mutations identifiees entrainent une perte de fonction du canal. De plus, notre equipe a pu demontrer que la perte de KCNK3 est commune a toutes les formes d’HTAP : idiopathique et heritable ainsi qu’experimentale. Durant ma these, mon projet principal a donc ete d’etudier l’implication de la dysfonction de KCNK3 dans le developpement de l’HTAP. Tout d’abord, nous avons pu demontrer que la perte d’expression/fonction de KCNK3 est egalement une caracteristique de l’hypertrophie / la dysfonction ventriculaire droite. Par la suite, en caracterisant un modele unique de rats mutes pour le gene Kcnk3, nous avons pu demontrer que l'inactivation genetique de Kcnk3 chez le rat conduit a une alteration vasculaire pulmonaire facilitant ainsi le developpement d’une hypertension pulmonaire (HTP). Faisant de ce modele un nouvel outil permettant de comprendre les mecanismes initiateurs de l’HTP et representerai un outil pertinent pour developper des cibles therapeutiques. Pour finir, nous avons montre que le developpement d’une HTP due a une insuffisance cardiaque gauche (via une ligature du l’aorte) est facilitee chez nos rats mutes pour Kcnk3.

  • loss of kcnk3 is a hallmark of rv hypertrophy dysfunction associated with pulmonary hypertension
    Cardiovascular Research, 2018
    Co-Authors: Mélanie Lambert, Catherine Ruckermartin, Angèle Boet, Véronique Capuano, Stéphane N. Hatem, P Mendesferreira, R Adao
    Abstract:

    Aims Mutations in the KCNK3 gene, which encodes for an outward-rectifier K+ channel, have been identified in patients suffering from pulmonary arterial hypertension (PAH), and constitute the first described channelopathy in PAH. In human PAH and experimental pulmonary hypertension (PH), we demonstrated that KCNK3 expression and function are severely reduced in pulmonary vascular cells, promoting PH-like phenotype at the morphologic and haemodynamic levels. Since KCNK3 channel is also expressed in both the human and rodent heart, we aimed to elucidate the pathophysiological role of KCNK3 channel in right ventricular (RV) hypertrophy (RVH) related to PH. Methods and results Using whole-cell Patch-clamp technique, we demonstrated that KCNK3 is predominantly expressed in adult rat RV cardiomyocytes compared to the left ventricle cardiomyocytes and participates in the repolarizing phase of the RV action potential. We revealed a reduction in KCNK3 function prior to development of RVH and the rise of pulmonary vascular resistance. KCNK3 function is severely reduced in RV cardiomyocytes during the development of RVH in several rat models of PH (exposure to monocrotaline, chronic hypoxia, and Sugen/hypoxia) and chronic RV pressure overload (pulmonary artery banding). In experimental PH, we revealed a reduction in KCNK3 function before any rise in pulmonary vascular resistance and the development of RVH. KCNK3 mRNA level is also reduced in human RV tissues from PAH patients compared to non-PAH patients. In line with these findings, chronic inhibition of KCNK3 in rats with the specific inhibitor (A293) induces RV hypertrophy which is associated with the re-expression of foetal genes, RV fibrosis, RV inflammation, and subsequent loss of RV performance as assessed by echocardiography. Conclusion Our data indicate that loss of KCNK3 function and expression is a hallmark of the RV hypertrophy/dysfunction associated with PH.

  • late breaking abstract kcnk3 dysfunction contributes to the development of pulmonary arterial hypertension characterization of kcnk3 deficient rats
    European Respiratory Journal, 2016
    Co-Authors: Fabrice Antigny, Mélanie Lambert, Aurélie Hautefort, Boris Manoury, Philippe Jourdon, Barbara Girerd, David Montani, Gérald Simonneau, Catherine Ruckermartin, Mars Humbert
    Abstract:

    Background: Inactivating mutations in the KCNK3 gene (K + channel) have been identified in heritable forms of pulmonary arterial hypertension (PAH). We discovered that KCNK3 dysfunction contributes actually to the development of both heritable and non-heritable PAH, and to experimental pulmonary hypertension (PH) ( Antigny et al. Circulation 2016 ). Methods and results: We generated Kcnk3 deficient rats using CRISPR-Cas9 technology and characterized at electrophysiological, hemodynamics, morphological and molecular levels the first genetically modified rat model linked to kcnk3 mutation: Kcnk3 Δ 94Ex1/+ (94pb deletion in exon 1 of Kcnk3 gene). Using a patch–clamp technique in freshly isolated pulmonary artery smooth muscle cells (PASMCs), we found that KCNK3-current is absent in heterozygous and homozygous Kcnk3 mutated rats and that isolated PASMC are significantly depolarized compared to WT rats. Kcnk3 mutation induced distal neomuscularization, abnormal pulmonary arteries vasoreactivity, elevated mean right ventricular systolic pressures and upregulation of MAP kinase signaling pathways. Heterozygous Kcnk3 -mutated rats developed more severe pulmonary hypertension than WT littermate under hypoxic condition. Conclusions: In this study we established the first Kcnk3 deficient rat model. We demonstrated that a genetic inactivation of KCNK3 in rats promoted the early signs of pulmonary hypertension, confirming that KCNK3 loss of function is a key event in PAH pathogenesis. The development and characterization of the first Kcnk3 deficient PH-rats model open new opportunities for testing relevant therapeutics molecules in context of heritable PH.

Rene Barrosoria - One of the best experts on this subject based on the ideXlab platform.

  • kcne1 and kcne3 modulate kcnq1 channels by affecting different gating transitions
    Proceedings of the National Academy of Sciences of the United States of America, 2017
    Co-Authors: Rene Barrosoria, Rosamary Ramentol, Sara I Liin, Marta E Perez, Robert S Kass, Peter H Larsson
    Abstract:

    Abstract KCNE β-subunits assemble with and modulate the properties of voltage-gated K+ channels. In the heart, KCNE1 associates with the α-subunit KCNQ1 to generate the slowly activating, voltage-dependent potassium current (IKs) in the heart that controls the repolarization phase of cardiac action potentials. By contrast, in epithelial cells from the colon, stomach, and kidney, KCNE3 coassembles with KCNQ1 to form K+ channels that are voltage-independent K+ channels in the physiological voltage range and important for controlling water and salt secretion and absorption. How KCNE1 and KCNE3 subunits modify KCNQ1 channel gating so differently is largely unknown. Here, we use voltage clamp fluorometry to determine how KCNE1 and KCNE3 affect the voltage sensor and the gate of KCNQ1. By separating S4 movement and gate opening by mutations or phosphatidylinositol 4,5-bisphosphate depletion, we show that KCNE1 affects both the S4 movement and the gate, whereas KCNE3 affects the S4 movement and only affects the gate in KCNQ1 if an intact S4-to-gate coupling is present. Further, we show that a triple mutation in the middle of the transmembrane (TM) segment of KCNE3 introduces KCNE1-like effects on the second S4 movement and the gate. In addition, we show that differences in two residues at the external end of the KCNE TM segments underlie differences in the effects of the different KCNEs on the first S4 movement and the voltage sensor-to-gate coupling.

  • kcne1 and kcne3 modulate kcnq1 channels by affecting different gating transitions
    Biophysical Journal, 2017
    Co-Authors: Rene Barrosoria, Rosamary Ramentol, Sara I Liin, Marta E Perez, Robert S Kass, Peter H Larsson
    Abstract:

    KCNE β subunits assemble with and modulate the properties of voltage-gated K+ channels. In the heart, KCNE1 associates with KCNQ1 to generate the slowly activating, voltage-dependent IKs current that controls the repolarization phase of cardiac action potentials. By contrast, in epithelial cells from the colon, stomach and kidney, KCNE3 coassembles with the α-subunit KCNQ1 to form apparent voltage-independent K+ channels important for controlling water and salt secretion. How KCNE1 and KCNE3 subunits modify KCNQ1 channel gating so differently is largely unknown. Different molecular mechanisms have been proposed to explain the effects of KCNE1 and KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometry to determine how KCNE1 and KCNE3 affect the voltage sensor and the gate of KCNQ1. By separating S4 movement and gate opening by a mutation, we show that KCNE1 affects both the S4 movement and the gate, whereas KCNE3 directly affects the S4 movement and only indirectly affects the gate in KCNQ1. Further, we show that a triple mutation in KCNE3 converts KCNQ1/KCNE3 channels into KCNQ1/KCNE1-like channels by introducing KCNE1-like effects on the KCNQ1 gate. Our results suggest that the difference between the effects of KCNE1 and KCNE3 on KCNQ1 is that KCNE1 affects both the voltage-sensing domain and the gate, whereas KCNE3 primarily affects the voltage-sensing domain and only indirectly affects the gate.

  • kcne3 stabilizes the voltage sensor s4 of kcnq1 channel kcne1 uncouples s4 and the gate
    Biophysical Journal, 2015
    Co-Authors: Rene Barrosoria, Robert S Kass, Kevin J Sampson, Gary Peng, Peter H Larsson
    Abstract:

    KCNEs are single-span transmembrane β-subunits that assemble with and modulate the biophysical properties of voltage-gated K+ (Kv) channels. In the heart, the pore forming α-subunit KCNQ1 associates with KCNE1 to form the slowly-activating, voltage-gated IKs channels that contribute to the repolarization of the cardiac action potential. In tissues such as the colon, stomach and kidney, KCNQ1 coassembles with the β-subunit KCNE3 to form voltage-independent K+ channels important for K+ and Cl- secretion. KCNE3 has also been shown to be expressed in the human heart, although its physiological function remains unknown. Different mechanisms have been proposed to explain how different KCNE subunits alter KCNQ1 gating and permeation. For instance, KCNE3 is assumed to lock the voltage sensor (S4) of KCNQ1 channel in the activated state, resulting in a constitutively open channel. Here, we use voltage clamp fluorometry (VCF) to understand how KCNE3 affects the voltage sensor and the gate of KCNQ1 channel. We show that KCNE3, contrary to what was previously assumed, allows S4 movement in KCNQ1/KCNE3 channels. KCNE3 shifts the closing and S4 movement of KCNQ1 to extreme hyperpolarized potentials, such that at physiological voltage range (−80 mV to +40 mV), the channel is always open. By decoupling S4 and the gate, either by mutations or PIP2 depletion, we show that KCNE3 mainly affects S4 movement in KCNQ1. Two negatively charged residues in the N-terminus of KCNE3 (D54 and D55) are, at least partly, responsible for stabilizing S4 in an outward position, therefore stabilizing KCNQ1/KCNE3 channels in the activated open state. Further, we unitized a triple mutation of KCNE3, previously shown to convert KCNQ1/KCNE1 channel to a KCNQ1/KCNE1-like current, and observe a decoupling of S4 and the gate