The Experts below are selected from a list of 1761 Experts worldwide ranked by ideXlab platform

David Fedida - One of the best experts on this subject based on the ideXlab platform.

  • the iks ion channel activator mefenamic acid requires kcne1 and modulates channel gating in a subunit dependent manner
    Molecular Pharmacology, 2020
    Co-Authors: Yundi Wang, Jodene Eldstrom, David Fedida
    Abstract:

    The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (IKs), which is partly responsible for cardiomyocyte repolarization and physiological shortening of the cardiac action potential. Mefenamic acid, an NSAID, has been identified as an IKs activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid9s effect on IKs. Using whole-cell patch-clamp, we show that mefenamic acid enhances IKs activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating IKs current into an almost linear current with instantaneous onset and slowed tail current decay, all of which are sensitive to the IKs blocker, HMR1556. Both single channels, which reveal no change in the maximum conductance, and whole-cell studies which reveal a dramatically altered G-V relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the IKs channel, and shifts channel gating kinetics towards more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose- and frequency-dependence of mefenamic acid action on IKs channels. Mutational analysis showed that mefenamic acid9s effect on IKs required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all IKs channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT The channels which generate the IKs current are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by IKs in heartbeat regulation, enhancing IKs current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most IKs activators unfortunately, only work on KCNQ1 alone and not the physiologically relevant IKs channel. We have demonstrated that mefenamic acid can enhance IKs in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.

  • the iks ion channel activator mefenamic acid requires kcne1 and modulates channel gating in a subunit dependent manner
    Molecular Pharmacology, 2020
    Co-Authors: Yundi Wang, Jodene Eldstrom, David Fedida
    Abstract:

    The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (IKs), which is partly responsible for cardiomyocyte repolarization and physiologic shortening of the cardiac action potential. Mefenamic acid, a nonsteroidal anti-inflammatory drug, has been identified as an IKs activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid’s effect on IKs. Using whole-cell patch clamp, we show that mefenamic acid enhances IKs activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating IKs current into an almost linear current with instantaneous onset and slowed tail current decay, sensitive to the IKs blocker (3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy) chroman-4-yl]-N-methylmethanesulfonamide (HMR1556). Both single channels, which reveal no change in the maximum conductance, and whole-cell studies, which reveal a dramatically altered conductance-voltage relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the IKs channel and shifts channel gating kinetics toward more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose and frequency dependence of mefenamic acid action on IKs channels. Mutational analysis showed that mefenamic acid’s effect on IKs required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all IKs channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT The channels which generate the cardiac slow delayed rectifier K+ current (IKs) are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by IKs in heartbeat regulation, enhancing IKs current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most IKs activators, unfortunately, only work on KCNQ1 alone and not the physiologically relevant IKs channel. We have demonstrated that mefenamic acid can enhance IKs in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.

  • iks ion channel pore conductance can result from individual voltage sensor movements
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Maartje Westhoff, Christopher I Murray, Jodene Eldstrom, Emely Thompson, David Fedida
    Abstract:

    The I Ks current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the I Ks complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of I Ks channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in I Ks current kinetics, including the nonlinear depolarization of the conductance–voltage (G–V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

  • the iks channel response to camp is modulated by the kcne1 kcnq1 stoichiometry
    Biophysical Journal, 2018
    Co-Authors: Emely Thompson, Jodene Eldstrom, Maartje Westhoff, Donald Mcafee, David Fedida
    Abstract:

    Abstract The delayed potassium rectifier current, IKs, is assembled from tetramers of KCNQ1 and varying numbers of KCNE1 accessory subunits in addition to calmodulin. This channel complex is important in the response of the cardiac action potential to sympathetic stimulation, during which IKs is enhanced. This is likely due to channels opening more quickly, more often, and to greater sublevel amplitudes during adrenergic stimulation. KCNQ1 alone is unresponsive to cyclic adenosine monophosphate (cAMP), and thus KCNE1 is required for a functional effect of protein kinase A phosphorylation. Here, we investigate the effect that KCNE1 has on the response to 8-4-chlorophenylthio (CPT)-cAMP, a membrane-permeable cAMP analog, by varying the number of KCNE1 subunits present using fusion constructs of IKs with either one (EQQQQ) or two (EQQ) KCNE1 subunits in the channel complex with KCNQ1. These experiments use both whole-cell and single-channel recording techniques. EQQ (2:4, E1:Q1) shows a significant shift in V1/2 of activation from 10.4 mV ± 2.2 in control to −2.7 mV ± 1.2 (p-value: 0.0024). EQQQQ (1:4, E1:Q1) shows a smaller change in response to 8-CPT-cAMP, 6.3 mV ± 2.3 to −3.2 mV ± 3.0 (p-value: 0.0435). As the number of KCNE1 subunits is reduced, the shift in the V1/2 of activation becomes smaller. At the single-channel level, a similar graded change in subconductance occupancy and channel activity is seen in response to 8-CPT-cAMP: the less E1, the smaller the response. However, both constructs show a significant reduction of a similar magnitude in the first latency to opening (EQQ control: 0.90 s ± 0.07 to 0.71 s ± 0.06, p-value: 0.0032 and EQQQQ control: 0.94 s ± 0.09 to 0.56 s ± 0.07, p-value

  • photo cross linking of iks demonstrates state dependent interactions between kcne1 and kcnq1
    Biophysical Journal, 2017
    Co-Authors: Maartje Westhoff, Christopher I Murray, Jodene Eldstrom, David Fedida
    Abstract:

    Abstract The slow delayed rectifier potassium current ( I Ks ) is a key repolarizing current during the cardiac action potential. It consists of four KCNQ1 α -subunits and up to four KCNE1 β -subunits, which are thought to reside within external clefts of the channel. The interaction of KCNE1 with KCNQ1 dramatically delays opening of the channel but the mechanisms by which this occur are not yet fully understood. Here, we have used unnatural amino acid photo-cross-linking to investigate the dynamic interactions that occur between KCNQ1 and KCNE1 during activation gating. The unnatural amino acid p -Benzoylphenylalanine was successfully incorporated into two residues within the transmembrane domain of KCNE1: F56 and F57. UV-induced cross-linking suggested that F56Bpa interacts with KCNQ1 in the open state, whereas F57Bpa interacts predominantly in resting channel conformations. When UV was applied at progressively more depolarized preopen holding potentials, cross-linking of F57Bpa with KCNQ1 was slowed, which indicates that KCNE1 is displaced within the channel's cleft early during activation, or that conformational changes in KCNQ1 alter its interaction with KCNE1. In E1R/R4E KCNQ1, a mutant with constitutively activated voltage sensors, F56Bpa and F57Bpa KCNE1 were cross-linked in open and closed states, respectively, which suggests that their actions are mediated mainly by modulation of KCNQ1 pore function.

Robert S Kass - One of the best experts on this subject based on the ideXlab platform.

  • regulation of iks potassium current by isoproterenol in adult cardiomyocytes requires type 9 adenylyl cyclase
    Cells, 2019
    Co-Authors: Thomas Hof, Robert S Kass, Tanya A Baldwin, Lei Chen, Carmen W. Dessauer
    Abstract:

    The subunits KCNQ1 and KCNE1 generate the slowly activating, delayed rectifier potassium current, IKs, that responds to sympathetic stimulation and is critical for human cardiac repolarization. The A-kinase anchoring protein Yotiao facilitates macromolecular complex formation between IKs and protein kinase A (PKA) to regulate phosphorylation of KCNQ1 and IKs currents following beta-adrenergic stimulation. We have previously shown that adenylyl cyclase Type 9 (AC9) is associated with a KCNQ1-Yotiao-PKA complex and facilitates isoproterenol-stimulated phosphorylation of KCNQ1 in an immortalized cell line. However, requirement for AC9 in sympathetic control of IKs in the heart was unknown. Using a transgenic mouse strain expressing the KCNQ1-KCNE1 subunits of IKs, we show that AC9 is the only adenylyl cyclase (AC) isoform associated with the KCNQ1-KCNE1-Yotiao complex in the heart. Deletion of AC9 resulted in the loss of isoproterenol-stimulated KCNQ1 phosphorylation in vivo, even though AC9 represents less than 3% of total cardiac AC activity. Importantly, a significant reduction of isoproterenol-stimulated IKs currents was also observed in adult cardiomyocytes from IKs-expressing AC9KO mice. AC9 and Yotiao co-localize with N-cadherin, a marker of intercalated disks and cell–cell junctions, in neonatal and adult cardiomyocytes, respectively. In conclusion, AC9 is necessary for sympathetic regulation of PKA phosphorylation of KCNQ1 in vivo and for functional regulation of IKs in adult cardiomyocytes.

  • 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

  • two kcnq1 mutations associated with familial atrial fibrillation s140g and v141m demonstrate distinct voltage sensor phenotypes
    Biophysical Journal, 2015
    Co-Authors: Gary Peng, Peter H Larsson, Rene Barrosoria, Kevin J Sampson, Robert S Kass
    Abstract:

    KCNQ1 is a voltage-dependent potassium channel that is expressed in the heart with the β-subunit KCNE1 to generate the slowly activating IKs current that plays a critical role in cardiac impulse conduction by allowing cardiac repolarization. Mutations in KCNQ1 leading to slowing of channel deactivation have been linked to arrhythmias including Short QT syndrome and familial atrial fibrillation. Two adjacent disease-linked mutations located in the voltage sensing domain (S1-S4) of KCNQ1, S140G and V141M, have been shown to drastically slow current deactivation. While their effects on IKs current deactivation kinetics are similar, their mechanisms may differ. For example, in the absence of KCNE1, S140G but not V141M slows current deactivation. Moreover, crosslinking studies suggest that while V141M can directly interact with KCNE1, S140G cannot. We explore the hypothesis that S140G and V141M, while exhibiting similar effects in IKs current gating, demonstrate distinct phenotypes on voltage sensor movement. Using voltage clamp fluorometry, we studied voltage sensor movement simultaneously with channel current. We found that in the absence of KCNE1, S140G but not V141M slows voltage sensor deactivation, consistent with their effects on current. Furthermore, in the presence of KCNE1, S140G slows voltage sensor movement, but V141M does not slow voltage sensor deactivation. This work shows that while S140G slows both the current deactivation and voltage sensor movement in the presence and absence of KCNE1, in contrast, V141M slows current deactivation only in the presence of KCNE1, without significantly slowing the kinetics of voltage sensor movement. This suggests that these two mutations slow the deactivation of KCNQ1/KCNE1 channels by different mechanisms, an observation made possible by the simultaneous measurement of both voltage sensor movement and channel current.

Jodene Eldstrom - One of the best experts on this subject based on the ideXlab platform.

  • the iks ion channel activator mefenamic acid requires kcne1 and modulates channel gating in a subunit dependent manner
    Molecular Pharmacology, 2020
    Co-Authors: Yundi Wang, Jodene Eldstrom, David Fedida
    Abstract:

    The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (IKs), which is partly responsible for cardiomyocyte repolarization and physiological shortening of the cardiac action potential. Mefenamic acid, an NSAID, has been identified as an IKs activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid9s effect on IKs. Using whole-cell patch-clamp, we show that mefenamic acid enhances IKs activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating IKs current into an almost linear current with instantaneous onset and slowed tail current decay, all of which are sensitive to the IKs blocker, HMR1556. Both single channels, which reveal no change in the maximum conductance, and whole-cell studies which reveal a dramatically altered G-V relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the IKs channel, and shifts channel gating kinetics towards more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose- and frequency-dependence of mefenamic acid action on IKs channels. Mutational analysis showed that mefenamic acid9s effect on IKs required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all IKs channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT The channels which generate the IKs current are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by IKs in heartbeat regulation, enhancing IKs current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most IKs activators unfortunately, only work on KCNQ1 alone and not the physiologically relevant IKs channel. We have demonstrated that mefenamic acid can enhance IKs in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.

  • the iks ion channel activator mefenamic acid requires kcne1 and modulates channel gating in a subunit dependent manner
    Molecular Pharmacology, 2020
    Co-Authors: Yundi Wang, Jodene Eldstrom, David Fedida
    Abstract:

    The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (IKs), which is partly responsible for cardiomyocyte repolarization and physiologic shortening of the cardiac action potential. Mefenamic acid, a nonsteroidal anti-inflammatory drug, has been identified as an IKs activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid’s effect on IKs. Using whole-cell patch clamp, we show that mefenamic acid enhances IKs activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating IKs current into an almost linear current with instantaneous onset and slowed tail current decay, sensitive to the IKs blocker (3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy) chroman-4-yl]-N-methylmethanesulfonamide (HMR1556). Both single channels, which reveal no change in the maximum conductance, and whole-cell studies, which reveal a dramatically altered conductance-voltage relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the IKs channel and shifts channel gating kinetics toward more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose and frequency dependence of mefenamic acid action on IKs channels. Mutational analysis showed that mefenamic acid’s effect on IKs required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all IKs channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT The channels which generate the cardiac slow delayed rectifier K+ current (IKs) are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by IKs in heartbeat regulation, enhancing IKs current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most IKs activators, unfortunately, only work on KCNQ1 alone and not the physiologically relevant IKs channel. We have demonstrated that mefenamic acid can enhance IKs in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.

  • iks ion channel pore conductance can result from individual voltage sensor movements
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Maartje Westhoff, Christopher I Murray, Jodene Eldstrom, Emely Thompson, David Fedida
    Abstract:

    The I Ks current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the I Ks complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of I Ks channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in I Ks current kinetics, including the nonlinear depolarization of the conductance–voltage (G–V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

  • the iks channel response to camp is modulated by the kcne1 kcnq1 stoichiometry
    Biophysical Journal, 2018
    Co-Authors: Emely Thompson, Jodene Eldstrom, Maartje Westhoff, Donald Mcafee, David Fedida
    Abstract:

    Abstract The delayed potassium rectifier current, IKs, is assembled from tetramers of KCNQ1 and varying numbers of KCNE1 accessory subunits in addition to calmodulin. This channel complex is important in the response of the cardiac action potential to sympathetic stimulation, during which IKs is enhanced. This is likely due to channels opening more quickly, more often, and to greater sublevel amplitudes during adrenergic stimulation. KCNQ1 alone is unresponsive to cyclic adenosine monophosphate (cAMP), and thus KCNE1 is required for a functional effect of protein kinase A phosphorylation. Here, we investigate the effect that KCNE1 has on the response to 8-4-chlorophenylthio (CPT)-cAMP, a membrane-permeable cAMP analog, by varying the number of KCNE1 subunits present using fusion constructs of IKs with either one (EQQQQ) or two (EQQ) KCNE1 subunits in the channel complex with KCNQ1. These experiments use both whole-cell and single-channel recording techniques. EQQ (2:4, E1:Q1) shows a significant shift in V1/2 of activation from 10.4 mV ± 2.2 in control to −2.7 mV ± 1.2 (p-value: 0.0024). EQQQQ (1:4, E1:Q1) shows a smaller change in response to 8-CPT-cAMP, 6.3 mV ± 2.3 to −3.2 mV ± 3.0 (p-value: 0.0435). As the number of KCNE1 subunits is reduced, the shift in the V1/2 of activation becomes smaller. At the single-channel level, a similar graded change in subconductance occupancy and channel activity is seen in response to 8-CPT-cAMP: the less E1, the smaller the response. However, both constructs show a significant reduction of a similar magnitude in the first latency to opening (EQQ control: 0.90 s ± 0.07 to 0.71 s ± 0.06, p-value: 0.0032 and EQQQQ control: 0.94 s ± 0.09 to 0.56 s ± 0.07, p-value

  • photo cross linking of iks demonstrates state dependent interactions between kcne1 and kcnq1
    Biophysical Journal, 2017
    Co-Authors: Maartje Westhoff, Christopher I Murray, Jodene Eldstrom, David Fedida
    Abstract:

    Abstract The slow delayed rectifier potassium current ( I Ks ) is a key repolarizing current during the cardiac action potential. It consists of four KCNQ1 α -subunits and up to four KCNE1 β -subunits, which are thought to reside within external clefts of the channel. The interaction of KCNE1 with KCNQ1 dramatically delays opening of the channel but the mechanisms by which this occur are not yet fully understood. Here, we have used unnatural amino acid photo-cross-linking to investigate the dynamic interactions that occur between KCNQ1 and KCNE1 during activation gating. The unnatural amino acid p -Benzoylphenylalanine was successfully incorporated into two residues within the transmembrane domain of KCNE1: F56 and F57. UV-induced cross-linking suggested that F56Bpa interacts with KCNQ1 in the open state, whereas F57Bpa interacts predominantly in resting channel conformations. When UV was applied at progressively more depolarized preopen holding potentials, cross-linking of F57Bpa with KCNQ1 was slowed, which indicates that KCNE1 is displaced within the channel's cleft early during activation, or that conformational changes in KCNQ1 alter its interaction with KCNE1. In E1R/R4E KCNQ1, a mutant with constitutively activated voltage sensors, F56Bpa and F57Bpa KCNE1 were cross-linked in open and closed states, respectively, which suggests that their actions are mediated mainly by modulation of KCNQ1 pore function.

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

  • two kcnq1 mutations associated with familial atrial fibrillation s140g and v141m demonstrate distinct voltage sensor phenotypes
    Biophysical Journal, 2015
    Co-Authors: Gary Peng, Peter H Larsson, Rene Barrosoria, Kevin J Sampson, Robert S Kass
    Abstract:

    KCNQ1 is a voltage-dependent potassium channel that is expressed in the heart with the β-subunit KCNE1 to generate the slowly activating IKs current that plays a critical role in cardiac impulse conduction by allowing cardiac repolarization. Mutations in KCNQ1 leading to slowing of channel deactivation have been linked to arrhythmias including Short QT syndrome and familial atrial fibrillation. Two adjacent disease-linked mutations located in the voltage sensing domain (S1-S4) of KCNQ1, S140G and V141M, have been shown to drastically slow current deactivation. While their effects on IKs current deactivation kinetics are similar, their mechanisms may differ. For example, in the absence of KCNE1, S140G but not V141M slows current deactivation. Moreover, crosslinking studies suggest that while V141M can directly interact with KCNE1, S140G cannot. We explore the hypothesis that S140G and V141M, while exhibiting similar effects in IKs current gating, demonstrate distinct phenotypes on voltage sensor movement. Using voltage clamp fluorometry, we studied voltage sensor movement simultaneously with channel current. We found that in the absence of KCNE1, S140G but not V141M slows voltage sensor deactivation, consistent with their effects on current. Furthermore, in the presence of KCNE1, S140G slows voltage sensor movement, but V141M does not slow voltage sensor deactivation. This work shows that while S140G slows both the current deactivation and voltage sensor movement in the presence and absence of KCNE1, in contrast, V141M slows current deactivation only in the presence of KCNE1, without significantly slowing the kinetics of voltage sensor movement. This suggests that these two mutations slow the deactivation of KCNQ1/KCNE1 channels by different mechanisms, an observation made possible by the simultaneous measurement of both voltage sensor movement and channel current.

  • kcne1 separates the main voltage sensor movement and channel opening in kcnq1 kcne1 channels
    Biophysical Journal, 2014
    Co-Authors: Rene Barrosoria, Sara I Liin, Marta E Perez, Santiago Rebolledo, Robert S Kass, Kevin J Sampson, Peter H Larsson
    Abstract:

    The IKs channel is a slowly activating potassium channel that generates one of the potassium currents that limits the duration of the cardiac action potential. The IKs channel consists of 4 pore forming alpha subunits (KCNQ1) and 2-4 beta subunits (KCNE1). The four KCNQ1 subunits form functional potassium channels, but the KCNE1 beta subunit is necessary to recapitulate the slow activation kinetics of the IKs channel. The mechanism by which KCNE1 slows the kinetics of KCNQ1 channels is a matter of current controversy. Here, we use a combination of voltage clamp fluorometry (VCF) and gating current measurements to show that IKs channel activation occurs in two steps: (1) mutually independent voltage sensor movements in the four KCNQ1 subunits generate the main gating charge movement and underlie the delay in the activation time course of the KCNQ1/KCNE1 currents, (2) a slower and concerted conformational change of all four voltage sensors and the gate, which opens the KCNQ1/KCNE1 channel. Gating currents develop with a similar time and voltage dependence as the first fluorescence component, as if the first component reports on the main S4 charge movement. In contrast to other Kv channels, the voltage dependences of the main voltage sensor movement and channel opening are separated by over 100 mV. The two activation steps in KCNQ1/KCNE1 channels can be farther separated by a disease-causing mutation in KCNE1. We determine rates and voltage dependence of the gating transitions to construct a model for KCNQ1/KCNE1 channels. Our model is consistent with that KCNQ1/KCNE1 channel has a fast S4 movement at negative voltages that moves the majority of gating charge and a slower second conformational change at positive voltages that moves a smaller amount of gating charge and opens the gate.

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

  • two kcnq1 mutations associated with familial atrial fibrillation s140g and v141m demonstrate distinct voltage sensor phenotypes
    Biophysical Journal, 2015
    Co-Authors: Gary Peng, Peter H Larsson, Rene Barrosoria, Kevin J Sampson, Robert S Kass
    Abstract:

    KCNQ1 is a voltage-dependent potassium channel that is expressed in the heart with the β-subunit KCNE1 to generate the slowly activating IKs current that plays a critical role in cardiac impulse conduction by allowing cardiac repolarization. Mutations in KCNQ1 leading to slowing of channel deactivation have been linked to arrhythmias including Short QT syndrome and familial atrial fibrillation. Two adjacent disease-linked mutations located in the voltage sensing domain (S1-S4) of KCNQ1, S140G and V141M, have been shown to drastically slow current deactivation. While their effects on IKs current deactivation kinetics are similar, their mechanisms may differ. For example, in the absence of KCNE1, S140G but not V141M slows current deactivation. Moreover, crosslinking studies suggest that while V141M can directly interact with KCNE1, S140G cannot. We explore the hypothesis that S140G and V141M, while exhibiting similar effects in IKs current gating, demonstrate distinct phenotypes on voltage sensor movement. Using voltage clamp fluorometry, we studied voltage sensor movement simultaneously with channel current. We found that in the absence of KCNE1, S140G but not V141M slows voltage sensor deactivation, consistent with their effects on current. Furthermore, in the presence of KCNE1, S140G slows voltage sensor movement, but V141M does not slow voltage sensor deactivation. This work shows that while S140G slows both the current deactivation and voltage sensor movement in the presence and absence of KCNE1, in contrast, V141M slows current deactivation only in the presence of KCNE1, without significantly slowing the kinetics of voltage sensor movement. This suggests that these two mutations slow the deactivation of KCNQ1/KCNE1 channels by different mechanisms, an observation made possible by the simultaneous measurement of both voltage sensor movement and channel current.

  • kcne1 separates the main voltage sensor movement and channel opening in kcnq1 kcne1 channels
    Biophysical Journal, 2014
    Co-Authors: Rene Barrosoria, Sara I Liin, Marta E Perez, Santiago Rebolledo, Robert S Kass, Kevin J Sampson, Peter H Larsson
    Abstract:

    The IKs channel is a slowly activating potassium channel that generates one of the potassium currents that limits the duration of the cardiac action potential. The IKs channel consists of 4 pore forming alpha subunits (KCNQ1) and 2-4 beta subunits (KCNE1). The four KCNQ1 subunits form functional potassium channels, but the KCNE1 beta subunit is necessary to recapitulate the slow activation kinetics of the IKs channel. The mechanism by which KCNE1 slows the kinetics of KCNQ1 channels is a matter of current controversy. Here, we use a combination of voltage clamp fluorometry (VCF) and gating current measurements to show that IKs channel activation occurs in two steps: (1) mutually independent voltage sensor movements in the four KCNQ1 subunits generate the main gating charge movement and underlie the delay in the activation time course of the KCNQ1/KCNE1 currents, (2) a slower and concerted conformational change of all four voltage sensors and the gate, which opens the KCNQ1/KCNE1 channel. Gating currents develop with a similar time and voltage dependence as the first fluorescence component, as if the first component reports on the main S4 charge movement. In contrast to other Kv channels, the voltage dependences of the main voltage sensor movement and channel opening are separated by over 100 mV. The two activation steps in KCNQ1/KCNE1 channels can be farther separated by a disease-causing mutation in KCNE1. We determine rates and voltage dependence of the gating transitions to construct a model for KCNQ1/KCNE1 channels. Our model is consistent with that KCNQ1/KCNE1 channel has a fast S4 movement at negative voltages that moves the majority of gating charge and a slower second conformational change at positive voltages that moves a smaller amount of gating charge and opens the gate.