The Experts below are selected from a list of 3105 Experts worldwide ranked by ideXlab platform
Robert S Kass - 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, 2017Co-Authors: Rene Barrosoria, Rosamary Ramentol, Sara I Liin, Marta E Perez, Robert S Kass, Peter H LarssonAbstract: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, 2017Co-Authors: Rene Barrosoria, Rosamary Ramentol, Sara I Liin, Marta E Perez, Robert S Kass, Peter H LarssonAbstract: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, 2015Co-Authors: Rene Barrosoria, Robert S Kass, Gary Peng, Kevin J Sampson, Peter H LarssonAbstract: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
-
long qt mutations at the interface between kcnq1 helix c and KCNE1 disrupt iks regulation by pka and pip2
Journal of Cell Science, 2014Co-Authors: Meidan Dvir, Robert S Kass, Inbal Bental Cohen, Yoni Haitin, Carmen W Dessauer, Joel A Hirsch, Roi Strulovich, Dana Sachyani, Olaf Pongs, Bernard AttaliAbstract:KCNQ1 and KCNE1 co-assembly generates the I KS K+ current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause long QT syndrome (LQT) and atrial fibrillation. The A-kinase anchor protein, yotiao (also known as AKAP9), brings the I KS channel complex together with signaling proteins to achieve regulation upon β1-adrenergic stimulation. Recently, we have shown that KCNQ1 helix C interacts with the KCNE1 distal C-terminus. We postulated that this interface is crucial for I KS channel modulation. Here, we examined the yet unknown molecular mechanisms of LQT mutations located at this intracellular intersubunit interface. All LQT mutations disrupted the internal KCNQ1–KCNE1 intersubunit interaction. LQT mutants in KCNQ1 helix C led to a decreased current density and a depolarizing shift of channel activation, mainly arising from impaired phosphatidylinositol-4,5-bisphosphate (PIP2) modulation. In the KCNE1 distal C-terminus, the LQT mutation P127T suppressed yotiao-dependent cAMP-mediated upregulation of the I KS current, which was caused by reduced KCNQ1 phosphorylation at S27. Thus, KCNQ1 helix C is important for channel modulation by PIP2, whereas the KCNE1 distal C-terminus appears essential for the regulation of I KS by yotiao-mediated PKA phosphorylation.
-
KCNE1 separates the main voltage sensor movement and channel opening in kcnq1 KCNE1 channels
Biophysical Journal, 2014Co-Authors: Rene Barrosoria, Sara I Liin, Marta E Perez, Robert S Kass, Kevin J Sampson, Santiago Rebolledo, Peter H LarssonAbstract: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.
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, 2017Co-Authors: Rene Barrosoria, Rosamary Ramentol, Sara I Liin, Marta E Perez, Robert S Kass, Peter H LarssonAbstract: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, 2017Co-Authors: Rene Barrosoria, Rosamary Ramentol, Sara I Liin, Marta E Perez, Robert S Kass, Peter H LarssonAbstract: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, 2015Co-Authors: Rene Barrosoria, Robert S Kass, Gary Peng, Kevin J Sampson, Peter H LarssonAbstract: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
-
KCNE1 separates the main voltage sensor movement and channel opening in kcnq1 KCNE1 channels
Biophysical Journal, 2014Co-Authors: Rene Barrosoria, Sara I Liin, Marta E Perez, Robert S Kass, Kevin J Sampson, Santiago Rebolledo, Peter H LarssonAbstract: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.
-
long qt mutations and KCNE1 β subunit modulate s4 movement in kcnq1 channel
Biophysical Journal, 2013Co-Authors: Rene Barrosoria, Marta E Perez, Robert S Kass, Kevin J Sampson, Jeremiah D Osteen, Peter H LarssonAbstract:The KCNQ1 potassium channel is expressed in many different cell types and plays widely different physiological roles. For example, in the heart KCNQ1 forms part of the voltage-gated IKs channels that contributes to limiting the duration of the cardiac action potential. The different functions of the KCNQ1 channel are mainly due to the co-assembly of the KCNQ1 channel with different beta subunits from the KCNE family. The IKS channel consists of 4 α-subunits (KCNQ1) which assemble with 2 to 4 β subunits (KCNE1). Mutations in either KCNQ1 or KCNE1 subunit cause multiple cardiac arrhythmia syndromes such as long QT syndrome, short QT syndrome, and familial atrial fibrillation. Here we use Voltage clamp fluorometry (VCF) to directly study the effects of wild type, mutant KCNQ1 and KCNE1 subunits on the voltage sensor movement in KCNQ1 channel. We assess the voltage sensor movement (fluorescence) and channel opening (current), in order to understand the coupling between the KCNQ1 voltage sensor and channel gate in the presence of KCNE1. Our data show that KCNE1 splits the voltage sensor movement in two separates phases. An early phase (1) involving voltage sensor movements to its active state upon changes in the membrane potential occurs at hyperpolarized potentials. A second phase (2) that happens at much more depolarized potentials involves an additional voltage sensor movement which is tightly coupled to channel opening. Mutations in KCNE1 that cause arrhythmia shift the voltage dependence of the voltage sensor movements of either phases 1 or 2 (or both), revealing some of the molecular mechanisms underlying the pathophysiology of these arrhythmia-causing mutations. Our data suggests a putative mechanism for how KCNE1 exerts its effects on the voltage sensor movement during IKs channel activation.
Gea-ny Tseng - One of the best experts on this subject based on the ideXlab platform.
-
adult ventricular myocytes segregate kcnq1 and KCNE1 to keep the iks amplitude in check until when larger iks is needed
Circulation-arrhythmia and Electrophysiology, 2017Co-Authors: Min Jiang, Yuhong Wang, Gea-ny TsengAbstract:Background— KCNQ1 and KCNE1 assemble to form the slow delayed rectifier ( I Ks ) channel critical for shortening ventricular action potentials during high β-adrenergic tone. However, too much I Ks under basal conditions poses an arrhythmogenic risk. Our objective is to understand how adult ventricular myocytes regulate the I Ks amplitudes under basal conditions and in response to stress. Methods and Results— We express fluorescently tagged KCNQ1 and KCNE1 in adult ventricular myocytes and follow their biogenesis and trafficking paths. We also study the distribution patterns of native KCNQ1 and KCNE1, and their relationship to I Ks amplitudes, in chronically stressed ventricular myocytes, and use COS-7 cell expression to probe the underlying mechanism. We show that KCNQ1 and KCNE1 are both translated in the perinuclear region but traffic by different routes, independent of each other, to their separate subcellular locations. KCNQ1 mainly resides in the jSR (junctional sarcoplasmic reticulum), whereas KCNE1 resides on the cell surface. Under basal conditions, only a small portion of KCNQ1 reaches the cell surface to support the I Ks function. However, in response to chronic stress, KCNQ1 traffics from jSR to the cell surface to boost the I Ks amplitude in a process depending on Ca binding to CaM (calmodulin). Conclusions— In adult ventricular myocytes, KCNE1 maintains a stable presence on the cell surface, whereas KCNQ1 is dynamic in its localization. KCNQ1 is largely in an intracellular reservoir under basal conditions but can traffic to the cell surface and boost the I Ks amplitude in response to stress.
-
Building KCNQ1/KCNE1 Channel Models and Probing their Interactions by Molecular-Dynamics Simulations
Biophysical Journal, 2013Co-Authors: Yu Xu, Mei Zhang, Yuhong Wang, Min Jiang, Xuan-yu Meng, Gea-ny TsengAbstract:The slow delayed rectifier (IKs) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of IKs-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in IKs channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance.
-
building kcnq1 KCNE1 channel models and probing their interactions by molecular dynamics simulations
Biophysical Journal, 2013Co-Authors: Yu Xu, Mei Zhang, Yuhong Wang, Min Jiang, Xuan-yu Meng, Gea-ny TsengAbstract:The slow delayed rectifier (IKs) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of IKs-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in IKs channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance.
-
Structural Basis for Differential KCNQ1 Interactions with KCNE1 and KCNE2 in the Extracellular Juxtamembrane Region
Biophysical Journal, 2013Co-Authors: Mei Zhang, Yuhong Wang, Min Jiang, Gea-ny TsengAbstract:KCNE1 and KCNE2 are both expressed in human heart and can associate with KCNQ1. KCNE1 and KCNE2 share transmembrane topology and sequence homology, yet they differ in KCNQ1 modulation (KCNE1 slows KCNQ1 Closed-to-Open transition, while KCNE2 slows KCNQ1 Open-to-Closed transition) and pharmacology (KCNQ1/KCNE2 is 5 and 10 fold less sensitive than KCNQ1/KCNE1 to niflumic acid, IKs activator, and azimilide, IKs suppressor). Previous work has shown that the extracellular juxtamembrane (EJM) region of KCNE1 interacts with the extracellular surface of KCNQ1 to modulate gating kinetics and to form IKs activator binding sites. This prompts us to compare the EJMs of the two KCNE subunits in terms of KCNQ1 interaction. We apply cysteine (Cys) scanning mutagenesis to EJMs of KCNE1 and KCNE2 and analyze the patterns of functional perturbation when coexpressed with KCNQ1. We use methanethiosulfonate (MTS) reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Finally we probe disulfide formation between Cys engineered into the EJMs of KCNE subunits and those engineered into extracellular surface of KCNQ1. The EJM of KCNE1 makes frequent contacts with KCNQ1, so that MTS modification of exposed Cys side chains can affect channel gating. KCNQ1/KCNE2 is largely indifferent to MTS modification of exposed Cys side chains engineered to EJM of KCNE2. However, MTS can slowly access 7 consecutive hydrophobic positions in the beginning of KCNE2 transmembrane domain, as if there is a crevice between KCNQ1 and KCNE2. Disulfide trapping experiments suggest that KCNE2 is leaning more toward KCNQ1 S2 than KCNE1. We propose that KCNE2 interferes with S2-S4 interactions during KCNQ1 Open-to-Closed transition and thus slows deactivation. This weakens sensitivity to niflumic acid and azimilide by affecting binding site directly or by an allosteric mechanism.
-
Probing the structural basis for differential KCNQ1 modulation by KCNE1 and KCNE2.
The Journal of General Physiology, 2012Co-Authors: Yuhong Wang, Mei Zhang, Min Jiang, Dimitar P. Zankov, Yu Xu, Gea-ny TsengAbstract:KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a “repolarization reserve” in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.
Changlin Tian - One of the best experts on this subject based on the ideXlab platform.
-
a distinct three helix centipede toxin ssd609 inhibits i ks channels by interacting with the KCNE1 auxiliary subunit
Scientific Reports, 2015Co-Authors: Fangming Wu, Changlin Tian, Longhua Zhang, Xingwang Yang, Chenyang Wang, Yiming Li, Shufang He, Yun ZhangAbstract:KCNE1 is a single-span transmembrane auxiliary protein that modulates the voltage-gated potassium channel KCNQ1. The KCNQ1/KCNE1 complex in cardiomyocytes exhibited slow activated potassium (Iks) currents. Recently, a novel 47-residue polypeptide toxin SSD609 was purified from Scolopendra subspinipes dehaani venom and showed Iks current inhibition. Here, chemically synthesized SSD609 was shown to exert Iks inhibition in extracted guinea pig cardiomyocytes and KCNQ1/KCNE1 current attenuation in CHO cells. The K+ current attenuation of SSD609 showed decent selectivity among different auxiliary subunits. Solution nuclear magnetic resonance analysis of SSD609 revealed a distinctive three-helix conformation that was stabilized by a new disulfide bonding pattern as well as segregated surface charge distribution. Structure-activity studies demonstrated that negatively charged Glu19 in the amphipathic extracellular helix of KCNE1 was the key residue that interacted with SSD609. The distinctive three-helix centipede toxin SSD609 is known to be the first polypeptide toxin acting on channel auxiliary subunit KCNE1, which suggests a new type of pharmacological regulation for ion channels in cardiomyocytes.
-
differential modulations of kcnq1 by auxiliary proteins KCNE1 and kcne2
Scientific Reports, 2015Co-Authors: Pan Li, Changlin Tian, Wenping Zeng, Fangming Wu, Longhua Zhang, Sheng Wang, Jiuping DingAbstract:KCNQ1 channels play vital roles in cardiovascular, gastric and other systems. The conductance and dynamics of KCNQ1 could be modulated by different single transmembrane helical auxiliary proteins (such as KCNE1, KCNE2 and others). In this study, detail KCNQ1 function modulations by different regions of KCNE1 or KCNE2 were examined using combinational methods of electrophysiology, immunofluorescence, solution NMR and related backbone flexibility analysis. In the presence of KCNE2 N-terminus, decreased surface expression and consequent low activities of KCNQ1 were observed. The transmembrane domains (TMDs) of KCNE1 and KCNE2 were illustrated to associate with the KCNQ1 channel in different modes: Ile64 in KCNE2-TMD interacting with Phe340 and Phe275 in KCNQ1, while two pairs of interacting residues (Phe340-Thr58 and Ala244-Tyr65) in the KCNQ1/KCNE1 complex. The KCNE1 C-terminus could modulate gating property of KCNQ1, whereas KCNE2 C-terminus had only minimal influences on KCNQ1. All of the results demonstrated different KCNQ1 function modulations by different regions of the two auxiliary proteins.
-
KCNE2 uses More Domains than KCNE1 to Modulate KCNQ1 Channel Function
Biophysical Journal, 2011Co-Authors: Yuhong Wang, Mei Zhang, Min Jiang, Pan Li, Xuan-yu Meng, Dimitar P. Zankov, Changlin Tian, Gea-ny TsengAbstract:KCNE1 & KCNE2 are both single membrane-passing peptides with amino- & carboxyl ends in extra- & intra-cellular compartments. They also share high sequence homology in the transmembrane (TM) and juxtamembranous regions. Yet the two have distinctly different effects when associated with the KCNQ1 channel. Understanding the structural basis for differential KCNQ1 modulation by the 2 KCNE subunits is prerequisite to drug design targeting such interactions. We use the techniques of NMR, cysteine (Cys)-scanning mutagenesis and voltage clamping to explore this issue. We learn the following: (1) KCNE2 differs from KCNE1 in the extracellular juxtamembranous region (helix vs unstructured loop). Cys substitution in this region has distinct impact on the gating kinetics and/or pore conductance of the KCNQ1/KCNE2 channel complex, but has little or no effects on the KCNQ1/KCNE1 channel function. (2) KCNE2 & KCNE1 sequences diverge in the carboxyl end. Truncating this region of KCNE1 (93 −129) does not interfere with its ability to modulate KCNQ1, while truncating the corresponding region of KCNE2 (98-123) abolishes its function as a KCNQ1 modulator. Intracellular application of a peptide corresponding to KCNE2 aa 98-123 reduces currents through KCNQ1, increases currents through KCNQ1/KCNE2, but has no effects on currents through KCNQ1/KCNE1. (3) Cys substitution along the TM helices of KCNE1 and KCNE2 affects the gating kinetics and/or pore conductance of the KCNQ1/KCNE channel complexes. Structural alignment suggests a rotation of KCNE2 relative to KCNE1 in terms of helical faces interacting with the pore domain and voltage-sensing domain of the KCNQ1 channel. We propose that while both KCNE subunits utilize their TM helices to interact with KCNQ1, the more rigid helical structure of KCNE2 allows it to allosterically modulate the KCNQ1 gating and ion permeation properties by the extracellular juxtamembranous and cytoplasmic carboxyl domains.
-
kcnq1 KCNE1 assembly co translation not required
Channels, 2010Co-Authors: Carlos G Vanoye, Changlin Tian, Charles R. Sanders, Richard C Welch, Alfred L GeorgeAbstract:Voltage-gated potassium channels are often assembled with accessory proteins which increases their functional diversity. KCNE proteins are small accessory proteins that modulate voltage-gated potassium (KV) channels. Although the functional effects of various KCNE proteins have been described, many questions remain regarding their assembly with the pore-forming subunits. For example, while previous experiments with some KV channels suggest that the association of the pore-subunit with the accessory subunits occurs co-translationally in the endoplasmic reticulum, it is not known whether KCNQ1 assembly with KCNE1 occurs in a similar manner to generate the medically important cardiac slow delayed rectifier current (IKs). In this study we used a novel approach to demonstrate that purified recombinant human KCNE1 protein (prKCNE1) modulates KCNQ1 channels heterologously expressed in Xenopus oocytes resulting in generation of IKs. Incubation of KCNQ1-expressing oocytes with cycloheximide did not prevent IKs exp...
-
KCNQ1/KCNE1 assembly, co-translation not required
Channels, 2010Co-Authors: Carlos G Vanoye, Changlin Tian, Charles R. Sanders, Richard C Welch, Alfred L GeorgeAbstract:Voltage-gated potassium channels are often assembled with accessory proteins which increases their functional diversity. KCNE proteins are small accessory proteins that modulate voltage-gated potassium (KV) channels. Although the functional effects of various KCNE proteins have been described, many questions remain regarding their assembly with the pore-forming subunits. For example, while previous experiments with some KV channels suggest that the association of the pore-subunit with the accessory subunits occurs co-translationally in the endoplasmic reticulum, it is not known whether KCNQ1 assembly with KCNE1 occurs in a similar manner to generate the medically important cardiac slow delayed rectifier current (IKs). In this study we used a novel approach to demonstrate that purified recombinant human KCNE1 protein (prKCNE1) modulates KCNQ1 channels heterologously expressed in Xenopus oocytes resulting in generation of IKs. Incubation of KCNQ1-expressing oocytes with cycloheximide did not prevent IKs exp...
Koichi Nakajo - One of the best experts on this subject based on the ideXlab platform.
-
stoichiometry of the kcnq1 KCNE1 ion channel complex
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Koichi Nakajo, Yoshihiro Kubo, Maximilian H Ulbrich, Ehud Y IsacoffAbstract:The KCNQ1 voltage-gated potassium channel and its auxiliary subunit KCNE1 play a crucial role in the regulation of the heartbeat. The stoichiometry of KCNQ1 and KCNE1 complex has been debated, with some results suggesting that the four KCNQ1 subunits that form the channel associate with two KCNE1 subunits (a 4∶2 stoichiometry), while others have suggested that the stoichiometry may not be fixed. We applied a single molecule fluorescence bleaching method to count subunits in many individual complexes and found that the stoichiometry of the KCNQ1 - KCNE1 complex is flexible, with up to four KCNE1 subunits associating with the four KCNQ1 subunits of the channel (a 4∶4 stoichiometry). The proportion of the various stoichiometries was found to depend on the relative expression densities of KCNQ1 and KCNE1. Strikingly, both the voltage-dependence and kinetics of gating were found to depend on the relative densities of KCNQ1 and KCNE1, suggesting the heart rhythm may be regulated by the relative expression of the auxiliary subunit and the resulting stoichiometry of the channel complex.
-
stoichiometry of kcnq1 KCNE1 ion channel complex is flexible and density dependent
Biophysical Journal, 2010Co-Authors: Koichi Nakajo, Yoshihiro Kubo, Maximilian H Ulbrich, Ehud Y IsacoffAbstract:Many membrane proteins including ion channels form multi-molecular complexes. Because the composition of a molecular complex may define its functional properties, it is important to know its stoichiometry. KCNQ1 encodes a voltage-gated potassium channel alpha subunit, and four KCNQ1 subunits form one ion channel. KCNQ1 channel forms a molecular complex with auxiliary subunit KCNE proteins. In the heart the KCNQ1-KCNE1 complex underlies slowly-activating IKs current, which plays a significant role in regulation of the cardiac action potential. Assuming a fixed KCNQ1-KCNE1 stoichiometry macroscopic current measurements led earlier investigators to the conclusion that each 4-subunit channel is associated with two KCNE1 subunits (4:2 subunit stoichiometry). We asked whether the KCNQ1-KCNE1 stoichiometry is indeed fixed by counting subunits in many individual complexes using TIRF microscopy (Ulbrich and Isacoff, 2007, 2008). We expressed GFP-tagged KCNQ1 or KCNE1 in Xenopus oocytes at low density and counted bleaching steps in many fluorescent spots corresponding to single channel complexes. First, we confirmed that KCNQ1 forms a tetramer. Next we counted GFP-tagged KCNE1 subunits co-expressed with mCherry-tagged KCNQ1. We observed up to four bleaching steps from GFP-KCNE1 co-localized with mCherry, indicating that up to four KCNE1 subunits can bind to one KCNQ1 tetrameric channel. We find that the number of KCNE1 subunits per complex increases as the expression of KCNE1 is raised relative to that of KCNQ1. Our results suggest that modulation of KCNQ channels may be regulated by the level of expression of KCNE subunits.
-
KCNE1 and kcne3 stabilize and or slow voltage sensing s4 segment of kcnq1 channel
The Journal of General Physiology, 2007Co-Authors: Koichi Nakajo, Yoshihiro KuboAbstract:KCNQ1 is a voltage-dependent K+ channel whose gating properties are dramatically altered by association with auxiliary KCNE proteins. For example, KCNE1, which is mainly expressed in heart and inner ear, markedly slows the activation kinetics of KCNQ1. Whether the voltage-sensing S4 segment moves differently in the presence of KCNE1 is not yet known, however. To address that question, we systematically introduced cysteine mutations, one at a time, into the first half of the S4 segment of human KCNQ1. A226C was found out as the most suited mutant for a methanethiosulfonate (MTS) accessibility analysis because it is located at the N-terminal end of S4 segment and its current was stable with repetitive stimuli in the absence of MTS reagent. MTS accessibility analysis revealed that the apparent second order rate constant for modification of the A226C mutant was state dependent, with faster modification during depolarization, and was 13 times slower in the presence of KCNE1 than in its absence. In the presence of KCNE3, on the other hand, the second order rate constant for modification was not state dependent, indicating that the C226 residue was always exposed to the extracellular milieu, even at the resting membrane potential. Taken together, these results suggest that KCNE1 stabilizes the S4 segment in the resting state and slows the rate of transition to the active state, while KCNE3 stabilizes the S4 segment in the active state. These results offer new insight into the mechanism of KCNQ1 channel modulation by KCNE1 and KCNE3.
-
KCNE1 and KCNE3 Stabilize and/or Slow Voltage Sensing S4 Segment of KCNQ1 Channel
The Journal of General Physiology, 2007Co-Authors: Koichi Nakajo, Yoshihiro KuboAbstract:KCNQ1 is a voltage-dependent K+ channel whose gating properties are dramatically altered by association with auxiliary KCNE proteins. For example, KCNE1, which is mainly expressed in heart and inner ear, markedly slows the activation kinetics of KCNQ1. Whether the voltage-sensing S4 segment moves differently in the presence of KCNE1 is not yet known, however. To address that question, we systematically introduced cysteine mutations, one at a time, into the first half of the S4 segment of human KCNQ1. A226C was found out as the most suited mutant for a methanethiosulfonate (MTS) accessibility analysis because it is located at the N-terminal end of S4 segment and its current was stable with repetitive stimuli in the absence of MTS reagent. MTS accessibility analysis revealed that the apparent second order rate constant for modification of the A226C mutant was state dependent, with faster modification during depolarization, and was 13 times slower in the presence of KCNE1 than in its absence. In the presence of KCNE3, on the other hand, the second order rate constant for modification was not state dependent, indicating that the C226 residue was always exposed to the extracellular milieu, even at the resting membrane potential. Taken together, these results suggest that KCNE1 stabilizes the S4 segment in the resting state and slows the rate of transition to the active state, while KCNE3 stabilizes the S4 segment in the active state. These results offer new insight into the mechanism of KCNQ1 channel modulation by KCNE1 and KCNE3.