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Charles R. Sanders - One of the best experts on this subject based on the ideXlab platform.
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upgraded molecular models of the human KCNQ1 Potassium Channel
PLOS ONE, 2019Co-Authors: Georg Kuenze, Charles R. Sanders, Carlos G Vanoye, Alfred L George, Amanda M Duran, Hope Woods, Kathryn R Brewer, Eli Fritz Mcdonald, Jens MeilerAbstract:The voltage-gated Potassium Channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and various resting voltage sensor structures. Using molecular dynamics (MD) simulations, the capacity of the models to describe experimentally established Channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor–pore domain interactions were validated. Rosetta energy calculations were applied to assess the utility of each model in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 experimentally characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants exhibited no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural basis for KCNQ1 function and can be used to generate hypotheses to explain KCNQ1 dysfunction.
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upgraded molecular models of the human KCNQ1 Potassium Channel
bioRxiv, 2019Co-Authors: Georg Kuenze, Charles R. Sanders, Carlos G Vanoye, Alfred L George, Amanda M Duran, Hope Woods, Kathryn R Brewer, Eli Fritz Mcdonald, Jens MeilerAbstract:Abstract The voltage-gated Potassium Channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and resting voltage sensor structures. Using molecular dynamics (MD) simulations, the models’ capability to describe experimentally established Channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor – pore domain interactions were validated. Rosetta energy calculations were applied to assess the models’ utility in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants had no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural mechanisms of KCNQ1 function and can be used to generate structure-based hypotheses to explain KCNQ1 dysfunction. Author Summary Cardiac rhythm is maintained by synchronized electrical impulses conducted throughout the heart. The Potassium ion Channel KCNQ1 is important for the repolarization phase of the cardiac action potential that underlies these electrical impulses. Heritable mutations in KCNQ1 can lead to Channel loss-of-function (LOF) and predisposition to a life-threatening cardiac arrhythmia. Knowledge of the three-dimensional structure of KCNQ1 is important to understand how mutations lead to LOF and to support structurally-guided design of new anti-arrhythmic drugs. In this work, we present the development and validation of molecular models of human KCNQ1 inferred by homology from the structure of frog KCNQ1. Models were developed for the open Channel state in which Potassium ions can pass through the Channel and the closed state in which the Channel is not conductive. Using molecular dynamics simulations, interactions in the voltage-sensing and pore domain of KCNQ1 and with the membrane lipid PIP2 were analyzed. Energy calculations for KCNQ1 mutations in the voltage-sensing domain reveled that most of the mutations that lead to LOF cause energetic destabilization of the KCNQ1 protein. The results support both the utility of the new models and growing evidence that mutation-induced protein destabilization is a common cause of KCNQ1 dysfunction.
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comprehensive assessment of disease mutant forms of the human KCNQ1 Potassium Channel
Biophysical Journal, 2017Co-Authors: Hui Huang, Keenan C Taylor, Charles R. SandersAbstract:The voltage-gated Potassium Channel KCNQ1 is critical for the cardiac action potential. Mutations in KCNQ1 and its accessory protein KCNE1 are the most common cause of congenital long-QT syndrome (LQTS). There are a variety of mechanisms by which a given mutation may cause KCNQ1 Channel dysfunction and prolonged activation potentials. The ideal treatment of patients harboring a KCNQ1 mutation is dependent on which specific mechanisms cause loss of function. In the present study, we have employed a multidisciplinary approach to systematically investigate the specific effects of 51 KCNQ1 mutations on the Channel structure, stability, trafficking, and (i.e. through collaboration) electrophysiological properties. The 51 mutations, located in the voltage sensor domain (VSD), are disease causing, benign, or of unknown significance. High quality NMR spectra of the isolated wild type VSD and of its mutant forms that are locked in fully activated state or in resting state serve as reference spectra. The 1H-15N TROSY spectrum of each mutant was collected and compared with the reference spectra to determine whether the mutation destabilizes the protein, or shifts the basal activated vs. resting state equilibrium. We have also expressed each mutant full length KCNQ1 in HEK293 cells and quantitatively assessed its total protein expression and cell surface expression using flow cytometry. These results will help elucidate the exact defects of each mutant associated with LQTS, potentially providing information that can be used to inform personalized treatment of LQTS subjects harboring KCNQ1 mutations.This work was supported by NIH Grant RO1 HL122010. We also thank the lab of Prof. Alfred George at Northwestern University for providing the cDNA for the KCNQ1-VSD mutants.
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Structural Analysis of KCNE1 Transmembrane Mutant Yielding KCNE3-Like Function
Biophysical Journal, 2016Co-Authors: Charles R. SandersAbstract:The KCNE family contains five single transmembrane-spanning proteins that modulate the voltage-gated Potassium Channel, KCNQ1 and provide functional diversity to the KCNQ1 Channel. For example, KCNE1 modulates the KCNQ1 Channel by slowing its activation and increasing its conductance. The KCNE3 protein also increases the conductance, but induces constitutive activation of the Channel. In 2001, Melman et al made a series of KCNE1 and KCNE3 chimeras, and identified the transmembrane region as key to distinct functions of KCNE1 and KCNE3. They showed that by swapping three transmembrane residues, T71V72G73, of KCNE3 for KCNE1, F57T58L59, to create a KCNE1/3 chimera they could create a current trace similar to KCNE3 and eliminate KCNE1's ability to delay opening. Thus, KCNE1 with TVG from KCNE3 yields a constitutively active Channel without a delay in opening. We hypothesize that this is due to changes in flexibility of the transmembrane-helix of KCNE1 when TVG triple mutation is present. By using NMR spectroscopy, biochemical studies, and computational docking, we aim to look at structural and conformational differences between KCNE1 and KCNE1TVG. We have expressed and purified KCNE1TVG for NMR studies and collected 2D-NMR spectra using a TROSY-based pulse sequence. Partial backbone assignments of KCNE1TVG have been determined by aligning and transfer assignments of the WT KCNE1 previous determined in our lab. Further 3D experiments have been carried out to complete assignments. Further NMR studied will be done to structurally determine KCNE1TVG. Computational models of KCNE1TVG/KCNQ1 in the open and closed state will be created. These models, along with working models for KCNQ1/KCNE1 will provide insight into how KCNE1TVG interacts with the Channel differently and similarly than both KCNE1 and KCNE3. This will provide insight into how KCNE1TVG interacts with the Channel differently and similarly than both KCNE1 and KCNE3.
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impact of mutations on the structure of the human Potassium Channel KCNQ1
Biophysical Journal, 2016Co-Authors: Hui Huang, Brett M Kroncke, Alfred L George, Keenan C Taylor, Charles R. SandersAbstract:Approximately one in 2000 newborns are affected by congenital long QT syndrome (LQTS), which is a life-threatening cardiac disorder. Mutations in the voltage-gated Potassium Channel KCNQ1 and its accessory protein KCNE1 cause 50% of congenital LQTS. Up to now, more than 400 mutations have been identified in KCNQ1 from LQTS subjects. The mechanistic effects of many of these mutations remain unknown. In the present study, we have investigated the specific effects of 32 KCNQ1 mutations on the structural properties of isolated voltage-sensor domain of KCNQ1 using solution nuclear magnetic resonance spectroscopy. The 32 mutations are disease causing, benign, or of unknown significance. The high quality NMR spectra of wild type and of one engineered double mutation E160R-S225E serve as reference spectra, representing the open state (active conformation) and closed state (inactive conformation) forms of the Channel, respectively. Mutants were labeled with 15N and purified into lysomyristoylphosphatidylglycerol (LMPG) detergent micelles. The 1H-15N TROSY-heteronuclear single quantum coherence spectroscopy spectrum of each mutant was collected and compared with the reference spectra. The effect of each mutation on the equilibrium between the Channel active conformation and inactive conformation was determined. These results will advance our understanding of the molecular basis of LQTS pathogenesis and will be important for the rational design of anti-arrhythmia therapeutics for patients harboring KCNQ1 mutations.
Carlos G Vanoye - One of the best experts on this subject based on the ideXlab platform.
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upgraded molecular models of the human KCNQ1 Potassium Channel
PLOS ONE, 2019Co-Authors: Georg Kuenze, Charles R. Sanders, Carlos G Vanoye, Alfred L George, Amanda M Duran, Hope Woods, Kathryn R Brewer, Eli Fritz Mcdonald, Jens MeilerAbstract:The voltage-gated Potassium Channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and various resting voltage sensor structures. Using molecular dynamics (MD) simulations, the capacity of the models to describe experimentally established Channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor–pore domain interactions were validated. Rosetta energy calculations were applied to assess the utility of each model in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 experimentally characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants exhibited no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural basis for KCNQ1 function and can be used to generate hypotheses to explain KCNQ1 dysfunction.
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upgraded molecular models of the human KCNQ1 Potassium Channel
bioRxiv, 2019Co-Authors: Georg Kuenze, Charles R. Sanders, Carlos G Vanoye, Alfred L George, Amanda M Duran, Hope Woods, Kathryn R Brewer, Eli Fritz Mcdonald, Jens MeilerAbstract:Abstract The voltage-gated Potassium Channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and resting voltage sensor structures. Using molecular dynamics (MD) simulations, the models’ capability to describe experimentally established Channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor – pore domain interactions were validated. Rosetta energy calculations were applied to assess the models’ utility in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants had no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural mechanisms of KCNQ1 function and can be used to generate structure-based hypotheses to explain KCNQ1 dysfunction. Author Summary Cardiac rhythm is maintained by synchronized electrical impulses conducted throughout the heart. The Potassium ion Channel KCNQ1 is important for the repolarization phase of the cardiac action potential that underlies these electrical impulses. Heritable mutations in KCNQ1 can lead to Channel loss-of-function (LOF) and predisposition to a life-threatening cardiac arrhythmia. Knowledge of the three-dimensional structure of KCNQ1 is important to understand how mutations lead to LOF and to support structurally-guided design of new anti-arrhythmic drugs. In this work, we present the development and validation of molecular models of human KCNQ1 inferred by homology from the structure of frog KCNQ1. Models were developed for the open Channel state in which Potassium ions can pass through the Channel and the closed state in which the Channel is not conductive. Using molecular dynamics simulations, interactions in the voltage-sensing and pore domain of KCNQ1 and with the membrane lipid PIP2 were analyzed. Energy calculations for KCNQ1 mutations in the voltage-sensing domain reveled that most of the mutations that lead to LOF cause energetic destabilization of the KCNQ1 protein. The results support both the utility of the new models and growing evidence that mutation-induced protein destabilization is a common cause of KCNQ1 dysfunction.
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a model of human Potassium Channel KCNQ1 modulation by accessory protein kcne3
Biophysical Journal, 2014Co-Authors: Brett M Kroncke, Carlos G Vanoye, Jens Meiler, Wade D Van Horn, D P Nannemann, Charles R. SandersAbstract:Human Potassium Channel KCNQ1 is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. To tune its current output, KCNQ1 complexes with several accessory proteins from the KCNE family. Each KCNE family member modulates KCNQ1 differently: KCNE1 causes the Channel to delay opening and become more conductive in the open state, KCNE3 causes the Channel to be constitutively open and more conductive, and KCNE4 causes the Channel to close. To extend previous efforts to characterize the spectrum of KCNQ1 structure and modulation, we used a hybrid experimental-computational approach to model the KCNQ1-KCNE3 complex. Our strategy was to determine the NMR structure of KCNE3 alone in lipid-mimicking bicelles, build a homology model of KCNQ1, generate in vivo restraints using electrophysiology, and dock the structure of KCNE3 onto the homology model of KCNQ1 using electrophysiology-based restraints to validate and refine the resulting models.The results from this method will be presented.
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working model for the structural basis for kcne1 modulation of the KCNQ1 Potassium Channel
Current Opinion in Structural Biology, 2011Co-Authors: Wade D Van Horn, Carlos G Vanoye, Charles R. SandersAbstract:The voltage-gated Potassium Channel KCNQ1 (Kv7.1) is modulated by KCNE1 (minK) to generate the I Ks current crucial to heartbeat. Defects in either protein result in serious cardiac arrhythmias. Recently developed structural models of the open and closed state KCNQ1/KCNE1 complexes offer a compelling explanation for how KCNE1 slows Channel opening and provides a platform from which to refine and test hypotheses for other aspects of KCNE1 modulation. These working models were developed using an integrative approach based on results from nuclear magnetic resonance spectroscopy, electrophysiology, biochemistry, and computational methods—an approach that can be applied iteratively for model testing and revision. We present a critical review of these structural models, illustrating the strengths and challenges of the integrative approach.
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structural underpinnings for modulation of the voltage gated Potassium Channel KCNQ1 by the kcne family of proteins
Biophysical Journal, 2010Co-Authors: Wade D Van Horn, Congbao Kang, Carlos G Vanoye, Alfred L George, R Welch, Charles R. SandersAbstract:The voltage-gated Potassium Channel KCNQ1 is modulated by KCNE1 to form the IKs current involved in cardiac repolarization. KCNE1 is the best characterized member of a family of modulatory proteins which impart distinct ion Channel physiology. Mutations in KCNQ1 and familial KCNE proteins have been linked to human diseases including congenital deafness and congenital long QT syndrome, which is an inherited predisposition to potentially life-threatening cardiac arrhythmias. The biophysical basis of the KCNE1 modulation of KCNQ1 has been previously characterized in our lab with an interdisciplinary approach utilizing nuclear magnetic resonance (NMR) spectroscopy, electrophysiology, biochemistry, and computational biology. In this work we extend the characterization to include two other family members; namely, KCNE3 and KCNE4. KCNQ1 homology models and the KCNE family proteins KCNE1, KCNE3, and KCNE4 are used as the basis of a comparative study to deduce the molecular mechanisms of voltage-gated Potassium Channel regulation by these accessory subunits. KCNE1 binds to KCNQ1 and causes delayed Channel activation and increased conductance, while, KCNE3 promotes rapid and increased conductance in KCNQ1. On the other hand, KCNE4 binding causes a strict inhibition of KCNQ1 conductance. In this work we present data that suggests the structural biological basis for how the homologous KCNE1, KCNE3, and KCNE4 proteins modulate KCNQ1 in such starkly contrasting manners. This work was supported by NIH grant R01DC007416.
Jens Meiler - One of the best experts on this subject based on the ideXlab platform.
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upgraded molecular models of the human KCNQ1 Potassium Channel
PLOS ONE, 2019Co-Authors: Georg Kuenze, Charles R. Sanders, Carlos G Vanoye, Alfred L George, Amanda M Duran, Hope Woods, Kathryn R Brewer, Eli Fritz Mcdonald, Jens MeilerAbstract:The voltage-gated Potassium Channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and various resting voltage sensor structures. Using molecular dynamics (MD) simulations, the capacity of the models to describe experimentally established Channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor–pore domain interactions were validated. Rosetta energy calculations were applied to assess the utility of each model in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 experimentally characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants exhibited no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural basis for KCNQ1 function and can be used to generate hypotheses to explain KCNQ1 dysfunction.
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upgraded molecular models of the human KCNQ1 Potassium Channel
bioRxiv, 2019Co-Authors: Georg Kuenze, Charles R. Sanders, Carlos G Vanoye, Alfred L George, Amanda M Duran, Hope Woods, Kathryn R Brewer, Eli Fritz Mcdonald, Jens MeilerAbstract:Abstract The voltage-gated Potassium Channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and resting voltage sensor structures. Using molecular dynamics (MD) simulations, the models’ capability to describe experimentally established Channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor – pore domain interactions were validated. Rosetta energy calculations were applied to assess the models’ utility in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants had no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural mechanisms of KCNQ1 function and can be used to generate structure-based hypotheses to explain KCNQ1 dysfunction. Author Summary Cardiac rhythm is maintained by synchronized electrical impulses conducted throughout the heart. The Potassium ion Channel KCNQ1 is important for the repolarization phase of the cardiac action potential that underlies these electrical impulses. Heritable mutations in KCNQ1 can lead to Channel loss-of-function (LOF) and predisposition to a life-threatening cardiac arrhythmia. Knowledge of the three-dimensional structure of KCNQ1 is important to understand how mutations lead to LOF and to support structurally-guided design of new anti-arrhythmic drugs. In this work, we present the development and validation of molecular models of human KCNQ1 inferred by homology from the structure of frog KCNQ1. Models were developed for the open Channel state in which Potassium ions can pass through the Channel and the closed state in which the Channel is not conductive. Using molecular dynamics simulations, interactions in the voltage-sensing and pore domain of KCNQ1 and with the membrane lipid PIP2 were analyzed. Energy calculations for KCNQ1 mutations in the voltage-sensing domain reveled that most of the mutations that lead to LOF cause energetic destabilization of the KCNQ1 protein. The results support both the utility of the new models and growing evidence that mutation-induced protein destabilization is a common cause of KCNQ1 dysfunction.
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a model of human Potassium Channel KCNQ1 modulation by accessory protein kcne3
Biophysical Journal, 2014Co-Authors: Brett M Kroncke, Carlos G Vanoye, Jens Meiler, Wade D Van Horn, D P Nannemann, Charles R. SandersAbstract:Human Potassium Channel KCNQ1 is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. To tune its current output, KCNQ1 complexes with several accessory proteins from the KCNE family. Each KCNE family member modulates KCNQ1 differently: KCNE1 causes the Channel to delay opening and become more conductive in the open state, KCNE3 causes the Channel to be constitutively open and more conductive, and KCNE4 causes the Channel to close. To extend previous efforts to characterize the spectrum of KCNQ1 structure and modulation, we used a hybrid experimental-computational approach to model the KCNQ1-KCNE3 complex. Our strategy was to determine the NMR structure of KCNE3 alone in lipid-mimicking bicelles, build a homology model of KCNQ1, generate in vivo restraints using electrophysiology, and dock the structure of KCNE3 onto the homology model of KCNQ1 using electrophysiology-based restraints to validate and refine the resulting models.The results from this method will be presented.
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an allosteric mechanism for drug block of the human cardiac Potassium Channel KCNQ1
Molecular Pharmacology, 2013Co-Authors: Tao Yang, Charles R. Sanders, Jarrod A. Smith, Jens Meiler, Brenda F. Leake, Dan M. RodenAbstract:The intracellular aspect of the sixth transmembrane segment within the ion-permeating pore is a common binding site for many voltage-gated ion Channel blockers. However, the exact site(s) at which drugs bind remain controversial. We used extensive site-directed mutagenesis coupled with molecular modeling to examine mechanisms in drug block of the human cardiac Potassium Channel KCNQ1. A total of 48 amino acid residues in the S6 segment, S4-S5 linker, and the proximal C-terminus of the KCNQ1 Channel were mutated individually to alanine; alanines were mutated to cysteines. Residues modulating drug block were identified when mutant Channels displayed <50% block on exposure to drug concentrations that inhibited wild-type current by ≥90%. Homology modeling of the KCNQ1 Channel based on the Kv1.2 structure unexpectedly predicted that the key residue modulating drug block (F351) faces away from the permeating pore. In the open-state Channel model, F351 lines a pocket that also includes residues L251 and V254 in S4-S5 linker. Docking calculations indicated that this pocket is large enough to accommodate quinidine. To test this hypothesis, L251A and V254A mutants were generated that display a reduced sensitivity to blockage with quinidine. Thus, our data support a model in which open state block of this Channel occurs not via binding to a site directly in the pore but rather by a novel allosteric mechanism: drug access to a side pocket generated in the open-state Channel configuration and lined by S6 and S4-S5 residues.
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An allosteric mechanism for drug block of the human cardiac Potassium Channel KCNQ1.
Molecular Pharmacology, 2012Co-Authors: Tao Yang, Charles R. Sanders, Jarrod A. Smith, Jens Meiler, Brenda F. Leake, Dan M. RodenAbstract:The intracellular aspect of the sixth transmembrane segment within the ion-permeating pore is a common binding site for many voltage-gated ion Channel blockers. However, the exact site(s) at which drugs bind remain controversial. We used extensive site-directed mutagenesis coupled with molecular modeling to examine mechanisms in drug block of the human cardiac Potassium Channel KCNQ1. A total of 48 amino acid residues in the S6 segment, S4-S5 linker, and the proximal C-terminus of the KCNQ1 Channel were mutated individually to alanine; alanines were mutated to cysteines. Residues modulating drug block were identified when mutant Channels displayed
Alfred L George - One of the best experts on this subject based on the ideXlab platform.
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upgraded molecular models of the human KCNQ1 Potassium Channel
PLOS ONE, 2019Co-Authors: Georg Kuenze, Charles R. Sanders, Carlos G Vanoye, Alfred L George, Amanda M Duran, Hope Woods, Kathryn R Brewer, Eli Fritz Mcdonald, Jens MeilerAbstract:The voltage-gated Potassium Channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and various resting voltage sensor structures. Using molecular dynamics (MD) simulations, the capacity of the models to describe experimentally established Channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor–pore domain interactions were validated. Rosetta energy calculations were applied to assess the utility of each model in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 experimentally characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants exhibited no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural basis for KCNQ1 function and can be used to generate hypotheses to explain KCNQ1 dysfunction.
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upgraded molecular models of the human KCNQ1 Potassium Channel
bioRxiv, 2019Co-Authors: Georg Kuenze, Charles R. Sanders, Carlos G Vanoye, Alfred L George, Amanda M Duran, Hope Woods, Kathryn R Brewer, Eli Fritz Mcdonald, Jens MeilerAbstract:Abstract The voltage-gated Potassium Channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and resting voltage sensor structures. Using molecular dynamics (MD) simulations, the models’ capability to describe experimentally established Channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor – pore domain interactions were validated. Rosetta energy calculations were applied to assess the models’ utility in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants had no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural mechanisms of KCNQ1 function and can be used to generate structure-based hypotheses to explain KCNQ1 dysfunction. Author Summary Cardiac rhythm is maintained by synchronized electrical impulses conducted throughout the heart. The Potassium ion Channel KCNQ1 is important for the repolarization phase of the cardiac action potential that underlies these electrical impulses. Heritable mutations in KCNQ1 can lead to Channel loss-of-function (LOF) and predisposition to a life-threatening cardiac arrhythmia. Knowledge of the three-dimensional structure of KCNQ1 is important to understand how mutations lead to LOF and to support structurally-guided design of new anti-arrhythmic drugs. In this work, we present the development and validation of molecular models of human KCNQ1 inferred by homology from the structure of frog KCNQ1. Models were developed for the open Channel state in which Potassium ions can pass through the Channel and the closed state in which the Channel is not conductive. Using molecular dynamics simulations, interactions in the voltage-sensing and pore domain of KCNQ1 and with the membrane lipid PIP2 were analyzed. Energy calculations for KCNQ1 mutations in the voltage-sensing domain reveled that most of the mutations that lead to LOF cause energetic destabilization of the KCNQ1 protein. The results support both the utility of the new models and growing evidence that mutation-induced protein destabilization is a common cause of KCNQ1 dysfunction.
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impact of mutations on the structure of the human Potassium Channel KCNQ1
Biophysical Journal, 2016Co-Authors: Hui Huang, Brett M Kroncke, Alfred L George, Keenan C Taylor, Charles R. SandersAbstract:Approximately one in 2000 newborns are affected by congenital long QT syndrome (LQTS), which is a life-threatening cardiac disorder. Mutations in the voltage-gated Potassium Channel KCNQ1 and its accessory protein KCNE1 cause 50% of congenital LQTS. Up to now, more than 400 mutations have been identified in KCNQ1 from LQTS subjects. The mechanistic effects of many of these mutations remain unknown. In the present study, we have investigated the specific effects of 32 KCNQ1 mutations on the structural properties of isolated voltage-sensor domain of KCNQ1 using solution nuclear magnetic resonance spectroscopy. The 32 mutations are disease causing, benign, or of unknown significance. The high quality NMR spectra of wild type and of one engineered double mutation E160R-S225E serve as reference spectra, representing the open state (active conformation) and closed state (inactive conformation) forms of the Channel, respectively. Mutants were labeled with 15N and purified into lysomyristoylphosphatidylglycerol (LMPG) detergent micelles. The 1H-15N TROSY-heteronuclear single quantum coherence spectroscopy spectrum of each mutant was collected and compared with the reference spectra. The effect of each mutation on the equilibrium between the Channel active conformation and inactive conformation was determined. These results will advance our understanding of the molecular basis of LQTS pathogenesis and will be important for the rational design of anti-arrhythmia therapeutics for patients harboring KCNQ1 mutations.
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structural underpinnings for modulation of the voltage gated Potassium Channel KCNQ1 by the kcne family of proteins
Biophysical Journal, 2010Co-Authors: Wade D Van Horn, Congbao Kang, Carlos G Vanoye, Alfred L George, R Welch, Charles R. SandersAbstract:The voltage-gated Potassium Channel KCNQ1 is modulated by KCNE1 to form the IKs current involved in cardiac repolarization. KCNE1 is the best characterized member of a family of modulatory proteins which impart distinct ion Channel physiology. Mutations in KCNQ1 and familial KCNE proteins have been linked to human diseases including congenital deafness and congenital long QT syndrome, which is an inherited predisposition to potentially life-threatening cardiac arrhythmias. The biophysical basis of the KCNE1 modulation of KCNQ1 has been previously characterized in our lab with an interdisciplinary approach utilizing nuclear magnetic resonance (NMR) spectroscopy, electrophysiology, biochemistry, and computational biology. In this work we extend the characterization to include two other family members; namely, KCNE3 and KCNE4. KCNQ1 homology models and the KCNE family proteins KCNE1, KCNE3, and KCNE4 are used as the basis of a comparative study to deduce the molecular mechanisms of voltage-gated Potassium Channel regulation by these accessory subunits. KCNE1 binds to KCNQ1 and causes delayed Channel activation and increased conductance, while, KCNE3 promotes rapid and increased conductance in KCNQ1. On the other hand, KCNE4 binding causes a strict inhibition of KCNQ1 conductance. In this work we present data that suggests the structural biological basis for how the homologous KCNE1, KCNE3, and KCNE4 proteins modulate KCNQ1 in such starkly contrasting manners. This work was supported by NIH grant R01DC007416.
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structure of kcne1 and implications for how it modulates the KCNQ1 Potassium Channel
Biochemistry, 2008Co-Authors: Congbao Kang, Frank D Sonnichsen, Jarrod A. Smith, Carlos G Vanoye, Alfred L George, Jens Meiler, Changlin Tian, Charles R. SandersAbstract:KCNE1 is a single-span membrane protein that modulates the voltage-gated Potassium Channel KCNQ1 (KV7.1) by slowing activation and enhancing Channel conductance to generate the slow delayed rectifier current (IKs) that is critical for the repolarization phase of the cardiac action potential. Perturbation of Channel function by inherited mutations in KCNE1 or KCNQ1 results in increased susceptibility to cardiac arrhythmias and sudden death with or without accompanying deafness. Here, we present the three-dimensional structure of KCNE1. The transmembrane domain (TMD) of KCNE1 is a curved α-helix and is flanked by intra- and extracellular domains comprised of α-helices joined by flexible linkers. Experimentally restrained docking of the KCNE1 TMD to a closed state model of KCNQ1 suggests that KCNE1 slows Channel activation by sitting on and restricting the movement of the S4−S5 linker that connects the voltage sensor to the pore domain. We postulate that this is an adhesive interaction that must be disrupted...
Wade D Van Horn - One of the best experts on this subject based on the ideXlab platform.
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a model of human Potassium Channel KCNQ1 modulation by accessory protein kcne3
Biophysical Journal, 2014Co-Authors: Brett M Kroncke, Carlos G Vanoye, Jens Meiler, Wade D Van Horn, D P Nannemann, Charles R. SandersAbstract:Human Potassium Channel KCNQ1 is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. To tune its current output, KCNQ1 complexes with several accessory proteins from the KCNE family. Each KCNE family member modulates KCNQ1 differently: KCNE1 causes the Channel to delay opening and become more conductive in the open state, KCNE3 causes the Channel to be constitutively open and more conductive, and KCNE4 causes the Channel to close. To extend previous efforts to characterize the spectrum of KCNQ1 structure and modulation, we used a hybrid experimental-computational approach to model the KCNQ1-KCNE3 complex. Our strategy was to determine the NMR structure of KCNE3 alone in lipid-mimicking bicelles, build a homology model of KCNQ1, generate in vivo restraints using electrophysiology, and dock the structure of KCNE3 onto the homology model of KCNQ1 using electrophysiology-based restraints to validate and refine the resulting models.The results from this method will be presented.
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working model for the structural basis for kcne1 modulation of the KCNQ1 Potassium Channel
Current Opinion in Structural Biology, 2011Co-Authors: Wade D Van Horn, Carlos G Vanoye, Charles R. SandersAbstract:The voltage-gated Potassium Channel KCNQ1 (Kv7.1) is modulated by KCNE1 (minK) to generate the I Ks current crucial to heartbeat. Defects in either protein result in serious cardiac arrhythmias. Recently developed structural models of the open and closed state KCNQ1/KCNE1 complexes offer a compelling explanation for how KCNE1 slows Channel opening and provides a platform from which to refine and test hypotheses for other aspects of KCNE1 modulation. These working models were developed using an integrative approach based on results from nuclear magnetic resonance spectroscopy, electrophysiology, biochemistry, and computational methods—an approach that can be applied iteratively for model testing and revision. We present a critical review of these structural models, illustrating the strengths and challenges of the integrative approach.
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structural underpinnings for modulation of the voltage gated Potassium Channel KCNQ1 by the kcne family of proteins
Biophysical Journal, 2010Co-Authors: Wade D Van Horn, Congbao Kang, Carlos G Vanoye, Alfred L George, R Welch, Charles R. SandersAbstract:The voltage-gated Potassium Channel KCNQ1 is modulated by KCNE1 to form the IKs current involved in cardiac repolarization. KCNE1 is the best characterized member of a family of modulatory proteins which impart distinct ion Channel physiology. Mutations in KCNQ1 and familial KCNE proteins have been linked to human diseases including congenital deafness and congenital long QT syndrome, which is an inherited predisposition to potentially life-threatening cardiac arrhythmias. The biophysical basis of the KCNE1 modulation of KCNQ1 has been previously characterized in our lab with an interdisciplinary approach utilizing nuclear magnetic resonance (NMR) spectroscopy, electrophysiology, biochemistry, and computational biology. In this work we extend the characterization to include two other family members; namely, KCNE3 and KCNE4. KCNQ1 homology models and the KCNE family proteins KCNE1, KCNE3, and KCNE4 are used as the basis of a comparative study to deduce the molecular mechanisms of voltage-gated Potassium Channel regulation by these accessory subunits. KCNE1 binds to KCNQ1 and causes delayed Channel activation and increased conductance, while, KCNE3 promotes rapid and increased conductance in KCNQ1. On the other hand, KCNE4 binding causes a strict inhibition of KCNQ1 conductance. In this work we present data that suggests the structural biological basis for how the homologous KCNE1, KCNE3, and KCNE4 proteins modulate KCNQ1 in such starkly contrasting manners. This work was supported by NIH grant R01DC007416.