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Activation Dynamics of Sodium Ion Channel
438a
Tuesday, March 1, 2016
membrane-spanning segments (S1-S6). S1-S4 form voltage-sensing domains (VSDs), and S5-S6 create the ion-conducting pore. The distinct therapeutic action of subclasses Ia, Ib, and Ic have been traditionally attributed to differences in NaV1.5 access and pore-binding rate. However, others have shown that lidocaine, a local anesthetic and Class Ib anti-arrhythmic interacts with the muscle Naþ channel, NaV1.4, DIII-VSD. Thus, we tested the hypothesis that Class I drug interaction with the NaV1.5 VSDs significantly determines the therapeutic phenotype. Methods: Previously, we created four NaV1.5 DNA constructs with a cysteine introduced to the extracellular S4 of individual VSDs. Channels encoded by these constructs are expressed in Xenopus oocytes and cysteine-labeled with the TAMRA-MTS fluorophore. Ionic current and fluorescence emission corresponding to changes in VSD conformation are then simultaneously recorded using the cut-open oocyte configuration. After control recordings, antiarrhythmics are administered to the internal solution. When currents are 80% blocked, VSD kinetics are measured. Results: We have not observed significant interaction of Class I drugs with the DI, DII, or DIV VSDs. In contrast, quinidine, lidocaine, and ranolazine all uniquely shift DIII-VSD activation. During control recordings, we observe DIII-VSD activation has V1/2=-108.9652.72mV. Lidocaine and ranolazine both induced a hyperpolarizing DIII-VSD activation shift (V1/2=147.2854.17mV, S.E.M., p=0.003, V1/2=-143.0951.94mV, S.E.M, p=0.001, respectively) while quinidine caused a large depolarizing shift (V1/2=80.6754.39mV, S.E.M, p=0.007). Conclusions: Drug interaction with the DIII-VSD has been tightly linked to use-dependent block of the late Naþ current, a hallmark class Ib drugs. In contrast, class Ia drugs typically reduce peak Naþ. Because the DIII-VSD is tightly coupled to NaV channel gating, we propose that the differences in DIII-VSD interaction are determining their unique therapeutic phenotypes. 2167-Pos Board B311 Biophysical Characterization of Two Nav1.4 Mutations Identified in Patients with Cold-Induced Myotonia and Periodic Paralysis Hugo Poulin1, Pascal Gosselin-Badaroudine1, Karima Habbout2, Savine Vicart3, Damien Syternberg4, Serena Giuliano2, Sophie Nicole4, Said Bendahhou2, Mohamed Chahine1. 1 Medicine, Laval University, Que´bec, QC, Canada, 2University Nice SophiaAntipolis, Nice, France, 3Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France, 4Sorbonne universite´s, Paris, France. Mutations in SCN4A gene encoding Nav1.4, the skeletal muscle voltage-gated Na channel underlie several skeletal muscle ion channelopathies whose two major phenotypes include enhanced excitability (myotonia), transient loss of excitability (periodic paralysis), a fluctuation between these two conditions or severe loss of function (myasthenia). Here, we report two novel dominant SCN4A missense mutations: R1451C, found in a patient with paramyotonia congenita (PMC) exhibiting coldinduced myotonia, and R1451L found in a patient diagnosed with periodic paralysis. These mutations are located in the transmembrane segment S4 of DIV domain. To elucidate the mechanism underlying the phenotypes caused by R1451C and R1451L, we used the whole-cell patch-clamp technique to study tsA201 cells expressing WT, R1451C or R1451L channels. Our results show that both mutations impaired fast inactivation kinetics at room temperature, with R1451L being the most affected. However, R1451C exhibited a unique temperature-dependent alteration of the fast inactivation kinetics that can explain the occurrence of myotonia in cold environment. Slow inactivation was assessed for both mutants, and revealed that the recovery from slow inactivation was only slowed for R1451L. This could explain the periodic paralysis phenotype of the patient. Homology modeling and in silico mutagenesis of R1451C and R1451L in the resting state based on a previous model of the channel was also used to probe the potential structural differences between these two mutations. The model revealed a reorganization of the hydrogen bonds between S4 and other segments in both mutations. We conclude from our investigation that the nature of the mutation is accountable for the clinical differences revealed in the patient carriers. 2168-Pos Board B312 Modeling Ion Channel Kinetics with Parameter Constraints Cynthia B. Lombardo, Marco A. Navarro, Autoosa Salari, Lorin S. Milescu. University of Missouri, Columbia, MO, USA. Ion channel gating mechanisms can be complex and difficult to extract from experimental data. A solution is to apply parameter constraints, which reflect prior knowledge or tested hypotheses and reduce model complexity and speed
up computation. Soft constraints balance the existing knowledge with the new experimental data and limit the parameter search engine to a smaller space of more acceptable values. In contrast, hard constraints enforce a mathematical relationship involving one or more parameters of the model. These constraints can be formulated as an invertible transformation between a set of model parameters and a set of ‘‘free’’ parameters. Each constraint reduces the number of free parameters by one. Linear constraints, such as microscopic reversibility or scaling between sequential transitions, can be conveniently obtained with the singular value decomposition. Here, we show how this method can be generalized to implement arbitrary linear constraints. We also show how to make these constraints depend on arbitrary model parameters. This can be applied, for example, to enforce allosteric constraints where the allosteric factor itself is a free parameter. Furthermore, we explore some useful ways for implementing soft constraints. 2169-Pos Board B313 Defining the Protein:Protein Interaction Interface of FGF14:Nav1.6 Complex Aditya K. Singh, Syed R. Ali, Fernanda Laezza. Department of Pharmacology & Toxicology, The University of Texas Medical Branch, Galveston, TX, USA. The voltage-gated Naþ (Nav) channel is composed of transmembrane spanning domains and of a cytosolic C-terminal tail which regulates channel function through protein:protein interactions (PPI) with auxiliary proteins, including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF (iFGF) family. In addition to binding to the Nav C-tail, FGF14 forms homodimers and previous structural studies have proposed a conserved surface common for the Nav channel and the iFGF homodimer formation. Seeking for potential differences between the FGF14:Nav1.6-C-tail complex and the FGF14:FGF14 dimer interface, we have engineered model-based amino acid residue mutations at predicted FGF14 hot-spots and begun to screen for their impact on the protein complex stability. Using the in-cell split-luciferase complementation assay to reconstitute the FGF14:Nav1.6-C-tail and the FGF14:FGF14 complex, we identified a point of divergence at the FGF14V160 residue whose mutation led to opposite effects on the relative binding affinity to the FGF14 monomer versus the Nav1.6-C-tail. Functional studies using whole-cell patch-clamp electrophysiology indicated that V160 is a critical residue for FGF14 modulation of Nav1.6-mediated currents that can be abolished by Ala mutation. Initial studies using intrinsic fluorescence showed efficient interaction between purified FGF14 and the Nav1.6 C-tail. Surface plasmon resonance and isothermal titration calorimetry measurements to evaluate the role of V160 in regulating binding affinity of FGF14 to the Nav1.6 C-tail are underway. With its unique role in the regulating the FGF14 binding and function, the V160 residue is well-positioned as target site for PPI-based drug development against Nav channels. Supported by: R01MH095995 (FL) and the Gulf Coast Consortia NIGMS Grant No.1 T32 GM089657-04 (SRA). 2170-Pos Board B314 Activation Dynamics of Sodium Ion Channel Matthew Harrigan, Vijay Pande. Stanford, Stanford, CA, USA. Voltage gated sodium channels initiate signaling in neurons and other cells which go on to become sensations of pleasure and pain, as well as thoughts and feelings. Sodium channel malfunction has been linked with cardiac arrhythmia, epilepsy, and neuropathic pain. We performed large-scale molecular dynamics simulations of the prokaryotic voltage gated sodium channel to probe dynamics and conformational change of channel activation. In collaboration with experimentalists, we propose a model for binding of batrachotoxin.
Voltage-gated Ca Channels 2171-Pos Board B315 Switchable Cardiac L Type Ca2D Channel Transcript by Mineralocorticoid Pathway Thassio Mesquita1, Gaelle Auguste1, Jessica Sabourin1, Gema Ruiz-Hurtado1, Vale´rie Rouffiac2, Florian Le-Billan3, Je´roˆme Fagart3, Florence Lefebvre1, Say Viengchareun3, Eric Morel1, Ana Maria Gomez1, Marc Lombe`s3, Jean Pierre benitah1. 1 UMR-S 1180, Inserm, Univ. Paris-Sud, Universite´ Paris-Saclay, ChatenayMalabry, France, 2Imaging and Cytometry Platform, UMR 8081 IR4M, Gustave Roussy Institute, Villejuif, France, 3UMR-S 1185, Inserm, Univ. Paris-Sud, Universite´ Paris-Saclay, Le Kremlin-Biceˆtre, France.
Tuesday, March 1, 2016
membrane-spanning segments (S1-S6). S1-S4 form voltage-sensing domains (VSDs), and S5-S6 create the ion-conducting pore. The distinct therapeutic action of subclasses Ia, Ib, and Ic have been traditionally attributed to differences in NaV1.5 access and pore-binding rate. However, others have shown that lidocaine, a local anesthetic and Class Ib anti-arrhythmic interacts with the muscle Naþ channel, NaV1.4, DIII-VSD. Thus, we tested the hypothesis that Class I drug interaction with the NaV1.5 VSDs significantly determines the therapeutic phenotype. Methods: Previously, we created four NaV1.5 DNA constructs with a cysteine introduced to the extracellular S4 of individual VSDs. Channels encoded by these constructs are expressed in Xenopus oocytes and cysteine-labeled with the TAMRA-MTS fluorophore. Ionic current and fluorescence emission corresponding to changes in VSD conformation are then simultaneously recorded using the cut-open oocyte configuration. After control recordings, antiarrhythmics are administered to the internal solution. When currents are 80% blocked, VSD kinetics are measured. Results: We have not observed significant interaction of Class I drugs with the DI, DII, or DIV VSDs. In contrast, quinidine, lidocaine, and ranolazine all uniquely shift DIII-VSD activation. During control recordings, we observe DIII-VSD activation has V1/2=-108.9652.72mV. Lidocaine and ranolazine both induced a hyperpolarizing DIII-VSD activation shift (V1/2=147.2854.17mV, S.E.M., p=0.003, V1/2=-143.0951.94mV, S.E.M, p=0.001, respectively) while quinidine caused a large depolarizing shift (V1/2=80.6754.39mV, S.E.M, p=0.007). Conclusions: Drug interaction with the DIII-VSD has been tightly linked to use-dependent block of the late Naþ current, a hallmark class Ib drugs. In contrast, class Ia drugs typically reduce peak Naþ. Because the DIII-VSD is tightly coupled to NaV channel gating, we propose that the differences in DIII-VSD interaction are determining their unique therapeutic phenotypes. 2167-Pos Board B311 Biophysical Characterization of Two Nav1.4 Mutations Identified in Patients with Cold-Induced Myotonia and Periodic Paralysis Hugo Poulin1, Pascal Gosselin-Badaroudine1, Karima Habbout2, Savine Vicart3, Damien Syternberg4, Serena Giuliano2, Sophie Nicole4, Said Bendahhou2, Mohamed Chahine1. 1 Medicine, Laval University, Que´bec, QC, Canada, 2University Nice SophiaAntipolis, Nice, France, 3Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France, 4Sorbonne universite´s, Paris, France. Mutations in SCN4A gene encoding Nav1.4, the skeletal muscle voltage-gated Na channel underlie several skeletal muscle ion channelopathies whose two major phenotypes include enhanced excitability (myotonia), transient loss of excitability (periodic paralysis), a fluctuation between these two conditions or severe loss of function (myasthenia). Here, we report two novel dominant SCN4A missense mutations: R1451C, found in a patient with paramyotonia congenita (PMC) exhibiting coldinduced myotonia, and R1451L found in a patient diagnosed with periodic paralysis. These mutations are located in the transmembrane segment S4 of DIV domain. To elucidate the mechanism underlying the phenotypes caused by R1451C and R1451L, we used the whole-cell patch-clamp technique to study tsA201 cells expressing WT, R1451C or R1451L channels. Our results show that both mutations impaired fast inactivation kinetics at room temperature, with R1451L being the most affected. However, R1451C exhibited a unique temperature-dependent alteration of the fast inactivation kinetics that can explain the occurrence of myotonia in cold environment. Slow inactivation was assessed for both mutants, and revealed that the recovery from slow inactivation was only slowed for R1451L. This could explain the periodic paralysis phenotype of the patient. Homology modeling and in silico mutagenesis of R1451C and R1451L in the resting state based on a previous model of the channel was also used to probe the potential structural differences between these two mutations. The model revealed a reorganization of the hydrogen bonds between S4 and other segments in both mutations. We conclude from our investigation that the nature of the mutation is accountable for the clinical differences revealed in the patient carriers. 2168-Pos Board B312 Modeling Ion Channel Kinetics with Parameter Constraints Cynthia B. Lombardo, Marco A. Navarro, Autoosa Salari, Lorin S. Milescu. University of Missouri, Columbia, MO, USA. Ion channel gating mechanisms can be complex and difficult to extract from experimental data. A solution is to apply parameter constraints, which reflect prior knowledge or tested hypotheses and reduce model complexity and speed
up computation. Soft constraints balance the existing knowledge with the new experimental data and limit the parameter search engine to a smaller space of more acceptable values. In contrast, hard constraints enforce a mathematical relationship involving one or more parameters of the model. These constraints can be formulated as an invertible transformation between a set of model parameters and a set of ‘‘free’’ parameters. Each constraint reduces the number of free parameters by one. Linear constraints, such as microscopic reversibility or scaling between sequential transitions, can be conveniently obtained with the singular value decomposition. Here, we show how this method can be generalized to implement arbitrary linear constraints. We also show how to make these constraints depend on arbitrary model parameters. This can be applied, for example, to enforce allosteric constraints where the allosteric factor itself is a free parameter. Furthermore, we explore some useful ways for implementing soft constraints. 2169-Pos Board B313 Defining the Protein:Protein Interaction Interface of FGF14:Nav1.6 Complex Aditya K. Singh, Syed R. Ali, Fernanda Laezza. Department of Pharmacology & Toxicology, The University of Texas Medical Branch, Galveston, TX, USA. The voltage-gated Naþ (Nav) channel is composed of transmembrane spanning domains and of a cytosolic C-terminal tail which regulates channel function through protein:protein interactions (PPI) with auxiliary proteins, including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF (iFGF) family. In addition to binding to the Nav C-tail, FGF14 forms homodimers and previous structural studies have proposed a conserved surface common for the Nav channel and the iFGF homodimer formation. Seeking for potential differences between the FGF14:Nav1.6-C-tail complex and the FGF14:FGF14 dimer interface, we have engineered model-based amino acid residue mutations at predicted FGF14 hot-spots and begun to screen for their impact on the protein complex stability. Using the in-cell split-luciferase complementation assay to reconstitute the FGF14:Nav1.6-C-tail and the FGF14:FGF14 complex, we identified a point of divergence at the FGF14V160 residue whose mutation led to opposite effects on the relative binding affinity to the FGF14 monomer versus the Nav1.6-C-tail. Functional studies using whole-cell patch-clamp electrophysiology indicated that V160 is a critical residue for FGF14 modulation of Nav1.6-mediated currents that can be abolished by Ala mutation. Initial studies using intrinsic fluorescence showed efficient interaction between purified FGF14 and the Nav1.6 C-tail. Surface plasmon resonance and isothermal titration calorimetry measurements to evaluate the role of V160 in regulating binding affinity of FGF14 to the Nav1.6 C-tail are underway. With its unique role in the regulating the FGF14 binding and function, the V160 residue is well-positioned as target site for PPI-based drug development against Nav channels. Supported by: R01MH095995 (FL) and the Gulf Coast Consortia NIGMS Grant No.1 T32 GM089657-04 (SRA). 2170-Pos Board B314 Activation Dynamics of Sodium Ion Channel Matthew Harrigan, Vijay Pande. Stanford, Stanford, CA, USA. Voltage gated sodium channels initiate signaling in neurons and other cells which go on to become sensations of pleasure and pain, as well as thoughts and feelings. Sodium channel malfunction has been linked with cardiac arrhythmia, epilepsy, and neuropathic pain. We performed large-scale molecular dynamics simulations of the prokaryotic voltage gated sodium channel to probe dynamics and conformational change of channel activation. In collaboration with experimentalists, we propose a model for binding of batrachotoxin.
Voltage-gated Ca Channels 2171-Pos Board B315 Switchable Cardiac L Type Ca2D Channel Transcript by Mineralocorticoid Pathway Thassio Mesquita1, Gaelle Auguste1, Jessica Sabourin1, Gema Ruiz-Hurtado1, Vale´rie Rouffiac2, Florian Le-Billan3, Je´roˆme Fagart3, Florence Lefebvre1, Say Viengchareun3, Eric Morel1, Ana Maria Gomez1, Marc Lombe`s3, Jean Pierre benitah1. 1 UMR-S 1180, Inserm, Univ. Paris-Sud, Universite´ Paris-Saclay, ChatenayMalabry, France, 2Imaging and Cytometry Platform, UMR 8081 IR4M, Gustave Roussy Institute, Villejuif, France, 3UMR-S 1185, Inserm, Univ. Paris-Sud, Universite´ Paris-Saclay, Le Kremlin-Biceˆtre, France.