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Ketamine Inhibition of Pentameric Ligand-Gated Ion Channels - Insights from Molecular Dynamics Simulations
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Wednesday, February 15, 2017
insights on the Noble gases binding location and affinity. Different gas:lipid ratios were used to characterize their effect on the physical properties of the membrane, as well as their affinity for the core of the lipid bilayer. Microsecond-long flooding simulations, combined with free energy calculations, were run to characterize access to binding sites and quantify binding affinities of Nobles Gases on the GLIC channel. This work extends and generalizes our previous study on bromoform action on GLIC, that revealed a complex network of interconnected binding sites, possibly all contributing in concert to the anesthetic effect [2]. We go beyond previous work by considering the measurable effect on lipid bilayer properties induced by the Noble gases therefore providing a better description of key pathways for anesthetic action. [1] Sauguet et al. Plos One. 2016. doi: 10.1371/ journal.pone.0149795 [2] Laurent, Murail et al. Structure. 2016. doi: 10.1016/j.str.2016.02.014 2728-Pos Board B335 Transmembrane Structural Determinants of Alcohol Binding and Modulation in a Model Ligand-Gated Ion Channel ¨ zge Yoluk3, Oliver Snow1,3, Rebecca J. Howard1,2, Stephanie A. Heusser1, O Go¨ran Klement1,3, Alex R. Mola2, Travers MD Ruel2, Erik Lindahl1,3. 1 Biochemistry & Biophysics, Stockholm University, Stockholm, Sweden, 2 Chemistry, Skidmore College, Saratoga Springs, NY, USA, 3Theoretical Physics, KTH Royal Institute of Technology, Stockholm, Sweden. Pentameric ligand-gated ion channels are heavily implicated in neurological effects of alcohol, yet a structural understanding of this process remains limited by a lack of high-resolution data for pharmacologically relevant receptors. The prokaryotic ligand-gated ion channel GLIC is a potentially valuable model system whose structure has been determined in multiple conformations and bound to various ligands. In particular, modification of a key transmembrane position in GLIC rendered it potently sensitive to potentiation, and enabled cocrystallization with ethanol as well as other anesthetic agents. To elucidate alcohol interactions with channel structure and function, we substituted a variety of amino acids in the ethanol site and channel pore, expressed the mutated channels in frog oocytes, and measured their gating and modulation properties by two-electrode voltage-clamp electrophysiology. We compared the resulting changes in agonist and modulator sensitivity with standard amino acid properties and molecular modeling of the predicted binding site and gating transitions. We identified structural determinants of binding that may reflect distinct properties of alcohols relative to classic drugs, and could provide insight into other low-affinity modulators such as anesthetics and lipids. We further found that modifying the ethanol pocket removed or even reversed effects of mutations in the ion channel pore, implicating tight coupling between the allosteric and active sites. Our ongoing work aims to integrate functional data with computational models of receptor binding and channel gating. 2729-Pos Board B336 Ketamine Inhibition of Pentameric Ligand-Gated Ion Channels - Insights from Molecular Dynamics Simulations Bogdan F. Ion, Marta M. Wells, Yan Xu, Pei Tang. Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA. The anesthetic ketamine is well known as a non-competitive antagonist of Nmethyl-D-aspartate receptors (NMDARs). However, not all the clinical effects of ketamine can be fully explained by its inhibition of NMDARs. An alternative mode of ketamine action is through inhibition of pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine receptors. Ketamine inhibits the bacterial pLGIC from Gloeobacter violaceus (GLIC) and the ketamine-GLIC co-crystallized structure was determined previously. Ketamine was bound to an intersubunit cavity that partially overlaps with the homologous antagonist-binding site. Here, we performed molecular dynamics simulations of GLIC crystal structures in the absence and presence of ketamine. Three parallel simulations were run for each GLIC system. Ketamine was parameterized using the Force Field Toolkit based on protocols from CGenFF. Simulations show that asymmetric binding (only one or two ketamine molecules) is preferred to the symmetric binding of five ketamine molecules observed in the crystal structure. Asymmetric ketamine binding introduces a series of structural changes starting from the binding site to the pore lining TM2 helices. The presence of ketamine stabilized intersubunit salt bridges between loop C and the adjacent complementary subunit but destabilized salt bridges and critical hydrophobic interactions at the interface between the extracellular and transmembrane domains (b1-b2$$$pre-TM1, b6-b7$$$TM2-M3). Interestingly, these interactions in the subunits where ketamine was not bound were weakened to a greater extent than in the subunits asymmetrically bound to ketamine. Ketamine binding also introduced a significant increase in the population of TM2 lateral tilting angles shifting towards a closed channel conformation when compared to apo GLIC. These conformational changes contributed to
the accelerated channel dehydration observed in the ketamine-bound system. The study provides a structural and dynamical basis for the inhibitory modulation of ketamine on pLGICs. Research supported by NIH R01GM066358. 2730-Pos Board B337 Structural and Functional Evidence for Multi-Site Allostery Mediated by General Anesthetics in a Model Ligand-Gated Ion Channel Stephanie A. Heusser1, Rebecca J. Howard1, Zeineb Fourati2, Marc Delarue2, Erik Lindahl1. 1 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden, 2Department of Structural Biology & Chemistry, Institut Pasteur, Paris, France. General anesthetics act as either positive or negative allosteric modulators of several pentameric ligand-gated ion channels, including physiologically important receptors for g-aminobutyric acid and acetylcholine. Although functional studies have implicated conserved sites of modulation in this channel family, the limited scope and resolution of structural data for human Cys-loop receptors have hampered mechanistic studies of anesthetic action. We previously showed the prokaryotic homolog GLIC to be a useful model system that recapitulates functional modulation of human ion channels, and enables structure determination both in apparent open and nonconducting states. Specifically, anesthetic inhibition of GLIC can be removed, reversed, or rendered bimodal by sitedirected mutations in the transmembrane domain (TMD). In this work, we provide crystallographic and electrophysiological evidence for a multi-site mechanism of bimodal modulation by anesthetizing agents, including the common surgical medication propofol. With the pore in an apparent nonconducting state, propofol bound at the intracellular (lower) end of the channel pore, similar to other inhibitors. Consistent with this result, single hydrophobic substitutions at the lower-pore site enhanced functional inhibition. Conversely, in the apparent open state, anesthetics bound to one or more contiguous sites in the extracellular-facing (upper) end of the TMD, particularly for variants in which anesthetic inhibition was reduced or reversed. In one novel variant exhibiting anesthetic potentiation, propofol binding in the upper-TMD converted the channel from an apparent nonconducting- to open-pore conformation under otherwise identical conditions, providing direct evidence for potentiation via specific sites in the upper TMD. Based on these findings, we propose a structural model for allostery in which anesthetic binding to spatially distinct TMD cavities differentially stabilizes opposing functional states of pentameric ligand-gated ion channels, providing valuable insights into channel modulation and drug development. 2731-Pos Board B338 Determinants of 5-Ht3A Intracellular Domain Oligomerization and RIC-3 Interaction Elham Pirayesh, Akash Pandhare, Michaela Jansen. Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA. The serotonin type 3A (5-HT3A) receptor is a homopentameric cation-selective member of the pentameric ligand-gated ion channel (pLGIC) superfamily. Members of this superfamily assemble from five subunits, each of which consists of three domains, extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). Recently, it was demonstrated that 5-HT3A-ICD fused to maltose binding protein (MBP) forms stable pentamers in solution (Pandhare, Grozdanov, Jansen, Sci. Rep. 2016). Also it has been shown that MBP-5-HT3A-ICD directly interacts with the chaperone protein resistance to inhibitors of choline esterase (RIC-3). To elucidate the nature of the oligomerization of the 5-HT3A-ICD and its interaction with the chaperone protein RIC-3 we developed different MBP-fused constructs of this domain by deletion of large portions of its amino acid sequence. We have expressed two mutants (MBP-5-HT3A-ICD-A and MBP-5-HT3A-ICD-B) in Escherichia coli and purified them to homogeneity. Additionally, we have also purified RIC-3 to be utilized in protein-protein interaction experiments. The oligomeric states of both deletion constructs is probed using size exclusion chromatography together with multi-angle light scattering. Additionally, using RIC-3 affinity pull down the interaction of MBP-5HT3A-ICD constructs and RIC-3 is investigated. Further studies are directed toward deciphering the molecular determinants for ICD oligomerization as well as RIC-3 protein-protein interaction. 2732-Pos Board B339 Characterizing the Intrinsic Assembly Behavior of the 5-HT3A Receptor Intracellular Domain Akash Pandhare, Michaela Jansen. Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
Wednesday, February 15, 2017
insights on the Noble gases binding location and affinity. Different gas:lipid ratios were used to characterize their effect on the physical properties of the membrane, as well as their affinity for the core of the lipid bilayer. Microsecond-long flooding simulations, combined with free energy calculations, were run to characterize access to binding sites and quantify binding affinities of Nobles Gases on the GLIC channel. This work extends and generalizes our previous study on bromoform action on GLIC, that revealed a complex network of interconnected binding sites, possibly all contributing in concert to the anesthetic effect [2]. We go beyond previous work by considering the measurable effect on lipid bilayer properties induced by the Noble gases therefore providing a better description of key pathways for anesthetic action. [1] Sauguet et al. Plos One. 2016. doi: 10.1371/ journal.pone.0149795 [2] Laurent, Murail et al. Structure. 2016. doi: 10.1016/j.str.2016.02.014 2728-Pos Board B335 Transmembrane Structural Determinants of Alcohol Binding and Modulation in a Model Ligand-Gated Ion Channel ¨ zge Yoluk3, Oliver Snow1,3, Rebecca J. Howard1,2, Stephanie A. Heusser1, O Go¨ran Klement1,3, Alex R. Mola2, Travers MD Ruel2, Erik Lindahl1,3. 1 Biochemistry & Biophysics, Stockholm University, Stockholm, Sweden, 2 Chemistry, Skidmore College, Saratoga Springs, NY, USA, 3Theoretical Physics, KTH Royal Institute of Technology, Stockholm, Sweden. Pentameric ligand-gated ion channels are heavily implicated in neurological effects of alcohol, yet a structural understanding of this process remains limited by a lack of high-resolution data for pharmacologically relevant receptors. The prokaryotic ligand-gated ion channel GLIC is a potentially valuable model system whose structure has been determined in multiple conformations and bound to various ligands. In particular, modification of a key transmembrane position in GLIC rendered it potently sensitive to potentiation, and enabled cocrystallization with ethanol as well as other anesthetic agents. To elucidate alcohol interactions with channel structure and function, we substituted a variety of amino acids in the ethanol site and channel pore, expressed the mutated channels in frog oocytes, and measured their gating and modulation properties by two-electrode voltage-clamp electrophysiology. We compared the resulting changes in agonist and modulator sensitivity with standard amino acid properties and molecular modeling of the predicted binding site and gating transitions. We identified structural determinants of binding that may reflect distinct properties of alcohols relative to classic drugs, and could provide insight into other low-affinity modulators such as anesthetics and lipids. We further found that modifying the ethanol pocket removed or even reversed effects of mutations in the ion channel pore, implicating tight coupling between the allosteric and active sites. Our ongoing work aims to integrate functional data with computational models of receptor binding and channel gating. 2729-Pos Board B336 Ketamine Inhibition of Pentameric Ligand-Gated Ion Channels - Insights from Molecular Dynamics Simulations Bogdan F. Ion, Marta M. Wells, Yan Xu, Pei Tang. Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA. The anesthetic ketamine is well known as a non-competitive antagonist of Nmethyl-D-aspartate receptors (NMDARs). However, not all the clinical effects of ketamine can be fully explained by its inhibition of NMDARs. An alternative mode of ketamine action is through inhibition of pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine receptors. Ketamine inhibits the bacterial pLGIC from Gloeobacter violaceus (GLIC) and the ketamine-GLIC co-crystallized structure was determined previously. Ketamine was bound to an intersubunit cavity that partially overlaps with the homologous antagonist-binding site. Here, we performed molecular dynamics simulations of GLIC crystal structures in the absence and presence of ketamine. Three parallel simulations were run for each GLIC system. Ketamine was parameterized using the Force Field Toolkit based on protocols from CGenFF. Simulations show that asymmetric binding (only one or two ketamine molecules) is preferred to the symmetric binding of five ketamine molecules observed in the crystal structure. Asymmetric ketamine binding introduces a series of structural changes starting from the binding site to the pore lining TM2 helices. The presence of ketamine stabilized intersubunit salt bridges between loop C and the adjacent complementary subunit but destabilized salt bridges and critical hydrophobic interactions at the interface between the extracellular and transmembrane domains (b1-b2$$$pre-TM1, b6-b7$$$TM2-M3). Interestingly, these interactions in the subunits where ketamine was not bound were weakened to a greater extent than in the subunits asymmetrically bound to ketamine. Ketamine binding also introduced a significant increase in the population of TM2 lateral tilting angles shifting towards a closed channel conformation when compared to apo GLIC. These conformational changes contributed to
the accelerated channel dehydration observed in the ketamine-bound system. The study provides a structural and dynamical basis for the inhibitory modulation of ketamine on pLGICs. Research supported by NIH R01GM066358. 2730-Pos Board B337 Structural and Functional Evidence for Multi-Site Allostery Mediated by General Anesthetics in a Model Ligand-Gated Ion Channel Stephanie A. Heusser1, Rebecca J. Howard1, Zeineb Fourati2, Marc Delarue2, Erik Lindahl1. 1 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden, 2Department of Structural Biology & Chemistry, Institut Pasteur, Paris, France. General anesthetics act as either positive or negative allosteric modulators of several pentameric ligand-gated ion channels, including physiologically important receptors for g-aminobutyric acid and acetylcholine. Although functional studies have implicated conserved sites of modulation in this channel family, the limited scope and resolution of structural data for human Cys-loop receptors have hampered mechanistic studies of anesthetic action. We previously showed the prokaryotic homolog GLIC to be a useful model system that recapitulates functional modulation of human ion channels, and enables structure determination both in apparent open and nonconducting states. Specifically, anesthetic inhibition of GLIC can be removed, reversed, or rendered bimodal by sitedirected mutations in the transmembrane domain (TMD). In this work, we provide crystallographic and electrophysiological evidence for a multi-site mechanism of bimodal modulation by anesthetizing agents, including the common surgical medication propofol. With the pore in an apparent nonconducting state, propofol bound at the intracellular (lower) end of the channel pore, similar to other inhibitors. Consistent with this result, single hydrophobic substitutions at the lower-pore site enhanced functional inhibition. Conversely, in the apparent open state, anesthetics bound to one or more contiguous sites in the extracellular-facing (upper) end of the TMD, particularly for variants in which anesthetic inhibition was reduced or reversed. In one novel variant exhibiting anesthetic potentiation, propofol binding in the upper-TMD converted the channel from an apparent nonconducting- to open-pore conformation under otherwise identical conditions, providing direct evidence for potentiation via specific sites in the upper TMD. Based on these findings, we propose a structural model for allostery in which anesthetic binding to spatially distinct TMD cavities differentially stabilizes opposing functional states of pentameric ligand-gated ion channels, providing valuable insights into channel modulation and drug development. 2731-Pos Board B338 Determinants of 5-Ht3A Intracellular Domain Oligomerization and RIC-3 Interaction Elham Pirayesh, Akash Pandhare, Michaela Jansen. Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA. The serotonin type 3A (5-HT3A) receptor is a homopentameric cation-selective member of the pentameric ligand-gated ion channel (pLGIC) superfamily. Members of this superfamily assemble from five subunits, each of which consists of three domains, extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). Recently, it was demonstrated that 5-HT3A-ICD fused to maltose binding protein (MBP) forms stable pentamers in solution (Pandhare, Grozdanov, Jansen, Sci. Rep. 2016). Also it has been shown that MBP-5-HT3A-ICD directly interacts with the chaperone protein resistance to inhibitors of choline esterase (RIC-3). To elucidate the nature of the oligomerization of the 5-HT3A-ICD and its interaction with the chaperone protein RIC-3 we developed different MBP-fused constructs of this domain by deletion of large portions of its amino acid sequence. We have expressed two mutants (MBP-5-HT3A-ICD-A and MBP-5-HT3A-ICD-B) in Escherichia coli and purified them to homogeneity. Additionally, we have also purified RIC-3 to be utilized in protein-protein interaction experiments. The oligomeric states of both deletion constructs is probed using size exclusion chromatography together with multi-angle light scattering. Additionally, using RIC-3 affinity pull down the interaction of MBP-5HT3A-ICD constructs and RIC-3 is investigated. Further studies are directed toward deciphering the molecular determinants for ICD oligomerization as well as RIC-3 protein-protein interaction. 2732-Pos Board B339 Characterizing the Intrinsic Assembly Behavior of the 5-HT3A Receptor Intracellular Domain Akash Pandhare, Michaela Jansen. Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA.