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Structural Mechanisms Underlying PUFA Modulation in Pentameric Ligand Gated Ion Channels
320a
Tuesday, February 14, 2017
1571-Plat Membrane Crowding and Complexity: Interplay between Protein-Lipid Interactions, Clustering and Diffusion Anna L. Duncan, Heidi Koldsø, Tyler J. Reddy, Jean He´lie, Mark S.P. Sansom. Department of Biochemistry, University of Oxford, Oxford, United Kingdom. It is well-understood that cell membranes are crowded and complex environments, containing up to 50 % protein by mass [1] and comprised of myriad types of lipid species [2]. Less well understood is the molecular detail of the effects of complexity and crowding on membrane organisation and dynamics. Advances in coarse-grained force fields and computational power mean that large-scale coarse-grained (CG) molecular dynamics (MD) simulations are increasingly being used to gain such understanding [3]. To investigate the role of protein crowding and lipid complexity on the organisation and dynamics of mammalian cell membranes, and particularly the effect of protein-lipid interactions on protein and lipid diffusion in crowded membranes, we have performed large CG MD simulations containing over 100 inward rectifier potassium (Kir) channels, which have specific lipid interactions with PIP2, other anionic lipid interactions and cholesterol. Lipid complexity has a marked effect on the clustering behaviour of channels, underlying which are the diffusive properties of different lipid mixtures. Subdiffusion of lipids tightly interacting with Kir channels is also observed, and this subdiffusion is further modulated by protein crowding. By simulating Kir channels in a complex lipid mixture, we can also examine the interplay between PIP2, other anionic lipids and cholesterol interactions. In simulating systems of >100 nm in dimension whilst retaining membrane complexity at a molecular level, we start to move further towards the use of simulation as a computational microscope, and can assess the possibilities using this approach. 1. Dupuy and Engelman. Proc Natl Acad Sci USA, 2008 105 (8): 2848–52 2. Coskun and Simons. Structure, 2011 19(11): 1543–48. 3. Chavent, Duncan and Sansom. Curr Op Struct Biol, 2016. 40: 8–16 1572-Plat The Effect of Propofol on Plasma Membrane Ultrastructure in the Intact Cells Weixiang Jin, Arnd Pralle. Physics, University at Buffalo, Buffalo, NY, USA. Despite many observed effects of anesthetic drugs, the mechanism of general anesthesia is still unknown.One well-studied drug used for human anesthesia, propofol, has been shown to interact with some ligand gated ion-channels. However, propofol also easily dissolves in the lipid bilayer and alters membrane fluidity. Which mechanism dominates in anesthesia or even how anesthesia arises are unclear. Here, we study the influence of propofol on plasma membrane (PM) ultrastructure in intact cells, which affect cell signaling. In the PM, transient submicroscopic nano-domains form by interactions between lipid-acyl-chains or lipid head groups, stabilized by cholesterol. In addition, membrane cytoskeleton may further regulate these nano-domains. These domains regulate receptor interactions and signaling.We study transient propofol affects on these domains from low to clinically relevant propofol concentrations by analyzing diffusion of GFP-tagged outer leaflet/inner leaflet membrane proteins.Using bimFCS we measure diffusion on multiple length scales simultaneously. We observe that at lower propofol concentrations (up to 2yM), the cholesterol nano-domains trap the GPI-mGFP less, which is consistent with the recent studies showing that propofoldecreases the phase transition temperature of plasma membrane derived vesicles. Interestingly, at higher concentrations of propofol (20yM to 150yM), the nanodomains trap the GPI-mGFP more strongly. This is only observed at physiological temperatures (37 C). By inhibiting myosin activity or actin filaments (de-)polymerization, we find that the activity of actin filaments further alters the behavior of cholesterol nano-domains due to propofol. We compare the effect of propofol and its analog confirming its specific effect as anesthetic drug. These results suggest that in intact cells, propofol induced cholesterol nano-domains changes are temperature dependent, regulated by cytoskeleton activities, and the effect is specific comparing to its analogs.
Platform: Ligand-gated Channels I 1573-Plat Structure and Mechanism of Neuronal Nicotinic Acetylcholine Receptors Claudio L. Morales-Perez, Colleen M. Noviello, Ryan E. Hibbs. Neuroscience and Biophysics, UTSW Medical Center, Dallas, TX, USA.
Nicotinic acetylcholine receptors are ligand gated ion channels that mediate fast chemical neurotransmission at the neuromuscular junction and play diverse signaling roles in the central nervous system. Here we describe a biochemical approach for characterization of subunit stoichiometry in heteromeric membrane proteins and present the first X-ray crystal structure of a nicotinic receptor. The a4b2 nicotinic receptor is the most abundant receptor subtype in the brain, is the principal target in nicotine addiction and its dysfunction is associated with familial epilepsy. The structure of the receptor in complex with the agonist nicotine reveals principles of ligand selectivity among different classes of subunit interfaces in the heteropentameric assembly. The receptor is stabilized by nicotine in a non-conducting, desensitized conformation. The constriction point in the permeation pathway is formed at the selectivity filter, located at the cytosolic end of the pore. The desensitized state of this channel provides a distinct structural reference point in the allosteric gating cycle of the larger Cys-loop receptor superfamily. 1574-Plat Photoaffinity Labeling of A4B2 Nicotinic Acetylcholine Receptor using [3H]-Labeled Positive Allosteric Modulators Gordon Ang1, Farah Deba1, Akash Pandhare2, Michael P. Blanton2, Jonathan B. Cohen3, Ayman K. Hamouda1. 1 Pharmaceutical Sciences, Texas A&M Health Science Center, Kingsville, TX, USA, 2Pharmacology and Neurosciences, Texas Tech University Health Science Center, Lubbock, TX, USA, 3Neurobiology, Harvard Medical School, Boston, MA, USA. The a4b2 nicotinic acetylcholine receptor (nAChR) is a potential drug target for treating neuropathological and tobacco use disorders. Positive allosteric modulators (PAMs), which bind o site(s) distinct from the ACh (agonist) binding sites, may provide the required specificity by binding to unique site(s) present only in the a4b2 nAChR. However, the molecular pharmacology of this class of compounds is unclear, and the diversity of PAM binding site(s) have not been determined. In previous work, two radiolabeled a4b2 nAChR PAMs, desformylflustrabromine; ([3H]dFBr, 76 Ci/mmol) and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H- pyrrazol-4-yl) isoxazole ([3H]CMPI, 16 Ci/mmol) have been prepared and their merits as photoprobes established using Torpedo nAChR as a model (Hamouda et al. 2015, Mol. Pharmacol. 88:1; Hamouda et al. 2016, Mol. Pharmacol. 89: 575). Here we expand these studies to a4b2 nAChR. Using membrane fractions isolated from a HEK-293 cell line stably expressing a4b2 nAChR (HEK-a4b2 nAChR), we are examining the effects of orthosteric ligands, ion channel blockers and other PAMs on the equilibrium binding of [3H] dFBr and [3H]CMPI. In addition, we purified a4b2 nAChRs from HEKa4b2 nAChR membranes using a bromo-acetylcholine bromide affinity column and performed initial [3H]dFBr and [3H]CMPI analytical photolabelings. When ~100 mg of affinity-purified a4b2 nAChR and 20 mCi of [3H]dFBr (76 Ci/mmol) or [3H]CMPI (16 Ci/mmol) were used, photolysis at 312 nm for 5 min ([3H]dFBr) or 3 min ([3H]CMPI) resulted in photolabeling efficiency of (pmol of 3H incorporated/pmol of a4b2 nAChR) of ~0.05% and 0.7%, respectively. Additional analytical [3H]dFBr and [3H] CMPI photolabeling in the presence of other nAChR ligands and preparative photolabeling experiments are in progress to identify amino acids contributing to dFBr and CMPI binding sites. 1575-Plat Structural Mechanisms Underlying PUFA Modulation in Pentameric Ligand Gated Ion Channels Yvonne W. Gicheru, Sandip Basak, Sudha Chakrapani. Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA. Pentameric ligand gated ion channels (pLGICs) mediate fast neurotransmission in the central and peripheral nervous systems. Modulators such as lipids, alcohols and general anesthetics allosterically modify the conformations of pentameric ligand gated ion channels (pLGICs) via the transmembrane domain. However, the molecular mechanisms underlying modulation are still unclear. GLIC is a prokaryotic homologue that has an overall conserved architecture and high sensitivity to clinically relevant compounds as the eukaryotic members of the family. Our study probed the functional and structural effect of acyl chain length and degree of unsaturation of polyunsaturated fatty acids (PUFAs) on GLIC function. A novel binding site for PUFAs on GLIC and the effect of perturbations on this site were investigated. By using a combination of electrophysiology, EPR spectroscopy, and X-ray crystallography we show a possible mechanism of PUFA modulation of pLGIC function through coupling of the outermost M4 helix with the channel pore.
Tuesday, February 14, 2017
1571-Plat Membrane Crowding and Complexity: Interplay between Protein-Lipid Interactions, Clustering and Diffusion Anna L. Duncan, Heidi Koldsø, Tyler J. Reddy, Jean He´lie, Mark S.P. Sansom. Department of Biochemistry, University of Oxford, Oxford, United Kingdom. It is well-understood that cell membranes are crowded and complex environments, containing up to 50 % protein by mass [1] and comprised of myriad types of lipid species [2]. Less well understood is the molecular detail of the effects of complexity and crowding on membrane organisation and dynamics. Advances in coarse-grained force fields and computational power mean that large-scale coarse-grained (CG) molecular dynamics (MD) simulations are increasingly being used to gain such understanding [3]. To investigate the role of protein crowding and lipid complexity on the organisation and dynamics of mammalian cell membranes, and particularly the effect of protein-lipid interactions on protein and lipid diffusion in crowded membranes, we have performed large CG MD simulations containing over 100 inward rectifier potassium (Kir) channels, which have specific lipid interactions with PIP2, other anionic lipid interactions and cholesterol. Lipid complexity has a marked effect on the clustering behaviour of channels, underlying which are the diffusive properties of different lipid mixtures. Subdiffusion of lipids tightly interacting with Kir channels is also observed, and this subdiffusion is further modulated by protein crowding. By simulating Kir channels in a complex lipid mixture, we can also examine the interplay between PIP2, other anionic lipids and cholesterol interactions. In simulating systems of >100 nm in dimension whilst retaining membrane complexity at a molecular level, we start to move further towards the use of simulation as a computational microscope, and can assess the possibilities using this approach. 1. Dupuy and Engelman. Proc Natl Acad Sci USA, 2008 105 (8): 2848–52 2. Coskun and Simons. Structure, 2011 19(11): 1543–48. 3. Chavent, Duncan and Sansom. Curr Op Struct Biol, 2016. 40: 8–16 1572-Plat The Effect of Propofol on Plasma Membrane Ultrastructure in the Intact Cells Weixiang Jin, Arnd Pralle. Physics, University at Buffalo, Buffalo, NY, USA. Despite many observed effects of anesthetic drugs, the mechanism of general anesthesia is still unknown.One well-studied drug used for human anesthesia, propofol, has been shown to interact with some ligand gated ion-channels. However, propofol also easily dissolves in the lipid bilayer and alters membrane fluidity. Which mechanism dominates in anesthesia or even how anesthesia arises are unclear. Here, we study the influence of propofol on plasma membrane (PM) ultrastructure in intact cells, which affect cell signaling. In the PM, transient submicroscopic nano-domains form by interactions between lipid-acyl-chains or lipid head groups, stabilized by cholesterol. In addition, membrane cytoskeleton may further regulate these nano-domains. These domains regulate receptor interactions and signaling.We study transient propofol affects on these domains from low to clinically relevant propofol concentrations by analyzing diffusion of GFP-tagged outer leaflet/inner leaflet membrane proteins.Using bimFCS we measure diffusion on multiple length scales simultaneously. We observe that at lower propofol concentrations (up to 2yM), the cholesterol nano-domains trap the GPI-mGFP less, which is consistent with the recent studies showing that propofoldecreases the phase transition temperature of plasma membrane derived vesicles. Interestingly, at higher concentrations of propofol (20yM to 150yM), the nanodomains trap the GPI-mGFP more strongly. This is only observed at physiological temperatures (37 C). By inhibiting myosin activity or actin filaments (de-)polymerization, we find that the activity of actin filaments further alters the behavior of cholesterol nano-domains due to propofol. We compare the effect of propofol and its analog confirming its specific effect as anesthetic drug. These results suggest that in intact cells, propofol induced cholesterol nano-domains changes are temperature dependent, regulated by cytoskeleton activities, and the effect is specific comparing to its analogs.
Platform: Ligand-gated Channels I 1573-Plat Structure and Mechanism of Neuronal Nicotinic Acetylcholine Receptors Claudio L. Morales-Perez, Colleen M. Noviello, Ryan E. Hibbs. Neuroscience and Biophysics, UTSW Medical Center, Dallas, TX, USA.
Nicotinic acetylcholine receptors are ligand gated ion channels that mediate fast chemical neurotransmission at the neuromuscular junction and play diverse signaling roles in the central nervous system. Here we describe a biochemical approach for characterization of subunit stoichiometry in heteromeric membrane proteins and present the first X-ray crystal structure of a nicotinic receptor. The a4b2 nicotinic receptor is the most abundant receptor subtype in the brain, is the principal target in nicotine addiction and its dysfunction is associated with familial epilepsy. The structure of the receptor in complex with the agonist nicotine reveals principles of ligand selectivity among different classes of subunit interfaces in the heteropentameric assembly. The receptor is stabilized by nicotine in a non-conducting, desensitized conformation. The constriction point in the permeation pathway is formed at the selectivity filter, located at the cytosolic end of the pore. The desensitized state of this channel provides a distinct structural reference point in the allosteric gating cycle of the larger Cys-loop receptor superfamily. 1574-Plat Photoaffinity Labeling of A4B2 Nicotinic Acetylcholine Receptor using [3H]-Labeled Positive Allosteric Modulators Gordon Ang1, Farah Deba1, Akash Pandhare2, Michael P. Blanton2, Jonathan B. Cohen3, Ayman K. Hamouda1. 1 Pharmaceutical Sciences, Texas A&M Health Science Center, Kingsville, TX, USA, 2Pharmacology and Neurosciences, Texas Tech University Health Science Center, Lubbock, TX, USA, 3Neurobiology, Harvard Medical School, Boston, MA, USA. The a4b2 nicotinic acetylcholine receptor (nAChR) is a potential drug target for treating neuropathological and tobacco use disorders. Positive allosteric modulators (PAMs), which bind o site(s) distinct from the ACh (agonist) binding sites, may provide the required specificity by binding to unique site(s) present only in the a4b2 nAChR. However, the molecular pharmacology of this class of compounds is unclear, and the diversity of PAM binding site(s) have not been determined. In previous work, two radiolabeled a4b2 nAChR PAMs, desformylflustrabromine; ([3H]dFBr, 76 Ci/mmol) and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H- pyrrazol-4-yl) isoxazole ([3H]CMPI, 16 Ci/mmol) have been prepared and their merits as photoprobes established using Torpedo nAChR as a model (Hamouda et al. 2015, Mol. Pharmacol. 88:1; Hamouda et al. 2016, Mol. Pharmacol. 89: 575). Here we expand these studies to a4b2 nAChR. Using membrane fractions isolated from a HEK-293 cell line stably expressing a4b2 nAChR (HEK-a4b2 nAChR), we are examining the effects of orthosteric ligands, ion channel blockers and other PAMs on the equilibrium binding of [3H] dFBr and [3H]CMPI. In addition, we purified a4b2 nAChRs from HEKa4b2 nAChR membranes using a bromo-acetylcholine bromide affinity column and performed initial [3H]dFBr and [3H]CMPI analytical photolabelings. When ~100 mg of affinity-purified a4b2 nAChR and 20 mCi of [3H]dFBr (76 Ci/mmol) or [3H]CMPI (16 Ci/mmol) were used, photolysis at 312 nm for 5 min ([3H]dFBr) or 3 min ([3H]CMPI) resulted in photolabeling efficiency of (pmol of 3H incorporated/pmol of a4b2 nAChR) of ~0.05% and 0.7%, respectively. Additional analytical [3H]dFBr and [3H] CMPI photolabeling in the presence of other nAChR ligands and preparative photolabeling experiments are in progress to identify amino acids contributing to dFBr and CMPI binding sites. 1575-Plat Structural Mechanisms Underlying PUFA Modulation in Pentameric Ligand Gated Ion Channels Yvonne W. Gicheru, Sandip Basak, Sudha Chakrapani. Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA. Pentameric ligand gated ion channels (pLGICs) mediate fast neurotransmission in the central and peripheral nervous systems. Modulators such as lipids, alcohols and general anesthetics allosterically modify the conformations of pentameric ligand gated ion channels (pLGICs) via the transmembrane domain. However, the molecular mechanisms underlying modulation are still unclear. GLIC is a prokaryotic homologue that has an overall conserved architecture and high sensitivity to clinically relevant compounds as the eukaryotic members of the family. Our study probed the functional and structural effect of acyl chain length and degree of unsaturation of polyunsaturated fatty acids (PUFAs) on GLIC function. A novel binding site for PUFAs on GLIC and the effect of perturbations on this site were investigated. By using a combination of electrophysiology, EPR spectroscopy, and X-ray crystallography we show a possible mechanism of PUFA modulation of pLGIC function through coupling of the outermost M4 helix with the channel pore.