FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Dynamics of the Ligand Binding Domain Layer during AMPA Receptor Activation
Baranovic, J., Chebli, M., Salazar, H., Carbone, A. L., Faelber(*), K., Lau(*), A. Y., Daumke(*), O.; Plested, A. J.
Biophys J, 110:896-911

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample.

Superactivation of AMPA receptors by auxiliary proteins
Carbone, A. L.; Plested, A. J.
Nat Commun, 7:10178

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Glutamate receptors form complexes in the brain with auxiliary proteins, which control their activity during fast synaptic transmission through a seemingly bewildering array of effects. Here we devise a way to isolate the activation of complexes using polyamines, which enables us to show that transmembrane AMPA receptor regulatory proteins (TARPs) exert their effects principally on the channel opening reaction. A thermodynamic argument suggests that because TARPs promote channel opening, receptor activation promotes AMPAR-TARP complexes into a superactive state with high open probability. A simple model based on this idea predicts all known effects of TARPs on AMPA receptor function. This model also predicts unexpected phenomena including massive potentiation in the absence of desensitization and supramaximal recovery that we subsequently detected in electrophysiological recordings. This transient positive feedback mechanism has implications for information processing in the brain, because it should allow activity-dependent facilitation of excitatory synaptic transmission through a postsynaptic mechanism.

Single-Channel Recording of Ligand-Gated Ion Channels
Plested, A. J.
Cold Spring Harb Protoc, 2016:pdb top087239

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Single-channel recordings reveal the microscopic properties of individual ligand-gated ion channels. Such recordings contain much more information than measurements of ensemble behavior and can yield structural and functional information about the receptors that participate in fast synaptic transmission in the brain. With a little care, a standard patch-clamp electrophysiology setup can be adapted for single-channel recording in a matter of hours. Thenceforth, it is a realistic aim to record single-molecule activity with microsecond resolution from arbitrary cell types, including cell lines and neurons.

Structural mechanisms of activation and desensitization in neurotransmitter-gated ion channels
Plested, A. J.
Nat Struct Mol Biol, 23:494-502

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Ion channels gated by neurotransmitters are present across metazoans, in which they are essential for brain function, sensation and locomotion; closely related homologs are also found in bacteria. Structures of eukaryotic pentameric cysteine-loop (Cys-loop) receptors and tetrameric ionotropic glutamate receptors in multiple functional states have recently become available. Here, I describe how these studies relate to established ideas regarding receptor activation and how they have enabled decades' worth of functional work to be pieced together, thus allowing previously puzzling aspects of receptor activity to be understood.

Single-Channel Recording of Glycine Receptors in Human Embryonic Kidney (HEK) Cells
Plested, A. J.; Baranovic, J.
Cold Spring Harb Protoc, 2016:pdb prot091652

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: This protocol describes how to record the single-channel activity of recombinant homomeric glycine receptors expressed in human embryonic kidney (HEK) cells. Cell-attached recordings readily reveal the large conductance (90 pS) and distinctive clusters of activations at high glycine concentration. This method for obtaining equilibrium recordings can be adapted to any ion channel receptor. The necessary extensions to outside-out patch for nonequilibrium recordings are also described, as are basic analyses of channel properties and activity.

Structural rearrangement of the intracellular domains during AMPA receptor activation
Zachariassen(*), L. G., Katchan, L., Jensen(*), A. G., Pickering(*), D. S., Plested, A. J.; Kristensen(*), A. S.
Proc Natl Acad Sci U S A, 113:E3950-3959

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.

How to build the fastest receptor on earth
Baranovic, J.; Plested, A. J. R.
Biol Chem, 397:195-205

Tags: (Molecular Neuroscience and Biophysics (Plested)

Abstract: In 2014, a slew of structures of glutamate receptors were published, based on crystallography and electron microscopy. Here we review these insights, integrate them with existing knowledge about receptor function and try to understand how the structures relate to the key property of the AMPA receptor - its speed.

Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies
Gupta(*), R., Lu(*), M., Hou(*), G., Caporini(*), M. A., Rosay(*), M., Maas(*), W., Struppe(*), J., Suiter(*), C., Ahn(*), J., Byeon(*), I. J., Franks, W. T., Orwick-Rydmark, M., Bertarello(*), A., Oschkinat, H., Lesage(*), A., Pintacuda(*), G., Gronenborn(*), A. M.; Polenova(*), T.
J Phys Chem B, 120:329-339

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

The progressive ankylosis protein ANK facilitates clathrin- and adaptor-mediated membrane traffic at the trans-Golgi network-to-endosome interface
Seifert(*), W., Posor, Y., Schu(*), P., Stenbeck(*), G., Mundlos(*), S., Klaassen(*), S., Nürnberg(*), P., Haucke, V., Kornak(*), U.; Kühnisch(*), J.
Hum Mol Genet, 25:3836-3848

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Dominant or recessive mutations in the progressive ankylosis gene ANKH have been linked to familial chondrocalcinosis (CCAL2), craniometaphyseal dysplasia (CMD), mental retardation, deafness and ankylosis syndrome (MRDA). The function of the encoded membrane protein ANK in cellular compartments other than the plasma membrane is unknown. Here, we show that ANK localizes to the trans-Golgi network (TGN), clathrin-coated vesicles and the plasma membrane. ANK functionally interacts with clathrin and clathrin associated adaptor protein (AP) complexes as loss of either protein causes ANK dispersion from the TGN to cytoplasmic endosome-like puncta. Consistent with its subcellular localization, loss of ANK results in reduced formation of tubular membrane carriers from the TGN, perinuclear accumulation of early endosomes and impaired transferrin endocytosis. Our data indicate that clathrin/AP-mediated cycling of ANK between the TGN, endosomes, and the cell surface regulates membrane traffic at the TGN/endosomal interface. These findings suggest that dysfunction of Golgi-endosomal membrane traffic may contribute to ANKH-associated pathologies.

Disruption of Kcc2-dependent inhibition of olfactory bulb output neurons suggests its importance in odour discrimination
Gödde, K., Gschwend(*), O., Puchkov, D., Pfeffer, C. K., Carleton(*), A.; Jentsch, T. J.
Nat Commun, 7:12043

Tags: Physiology and Pathology of Ion Transport (Jentsch), Cellular Imaging (Wiesner/Puchkov)

Abstract: Synaptic inhibition in the olfactory bulb (OB), the first relay station of olfactory information, is believed to be important for odour discrimination. We interfered with GABAergic inhibition of mitral and tufted cells (M/T cells), the principal neurons of the OB, by disrupting their potassium-chloride cotransporter 2 (Kcc2). Roughly, 70% of mice died around 3 weeks, but surviving mice appeared normal. In these mice, the resulting increase in the intracellular Cl(-) concentration nearly abolished GABA-induced hyperpolarization of mitral cells (MCs) and unexpectedly increased the number of perisomatic synapses on MCs. In vivo analysis of odorant-induced OB electrical activity revealed increased M/T cell firing rate, altered phasing of action potentials in the breath cycle and disrupted separation of odour-induced M/T cell activity patterns. Mice also demonstrated a severely impaired ability to discriminate chemically similar odorants or odorant mixtures. Our work suggests that precisely tuned GABAergic inhibition onto M/T cells is crucial for M/T cell spike pattern separation needed to distinguish closely similar odours.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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