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Intra and Interdomain Motions of the NMDA Receptor using Single Molecule FRET
288a
Monday, February 29, 2016
P2X7R-dependent ionic currents differ in their activation kinetics by an absence of desensitization and induction of large membrane pores by long lasting agonist applications, i.e. the gating and permeation mechanisms of P2X7 receptors are not fully understood. G338 within the second transmembrane domain (TM2) is part of the narrow ion channel pore domain. Substitution of G338 to cysteine reversed the gating behaviour of the hP2X7R channel, i.e. hP2X7G338C expressing Xenopus oocytes displayed a leak conductance which is decreased by application of ATP or by the P2X7R agonist, BzATP. Substitution of extracellular Naþ by the larger organic Trisþ significantly decreased the ion conductance and shifted the reversal potential of the ATP-induced current to more negative potentials indicating that the leak current was carried by cations. Modeling the structural changes of hP2X7G338C during ATP application with the recently published truncated zebrafish P2X4 receptor model as template we found that G338 is located at the narrowest part of the closed ion channel pore and substitution by cysteine pushes the TM2 of the three subunits apart leading to an incomplete channel closure. ATP application leads to a conformation switch of hP2X7G338C which allows an interaction of C338 with Y40 and Y343 decreasing the ion channel pore diameter and leading to a reduced ion current. These findings point to a critical role of G338 in gating the P2X7 receptor. This work was supported by the German research foundation (DFG, Ma 1581/ 15-1, Schm 536/9-1 1419-Pos Board B396 Plant Glutamate Receptors: Electrophysiological Characterization and Evolutionary Perspectives Erwan Michard1, Michael M. Wudick1, Michael A. Lizzio1, Carlos Ortiz Ramı´rez2, Cla´udia Campos2, Jose´ A. Feijo´1. 1 UMD, College Park, MD, USA, 2IGC, Oeiras, Portugal. Ionotropic glutamate receptors are ligand-gated, multimeric cation channels involved in neuronal transmission in vertebrates. In animals, ligand selectivity is highly specific, and depending on the subunit, channels open in response to glycine/D-serine or glutamate. The molecular evolution of the animal glutamate receptor family is an important element in the evolution of the central nervous system. Interestingly, glutamate receptor homologous channels (GLRs) can also be found in plants where they are, for instance, involved in calcium signaling during pollen tube growth or in long distance signaling. While the genome of the moss Physcomitrella patens codes for two GLRs, the GLR family largely diversified in flowering plants. The genome of Arabidopsis thaliana encodes 20 GLRs that subgroup in three clades, one of which is homologous to the moss GLRs, and two clades that are specific for higher plants. Our work consists of studying the molecular specificities of plant GLRs by addressing two main questions: (1) What is the evolutionary meaning of plant GLR diversification? (2) In which signaling pathways are GLRs from the different clades involved in? To address these questions we performed an electrophysiological characterization of GLRs by studying their activity in protoplasts from moss and Arabidopsis GLR knock-plants. To further characterize the function of GLRs, we also expressed and studied them in heterologous systems. Our work reveals unique gating properties of plant GLRs and gives novel insights into the specificity of plant GLRs compared to the animal ones. 1420-Pos Board B397 Functional Roles of a Conserved Tryptophan at Subunit-Subunit Interfaces in NMDA Receptor Membrane Regions Madeleine R. Wilcox, Nathan G. Glasgow, Jon W. Johnson. Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA. NMDA receptors (NMDARs) are ionotropic glutamate receptors (iGluRs) characterized by channel block by extracellular magnesium (Mg2þo) and slow deactivation kinetics. NMDARs are typically composed of two GluN1 and two GluN2 (GluN2A-GluN2D) subunits. The transmembrane domain (TMD) of each subunit contains three membrane-spanning regions (M1, M3, and M4) and a pore lining re-entrant loop (M2). The M2 region of each NMDAR subunit contains a tryptophan (W) that is highly conserved: nearly all known mammalian iGluR subunits, several potassium channels, and iGluR-like proteins in phylogenetically distant organisms contain W at the homologous site. This highly conserved W is located in NMDARs at the interface between the M2 region and the adjacent M3 region. Previous work demonstrated that mutation of this highly-conserved W in the GluN2B subunit (W607) drastically decreased block by Mg2þo, while mutation of the homologous residue in the GluN2A subunit (W606) had a relatively minor effect (Williams et al., 1998 Mol Pharmacol 53, 933). Interestingly, mutating the homologous residue in GluN1 subunits (W608) also has little effect on
Mg2þo block (Siegler Retchless et al., 2012 Nat Neurosci 15, 406). To further explore the function of the conserved W at subunit-subunit interfaces we used whole-cell recordings from recombinant receptors expressed in tsA201 cells. We examined GluN2A(W606) and GluN2B(W607) mutants expressed with wild-type GluN1 subunits or GluN1 subunits with mutations of M3 residues at the M2-M3 interface. We found that receptors containing mutations at both GluN2A(W606) and a residue in the adjacent interface-lining GluN1 M3 region dramatically decreased block by Mg2þo. We also examined GluN1(W608) mutants and observed profoundly slowed deactivation kinetics. Overall, these results suggest that the conserved W is broadly involved in channel properties and gating of NMDARs. 1421-Pos Board B398 Intra and Interdomain Motions of the NMDA Receptor using Single Molecule FRET Drew Dolino1, Sudeshna Chatterjee2, David Cooper2, Henriette Jaurich2, Swarna Ramaswamy1, Soheila Rezaei Adariani3, Hugo Sanabria3, Christy Landes2, Vasanthi Jayaraman1. 1 Biochemistry & Molecular Biology, UTHealth, Houston, TX, USA, 2 Chemistry, Rice University, Houston, TX, USA, 3Physics and Astronomy, Clemson University, Clemson, SC, USA. Ionotropic glutamate receptors are tetrameric ligand-gated ion channels which mediate the majority of excitatory neurotransmission in the central nervous system. The NMDA receptor (NMDAR) in particular is unique in that it is an obligate heterotetramer that also requires a co-agonist glycine or D-serine. The amino-terminal domains (ATD) and ligand-binding domains (LBD) of the NMDAR have been shown to fold into a clamshell-like shape that close upon the introduction of their respective ligand. At the LBD, extent of cleft closure of the agonist-binding domain is one mechanism by which the agonist mediates channel activity. However, only an open-cleft or a closed-cleft conformation has been seen in crystal structures of the glycine-binding GluN1 subunit of the NMDAR, and no partially-closed cleft states have been observed, leaving unanswered the mechanisms of partial agonism. Similarly, it is still unclear how binding of allosteric modulators at the NMDAR ATD is propagated downward through the LBD and transmembrane domain to affect channel activity. Here, we have used single molecule FRET to examine NMDAR dynamics in order to address these questions. Unnatural amino acids were used to achieve specific or directional labeling of the protein. Our studies in the LBD reveal experimental evidence of the two-state model, with a common closed cleft conformation among different agonists with agonist efficacy dictated by its ability to stabilize that closed cleft. We also see that this stability, as measured by the fractional occupancy of the isolated domain in cleft-closure states below a threshold, correlates linearly with agonist efficacy, providing a link between LBD dynamics, cleft closure, and channel activity. Our interdomain studies on the full-length receptor reveal large-scale intersubunit motions and striking differences in the conformational landscape when bound to agonist and allosteric modulators. 1422-Pos Board B399 MPX-004: A New Pharmacological Tool to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit Robert A. Volkmann, Christopher Fanger, David R. Anderson, Frank S. Menniti. Mnemosyne Pharmaceuticals, Providence, RI, USA. GluN2A subunit-containing NMDA receptors are the most abundant in the mammalian CNS and physiological and genetic evidence implicate these receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2Aselective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl) methyl)pyrazine-2-carboxamide). MPX-004 inhibits hGluN2A-containing NMDA receptors expressed in HEK cells and Xenopus oocytes with IC50s of 80 nM and 219 nM, respectively. In contrast, MPX-004 at concentrations that completely inhibited GluN2A activity has no inhibitory effect on GluN2B, C, or D receptor-mediated responses in these same heterologous expression systems. Maximal concentrations of MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices and 25% of the pyramidal cell NMDA receptor-mediated response in mouse visual cortex slices. Significantly, the compound had no effect in visual cortex slices from GRIN2A KO mice, confirming selectivity for native NMDA receptors. Thus, MPX-004 offers a highly selective pharmacological tool to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.
Monday, February 29, 2016
P2X7R-dependent ionic currents differ in their activation kinetics by an absence of desensitization and induction of large membrane pores by long lasting agonist applications, i.e. the gating and permeation mechanisms of P2X7 receptors are not fully understood. G338 within the second transmembrane domain (TM2) is part of the narrow ion channel pore domain. Substitution of G338 to cysteine reversed the gating behaviour of the hP2X7R channel, i.e. hP2X7G338C expressing Xenopus oocytes displayed a leak conductance which is decreased by application of ATP or by the P2X7R agonist, BzATP. Substitution of extracellular Naþ by the larger organic Trisþ significantly decreased the ion conductance and shifted the reversal potential of the ATP-induced current to more negative potentials indicating that the leak current was carried by cations. Modeling the structural changes of hP2X7G338C during ATP application with the recently published truncated zebrafish P2X4 receptor model as template we found that G338 is located at the narrowest part of the closed ion channel pore and substitution by cysteine pushes the TM2 of the three subunits apart leading to an incomplete channel closure. ATP application leads to a conformation switch of hP2X7G338C which allows an interaction of C338 with Y40 and Y343 decreasing the ion channel pore diameter and leading to a reduced ion current. These findings point to a critical role of G338 in gating the P2X7 receptor. This work was supported by the German research foundation (DFG, Ma 1581/ 15-1, Schm 536/9-1 1419-Pos Board B396 Plant Glutamate Receptors: Electrophysiological Characterization and Evolutionary Perspectives Erwan Michard1, Michael M. Wudick1, Michael A. Lizzio1, Carlos Ortiz Ramı´rez2, Cla´udia Campos2, Jose´ A. Feijo´1. 1 UMD, College Park, MD, USA, 2IGC, Oeiras, Portugal. Ionotropic glutamate receptors are ligand-gated, multimeric cation channels involved in neuronal transmission in vertebrates. In animals, ligand selectivity is highly specific, and depending on the subunit, channels open in response to glycine/D-serine or glutamate. The molecular evolution of the animal glutamate receptor family is an important element in the evolution of the central nervous system. Interestingly, glutamate receptor homologous channels (GLRs) can also be found in plants where they are, for instance, involved in calcium signaling during pollen tube growth or in long distance signaling. While the genome of the moss Physcomitrella patens codes for two GLRs, the GLR family largely diversified in flowering plants. The genome of Arabidopsis thaliana encodes 20 GLRs that subgroup in three clades, one of which is homologous to the moss GLRs, and two clades that are specific for higher plants. Our work consists of studying the molecular specificities of plant GLRs by addressing two main questions: (1) What is the evolutionary meaning of plant GLR diversification? (2) In which signaling pathways are GLRs from the different clades involved in? To address these questions we performed an electrophysiological characterization of GLRs by studying their activity in protoplasts from moss and Arabidopsis GLR knock-plants. To further characterize the function of GLRs, we also expressed and studied them in heterologous systems. Our work reveals unique gating properties of plant GLRs and gives novel insights into the specificity of plant GLRs compared to the animal ones. 1420-Pos Board B397 Functional Roles of a Conserved Tryptophan at Subunit-Subunit Interfaces in NMDA Receptor Membrane Regions Madeleine R. Wilcox, Nathan G. Glasgow, Jon W. Johnson. Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA. NMDA receptors (NMDARs) are ionotropic glutamate receptors (iGluRs) characterized by channel block by extracellular magnesium (Mg2þo) and slow deactivation kinetics. NMDARs are typically composed of two GluN1 and two GluN2 (GluN2A-GluN2D) subunits. The transmembrane domain (TMD) of each subunit contains three membrane-spanning regions (M1, M3, and M4) and a pore lining re-entrant loop (M2). The M2 region of each NMDAR subunit contains a tryptophan (W) that is highly conserved: nearly all known mammalian iGluR subunits, several potassium channels, and iGluR-like proteins in phylogenetically distant organisms contain W at the homologous site. This highly conserved W is located in NMDARs at the interface between the M2 region and the adjacent M3 region. Previous work demonstrated that mutation of this highly-conserved W in the GluN2B subunit (W607) drastically decreased block by Mg2þo, while mutation of the homologous residue in the GluN2A subunit (W606) had a relatively minor effect (Williams et al., 1998 Mol Pharmacol 53, 933). Interestingly, mutating the homologous residue in GluN1 subunits (W608) also has little effect on
Mg2þo block (Siegler Retchless et al., 2012 Nat Neurosci 15, 406). To further explore the function of the conserved W at subunit-subunit interfaces we used whole-cell recordings from recombinant receptors expressed in tsA201 cells. We examined GluN2A(W606) and GluN2B(W607) mutants expressed with wild-type GluN1 subunits or GluN1 subunits with mutations of M3 residues at the M2-M3 interface. We found that receptors containing mutations at both GluN2A(W606) and a residue in the adjacent interface-lining GluN1 M3 region dramatically decreased block by Mg2þo. We also examined GluN1(W608) mutants and observed profoundly slowed deactivation kinetics. Overall, these results suggest that the conserved W is broadly involved in channel properties and gating of NMDARs. 1421-Pos Board B398 Intra and Interdomain Motions of the NMDA Receptor using Single Molecule FRET Drew Dolino1, Sudeshna Chatterjee2, David Cooper2, Henriette Jaurich2, Swarna Ramaswamy1, Soheila Rezaei Adariani3, Hugo Sanabria3, Christy Landes2, Vasanthi Jayaraman1. 1 Biochemistry & Molecular Biology, UTHealth, Houston, TX, USA, 2 Chemistry, Rice University, Houston, TX, USA, 3Physics and Astronomy, Clemson University, Clemson, SC, USA. Ionotropic glutamate receptors are tetrameric ligand-gated ion channels which mediate the majority of excitatory neurotransmission in the central nervous system. The NMDA receptor (NMDAR) in particular is unique in that it is an obligate heterotetramer that also requires a co-agonist glycine or D-serine. The amino-terminal domains (ATD) and ligand-binding domains (LBD) of the NMDAR have been shown to fold into a clamshell-like shape that close upon the introduction of their respective ligand. At the LBD, extent of cleft closure of the agonist-binding domain is one mechanism by which the agonist mediates channel activity. However, only an open-cleft or a closed-cleft conformation has been seen in crystal structures of the glycine-binding GluN1 subunit of the NMDAR, and no partially-closed cleft states have been observed, leaving unanswered the mechanisms of partial agonism. Similarly, it is still unclear how binding of allosteric modulators at the NMDAR ATD is propagated downward through the LBD and transmembrane domain to affect channel activity. Here, we have used single molecule FRET to examine NMDAR dynamics in order to address these questions. Unnatural amino acids were used to achieve specific or directional labeling of the protein. Our studies in the LBD reveal experimental evidence of the two-state model, with a common closed cleft conformation among different agonists with agonist efficacy dictated by its ability to stabilize that closed cleft. We also see that this stability, as measured by the fractional occupancy of the isolated domain in cleft-closure states below a threshold, correlates linearly with agonist efficacy, providing a link between LBD dynamics, cleft closure, and channel activity. Our interdomain studies on the full-length receptor reveal large-scale intersubunit motions and striking differences in the conformational landscape when bound to agonist and allosteric modulators. 1422-Pos Board B399 MPX-004: A New Pharmacological Tool to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit Robert A. Volkmann, Christopher Fanger, David R. Anderson, Frank S. Menniti. Mnemosyne Pharmaceuticals, Providence, RI, USA. GluN2A subunit-containing NMDA receptors are the most abundant in the mammalian CNS and physiological and genetic evidence implicate these receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2Aselective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl) methyl)pyrazine-2-carboxamide). MPX-004 inhibits hGluN2A-containing NMDA receptors expressed in HEK cells and Xenopus oocytes with IC50s of 80 nM and 219 nM, respectively. In contrast, MPX-004 at concentrations that completely inhibited GluN2A activity has no inhibitory effect on GluN2B, C, or D receptor-mediated responses in these same heterologous expression systems. Maximal concentrations of MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices and 25% of the pyramidal cell NMDA receptor-mediated response in mouse visual cortex slices. Significantly, the compound had no effect in visual cortex slices from GRIN2A KO mice, confirming selectivity for native NMDA receptors. Thus, MPX-004 offers a highly selective pharmacological tool to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.