Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors

BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are the most complex of ionotropic glutamate receptors (iGluRs). Subunits of this subfamily assemble into heteromers, which – depending on the subunit combination – may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity. RESULTS: Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg(2+). Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants – possibly via disturbing the "proton shield" of NR1b splice variants. CONCLUSION: While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors

Die elektrorezeptiven Foveae von <i>Gnathonemus petersii</i> : Rezeptorverteilung, Rezeptormorphologie und Futtersuchverhalten

Afrikanische schwach elektrische Fische der Familie der Mormyridae, so auch der Elefantenrüsselfisch Gnathonemus petersii, sind in der Lage, ihre Umwelt über aktive Elektroortung wahrzunehmen. Mit Hilfe eines elektrischen Organs in ihrem Schwanzstiel senden sie kurze schwachelektrische Pulse aus. Durch diese Pulse (electric organ discharges, EODs) bauen sie ein bipolares, dreidimensionales Feld um ihren Körper auf. Änderungen in diesem Feld werden durch spezialisierte Elektrorezeptororgane, die Mormyromasten, wahrgenommen. Die Elektrorezeptorepidermis, auf der sich die Mormyromasten befinden, erstreckt sich über Kopf, Bauch und Rücken der Fische. Neben diesen Elektrorezeptororganen tragen Mormyriden ampulläre Rezeptoren zur passiven Elektroortung und Knollenorgane zur elektrischen Kommunikation auf ihrer Haut. In dieser Arbeit wurden einige Gesichtspunkte der „Fovea-Hypothese“ bei Gnathonemus petersii bearbeitet. Demnach reagieren zwei Körperregionen des Fischs auf bestimmte Aspekte des elektrischen Feldes und besitzen eine höhere räumliche Auflösung. Diese beiden Regionen sind das verlängerte Kinn, das so genannte Schnauzenorgan und die Nasalregion, zwischen Maul und Nasenlöchern. Für diese beiden Regionen wurde postuliert, dass sie elektrofoveale Regionen darstellen, also funktionell der Fovea im Auge vieler Wirbeltiere ähneln, aber unterschiedliche Aufgaben erfüllen. Um als Fovea bezeichnet zu werden, müssen unter anderem 3 Bedingungen erfüllt werden: 1. eine überdurchschnittlich hohe Dichte an Rezeptororganen, 2. eine besondere Morphologie der Rezeptorstrukturen und 3. ein besonderes Fixierungsverhalten, bei dem die Region auf bestimmte Reize ausgerichtet wird. In der vorliegenden Arbeit konnte gezeigt werden, dass auf dem Schnauzenorgan, besonders auf der Spitze, sehr hohe Dichten an Mormyromasten auftreten (65 Mormyromasten/mm2). Zur Basis sinkt die Dichte, ist aber immer noch höher als auf anderen Regionen. Auch die Mormyromastendichte auf der Nasalregion ist mit 4,6/mm2 noch beachtlich hoch, verglichen mit anderen Körperregionen, wie dem Rücken, wo im Schnitt nur noch 1,4 Mormyromasten/mm2 gefunden werden konnten. Auch der Durchmesser der Elektrorezeptorporen ändert sich zwischen den untersuchten Regionen, allerdings in entgegen gesetzter Richtung. Je höher die Dichte der Mormyromasten, desto geringer ist der Durchmesser der Poren, die auf der Hautoberfläche erkennbar sind. Dieser Unterschied konnte in histologischen Untersuchungen der Mormyromasten der gleichen Körperregionen verifiziert werden und darüber hinaus weitere Unterschiede in der Morphologie der Elektrorezeptororgane gefunden werden. So ändert sich die Größe der meisten Kompartimente der Mormyromasten. Die Größe der sensorischen Zellen ändert sich kaum. Diese als A- und B-Zellen bezeichneten Rezeptorzellen variieren in ihrer Anzahl pro Mormyromast. Sie steigt mit größer werdenden Rezeptororgandurchmessern an, ist also auf dem Schnauzenorgan am geringsten und auf dem Rücken am höchsten. Verrechnet mit der Dichte der Mormyromasten ergibt sich dennoch die höchste Dichte sensorischer Zellen auf dem Schnauzenorgan, gefolgt von der Nasalregion und dem Rücken. Dies spricht für eine höhere Sensitivität der einzelnen Mormyromasten auf dem Rücken, das System allerdings sollte die höchste Sensitivität und die höchste räumliche Auflösung auf dem Schnauzenorgan haben. Als weiterer Punkt wurde untersucht, welche Aufgaben die beiden fovealen Regionen im Verhalten der Fische erfüllen. Verhaltensversuche zeigten, dass der Fisch bei der Futtersuche den Boden mit seinem Schnauzenorgan absucht und damit unbekannte Objekte untersuchte. Mit den Pendelbewegungen des Schnauzenorgans die er bei der Futtersuche durchführt, deckt er einen sehr großen Bereich des überschwommenen Bodens ab (etwa 750° pro Sekunde). Das Schnauzenorgan kann so als Nahbereichs- Detektionssystem zum Aufspüren von Futter und zur Untersuchung von Objekten bezeichnet werden. Die Nasalregion wird bei solchen Suchbewegungen immer in einem konstanten Winkel in Schwimmrichtung gehalten (40° nach vorne). Die Nasalregion kann somit als Fernbereichs-Detektionssystem zur Vermeidung von Hindernissen angesehen werden. Diese Ergebnisse liefern gute Hinweise zur Bestätigung der Hypothese zur Existenz zweier elektrofovealer Regionen auf der Haut von Gnathonemus petersii.</p

The Transmembrane Domain C of AMPA Receptors is Critically Involved in Receptor Function and Modulation

Ionotropic glutamate receptors are major players in synaptic transmission and are critically involved in many cognitive events. Although receptors of different subfamilies serve different functions, they all show a conserved domain topology. For most of these domains, structure–function relationships have been established and are well understood. However, up to date the role of the transmembrane domain C in receptor function has been investigated only poorly. We have constructed a series of receptor chimeras and point mutants designed to shed light on the structural and/or functional importance of this domain. We here present evidence that the role of transmembrane domain C exceeds that of a mere scaffolding domain and that several amino acid residues located within the domain are crucial for receptor gating and desensitization. Furthermore, our data suggest that the domain may be involved in receptor interaction with transmembrane AMPA receptor regulatory proteins

Residues at the tip of the pore loop of NR3B-containing NMDA receptors determine Ca2+ permeability and Mg2+ block

<p>Abstract</p> <p>Background</p> <p>Members of the complex N-methyl-D-aspartate receptor (NMDAR) subfamily of ionotropic glutamate receptors (iGluRs) conventionally assemble from NR1 and NR2 subunits, the composition of which determines receptor properties. Hallmark features of conventional NMDARs include the requirement for a coagonist, voltage-dependent block by Mg²⁺, and high permeability for Ca²⁺. Both Mg²⁺sensitivity and Ca²⁺permeability are critically dependent on the amino acids at the N and N+1 positions of NR1 and NR2. The recently discovered NR3 subunits feature an unprecedented glycine-arginine combination at those critical sites within the pore. Diheteromers assembled from NR1 and NR3 are not blocked by Mg²⁺and are not permeable for Ca²⁺.</p> <p>Results</p> <p>Employing site-directed mutagenesis of receptor subunits, electrophysiological characterization of mutants in a heterologous expression system, and molecular modeling of the NMDAR pore region, we have investigated the contribution of the unusual NR3 N and N+1 site residues to the unique functional characteristics of receptors containing these subunits. Contrary to previous studies, we provide evidence that both the NR3 N and N+1 site amino acids are critically involved in mediating the unique pore properties. Ca²⁺permeability could be rescued by mutating the NR3 N site glycine to the NR1-like asparagine. Voltage-dependent Mg²⁺block could be established by providing an Mg²⁺coordination site at either the NR3 N or N+1 positions. Conversely, "conventional" receptors assembled from NR1 and NR2 could be made Mg²⁺insensitive and Ca²⁺impermeable by equipping either subunit with the NR3-like glycine at their N positions, with a stronger contribution of the NR1 subunit.</p> <p>Conclusions</p> <p>This study sheds light on the structure-function relationship of the least characterized member of the NMDAR subfamily. Contrary to previous reports, we provide evidence for a critical functional involvement of the NR3 N and N+1 site amino acids, and propose them to be the essential determinants for the unique pore properties mediated by this subunit.</p

System Simulation and Analysis of an LNG-Fueled SOFC System Using Additively Manufactured High Temperature Heat Exchangers

A laboratory-scale solid oxide fuel cell (SOFC) system using liquefied natural gas (LNG) as a fuel is designed to be used as an energy converter on seagoing vessels (MultiSchIBZ project). The presented system design phase is supported by thermodynamic system simulation. As heat integration plays a crucial role with regard to fuel recirculation and endothermic pre-reforming, the heat exchanger and pre-reforming component models need to exhibit a high degree of accuracy throughout the entire operating range. Compact additively manufactured tube-bundle and plate-fin heat exchangers are designed to achieve high heat exchange efficiencies at low pressure losses. Their heat transfer correlations are derived from experimental component tests under operating conditions. A simulation study utilizing these heat exchanger characteristics is carried out for four configuration variants of pre-reforming and heat integration. Their system behaviour is analyzed with regard to the degree of pre-reforming and the outlet temperature of the fuel processing module. The combination of allothermal pre-reforming with additively manufactured plate-fin heat exchangers exhibits the best heat integration performance at nominal full load and yields a partial load capability to up to 60% electrical load at net electrical efficiencies of 58 to 60% (LHV). © 2022 by the authors. Licensee MDPI, Basel, Switzerland

The rigid amphipathic fusion Inhibitor dUY11 acts through photosensitization of viruses

Copyright © 2014, American Society for Microbiology. All Rights Reserved.Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JVI.02907-13.Rigid amphipathic fusion inhibitors (RAFIs) are lipophilic inverted-cone-shaped molecules thought to antagonize the membrane curvature transitions that occur during virus-cell fusion and are broad-spectrum antivirals against enveloped viruses (Broad-SAVE). Here, we show that RAFIs act like membrane-binding photosensitizers: their antiviral effect is dependent on light and the generation of singlet oxygen (1O2), similar to the mechanistic paradigm established for LJ001, a chemically unrelated class of Broad-SAVE. Photosensitization of viral membranes is a common mechanism that underlies these Broad-SAVE.This work was supported by NIH grants U01 AI070495, U01 AI082100, R01 AI069317, and U54 AI065359 (PSWRCE) (to B.L.) and by Fundação para a Ciência e a Tecnologia-Ministério da Educação e Ciência (Portugal) project DELIN-HIVERA/0002/2013 and fellowship SFRH/BPD/72037/2010 (to N.C.S. and A.H., respectively

Optical control of AMPA receptors using a photoswitchable quinoxaline-2,3-dione antagonist

AMPA receptors respond to the neurotransmitter glutamate and play a critical role in excitatory neurotransmission. They have been implicated in several psychiatric disorders and have rich pharmacology. Antagonists of AMPA receptors have been explored as drugs and one has even reached the clinic. We now introduce a freely diffusible photoswitchable antagonist that is selective for AMPA receptors and endows them with light-sensitivity. Our photoswitch, ShuBQX-3, is active in its dark-adapted trans-isoform but is significantly less active as its cis-isoform. ShuBQX-3 exhibits a remarkable red-shifting of its photoswitching properties through interactions with the AMPA receptor ligand binding site. Since it can be used to control action potential firing with light, it could emerge as a powerful tool for studying synaptic transmission with high spatial and temporal precision

Enhanced mGlu5 Signaling in Excitatory Neurons Promotes Rapid Antidepressant Effects via AMPA Receptor Activation

Conventional antidepressants have limited efficacy and many side effects, highlighting the need for fast-acting and specific medications. Induction of the synaptic protein Homer1a mediates the effects of different antidepressant treatments, including the rapid action of ketamine and sleep deprivation (SD). We show here that mimicking Homer1a upregulation via intravenous injection of cell-membrane-permeable TAT-Homer1a elicits rapid antidepressant effects in various tests. Similar to ketamine and SD, in vitro and in vivo application of TAT-Homer1a enhances mGlu5 signaling, resulting in increased mTOR pathway phosphorylation, and upregulates synaptic AMPA receptor expression and activity. The antidepressant action of SD and Homer1a induction depends on mGlu5 activation specifically in excitatory CaMK2a neurons and requires enhanced AMPA receptor activity, translation, and trafficking. Moreover, our data demonstrate a pronounced therapeutic potential of different TAT-fused peptides that directly modulate mGlu5 and AMPA receptor activity and thus might provide a novel strategy for rapid and effective antidepressant treatment

Ionotropic glutamate receptors in GtoPdb v.2021.3

The ionotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (&#945;-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes, named originally according to their preferred, synthetic, agonist [35, 92, 155]. Receptor heterogeneity within each class arises from the homo-oligomeric, or hetero-oligomeric, assembly of distinct subunits into cation-selective tetramers. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand binding domain (LBD), 3 TM domains (M1, M3 and M4), a channel lining re-entrant 'p-loop' (M2) located between M1 and M3 and an intracellular carboxy- terminal domain (CTD) [99, 68, 107, 155, 82]. The X-ray structure of a homomeric ionotropic glutamate receptor (GluA2- see below) has recently been solved at 3.6&#197; resolution [143] and although providing the most complete structural information current available may not representative of the subunit arrangement of, for example, the heteromeric NMDA receptors [71]. It is beyond the scope of this supplement to discuss the pharmacology of individual ionotropic glutamate receptor isoforms in detail; such information can be gleaned from [35, 66, 31, 77, 42, 114, 24, 65, 155, 112, 113, 162]. Agents that discriminate between subunit isoforms are, where appropriate, noted in the tables and additional compounds that distinguish between receptor isoforms are indicated in the text below.The classification of glutamate receptor subunits has been re-addressed by NC-IUPHAR [28]. The scheme developed recommends a nomenclature for ionotropic glutamate receptor subunits that is adopted here.NMDA receptorsNMDA receptors assemble as obligate heteromers that may be drawn from GluN1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A and GluN3B subunits. Alternative splicing can generate eight isoforms of GluN1 with differing pharmacological properties. Various splice variants of GluN2B, 2C, 2D and GluN3A have also been reported. Activation of NMDA receptors containing GluN1 and GluN2 subunits requires the binding of two agonists, glutamate to the S1 and S2 regions of the GluN2 subunit and glycine to S1 and S2 regions of the GluN1 subunit [41, 25]. The minimal requirement for efficient functional expression of NMDA receptors in vitro is a di-heteromeric assembly of GluN1 and at least one GluN2 subunit variant, as a dimer of heterodimers arrangement in the extracellular domain [48, 99, 71]. However, more complex tri-heteromeric assemblies, incorporating multiple subtypes of GluN2 subunit, or GluN3 subunits, can be generated in vitro and occur in vivo. The NMDA receptor channel commonly has a high relative permeability to Ca2+ and is blocked, in a voltage-dependent manner, by Mg2+ such that at resting potentials the response is substantially inhibited.AMPA and Kainate receptorsAMPA receptors assemble as homomers, or heteromers, that may be drawn from GluA1, GluA2, GluA3 and GluA4 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I (i.e. &#947;2, &#947;3, &#947;4 and &#947;8) act, with variable stoichiometry, as auxiliary subunits to AMPA receptors and influence their trafficking, single channel conductance gating and pharmacology (reviewed in [43, 103, 153, 64]). Functional kainate receptors can be expressed as homomers of GluK1, GluK2 or GluK3 subunits. GluK1-3 subunits are also capable of assembling into heterotetramers (e.g. GluK1/K2; [87, 119, 118]). Two additional kainate receptor subunits, GluK4 and GluK5, when expressed individually, form high affinity binding sites for kainate, but lack function, but can form heteromers when expressed with GluK1-3 subunits (e.g. GluK2/K5; reviewed in [119, 65, 118]). Kainate receptors may also exhibit 'metabotropic' functions [87, 131]. As found for AMPA receptors, kainate receptors are modulated by auxiliary subunits (Neto proteins, [118, 88]). An important function difference between AMPA and kainate receptors is that the latter require extracellular Na+ and Cl- for their activation [11, 120]. RNA encoding the GluA2 subunit undergoes extensive RNA editing in which the codon encoding a p-loop glutamine residue (Q) is converted to one encoding arginine (R). This Q/R site strongly influences the biophysical properties of the receptor. Recombinant AMPA receptors lacking RNA edited GluA2 subunits are: (1) permeable to Ca2+; (2) blocked by intracellular polyamines at depolarized potentials causing inward rectification (the latter being reduced by TARPs); (3) blocked by extracellular argiotoxin and joro spider toxins and (4) demonstrate higher channel conductances than receptors containing the edited form of GluA2 [139, 63]. GluK1 and GluK2, but not other kainate receptor subunits, are similarly edited and broadly similar functional characteristics apply to kainate receptors lacking either an RNA edited GluK1, or GluK2, subunit [87, 118]. Native AMPA and kainate receptors displaying differential channel conductances, Ca2+ permeabilites and sensitivity to block by intracellular polyamines have been identified [30, 63, 91]. GluA1-4 can exist as two variants generated by alternative splicing (termed &#8216;flip&#8217; and &#8216;flop&#8217;) that differ in their desensitization kinetics and their desensitization in the presence of cyclothiazide which stabilises the nondesensitized state. TARPs also stabilise the non-desensitized conformation of AMPA receptors and facilitate the action of cyclothiazide [103]. Splice variants of GluK1-3 also exist which affects their trafficking [87, 118]