The ionotropic glutamate delta receptor GluD1, encoded by the GRID1 gene, is involved in synapse formation, function, and plasticity. GluD1 does not bind glutamate, but instead cerebellin and D-serine, which allow the formation of trans-synaptic bridges, and trigger transmembrane signaling. Despite wide expression in the nervous system, pathogenic GRID1 variants have not been characterized in humans so far. We report homozygous missense GRID1 variants in five individuals from two unrelated consanguineous families presenting with intellectual disability and spastic paraplegia, without (p.Thr752Met) or with (p.Arg161His) diagnosis of glaucoma, a threefold phenotypic association whose genetic bases had not been elucidated previously. Molecular modeling and electrophysiological recordings indicated that Arg161His and Thr752Met mutations alter the hinge between GluD1 cerebellin and D-serine binding domains and the function of this latter domain, respectively. Expression, trafficking, physical interaction with metabotropic glutamate receptor mGlu1, and cerebellin binding of GluD1 mutants were not conspicuously altered. Conversely, upon expression in neurons of dissociated or organotypic slice cultures, we found that both GluD1 mutants hampered metabotropic glutamate receptor mGlu1/5 signaling via Ca 2+ and the ERK pathway and impaired dendrite morphology and excitatory synapse density. These results show that the clinical phenotypes are distinct entities segregating in the families as an autosomal recessive trait, and caused by pathophysiological effects of GluD1 mutants involving metabotropic glutamate receptor signaling and neuronal connectivity. Our findings unravel the importance of GluD1 receptor signaling in sensory, cognitive and motor functions of the human nervous system.

Ionotropic glutamate receptors are ligand‐gated ion channels essential for fast excitatory neurotransmission in the brain. In contrast to most other members of the iGluR family, the subfamily of delta receptors, GluD1 and GluD2, does not bind glutamate but glycine/D‐serine. GluD1 is widely expressed in the brain and the inner ear, where it is required for high‐frequency hearing. Furthermore, it has been associated with schizophrenia, autism and depression. X‐ray structures of the ligand‐binding domain (LBD) of GluD2 have been published; however, no high‐resolution structure is available for the ligand‐binding domain of GluD1 (GluD1‐LBD). Here, we report the X‐ray crystal structure of the GluD1‐LBD in its apo form at 2.57 Å resolution. Using isothermal titration calorimetry, we show that D‐serine binds to the GluD1‐LBD in an exothermic manner with a Kd of 160 μm, which is approximately five‐fold greater than at GluD2. Furthermore, we identify Glu822 as a critical determinant of receptor activation in GluD1 A654T. In contrast to studies on the GluD2 lurcher mutant A654T, we did not observe any effect of 1 mm D‐serine on the spontaneous currents at mouse GluD1 A654T by electrophysiological recordings of Xenopus laevis oocytes as previously also reported by others. These results point towards differences in the structure and dynamics between GluD1 and GluD2. Molecular dynamics simulations were employed to address this observation, suggesting that the apo structure of GluD1 is less flexible than the apo structure of GluD2 and that Pro725 in GluD1 may affect the interlobe closure of the ligand‐binding domain of GluD1.

Alzheimer's disease (AD) is a progressive brain disorder associated with slow loss of brain functions leading to memory failure and modest changes in behavior. The multifactorial neuropathological condition is due to a depletion of cholinergic neurons and accumulation of amyloid-beta (Aβ) plaques. Recently, a multi-target-directed ligand (MTDL) strategy has emerged as a robust drug discovery tool to overcome current challenges. In this research work, we aimed to design and develop a library of triazole-bridged aryl adamantane analogs for the treatment of AD. All synthesized analogs were characterized and evaluated through various in vitro and in vivo biological studies. The optimal compounds 32 and 33 exhibited potent inhibitory activities against acetylcholinesterase (AChE) (32 - IC50 = 0.086 μM; 33 - 0.135 μM), and significant Aβ aggregation inhibition (20 μM). N-methyl-d-aspartate (NMDA) receptor (GluN1-1b/GluN2B subunit combination) antagonistic activity of compounds 32 and 33 measured upon heterologous expression in Xenopus laevis oocytes showed IC50 values of 3.00 μM and 2.86 μM, respectively. The compounds possessed good blood-brain barrier permeability in the PAMPA assay and were safe for SH-SY5Y neuroblastoma (10 μM) and HEK-293 cell lines (30 μM). Furthermore, in vivo behavioral studies in rats demonstrated that both compounds improved cognitive and spatial memory impairment at a dose of 10 mg/kg oral administration. Together, our findings suggest triazole-bridged aryl adamantane as a promising new scaffold for the development of anti-Alzheimer's drugs.

Circulating autoantibodies (AB) of different immunoglobulin classes (IgM, IgA, and IgG), directed against the obligatory N-methyl- d -aspartate-receptor subunit NR1 (NMDAR1-AB), belong to the mammalian autoimmune repertoire, and appear with age-dependently high seroprevalence across health and disease. Upon access to the brain, they can exert NMDAR-antagonistic/ketamine-like actions. Still unanswered key questions, addressed here, are conditions of NMDAR1-AB formation/boosting, intraindividual persistence/course in serum over time, and (patho)physiological significance of NMDAR1-AB in modulating neuropsychiatric phenotypes. We demonstrate in a translational fashion from mouse to human that (1) serum NMDAR1-AB fluctuate upon long-term observation, independent of blood–brain barrier (BBB) perturbation; (2) a standardized small brain lesion in juvenile mice leads to increased NMDAR1-AB seroprevalence (IgM + IgG), together with enhanced Ig-class diversity; (3) CTLA4 (immune-checkpoint) genotypes, previously found associated with autoimmune disease, predispose to serum NMDAR1-AB in humans; (4) finally, pursuing our prior findings of an early increase in NMDAR1-AB seroprevalence in human migrants, which implicated chronic life stress as inducer, we independently replicate these results with prospectively recruited refugee minors. Most importantly, we here provide the first experimental evidence in mice of chronic life stress promoting serum NMDAR1-AB (IgA). Strikingly, stress-induced depressive-like behavior in mice and depression/anxiety in humans are reduced in NMDAR1-AB carriers with compromised BBB where NMDAR1-AB can readily reach the brain. To conclude, NMDAR1-AB may have a role as endogenous NMDAR antagonists, formed or boosted under various circumstances, ranging from genetic predisposition to, e.g., tumors, infection, brain injury, and stress, altogether increasing over lifetime, and exerting a spectrum of possible effects, also including beneficial functions.

ABSTRACTHistamine (HA) is a neurotransmitter in arthropod photoreceptors. It is recycled via conjugation to β-alanine to form β-alanylhistamine (carcinine). Conjugation occurs in epithelial glia that surround photoreceptor terminals in the first optic neuropil, and carcinine (CA) is then transported back to photoreceptors and cleaved to liberate HA and β-alanine. The gene Inebriated (Ine) encodes an Na+/Cl−-dependent SLC6 family transporter translated as two protein isoforms, long (P1) and short (P2). Photoreceptors specifically express Ine-P2 whereas Ine-P1 is expressed in non-neuronal cells. Both ine1 and ine3 have significantly reduced head HA contents compared with wild type, and a smaller increase in head HA after drinking 1% CA. Similarly, uptake of 0.1% CA was reduced in ine1 and ine3 mutant synaptosomes, but increased by 90% and 84% respectively for fractions incubated in 0.05% β-Ala, compared with wild type. Screening potential substrates in Ine expressing Xenopus oocytes revealed very little response to carcinine and β-Ala but increased conductance with glycine. Both ine1 and ine3 mutant responses in light-dark phototaxis did not differ from wild-type. Collectively our results suggest that Inebriated functions in an adjunct role as a transporter to the previously reported carcinine transporter CarT.

Autoantibodies of the IgG class against N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1-AB) were considered pathognomonic for anti-NMDAR encephalitis. This view has been challenged by the age-dependent seroprevalence (up to >20%) of functional NMDAR1-AB of all immunoglobulin classes found in >5000 individuals, healthy or affected by different diseases. These findings question a merely encephalitogenic role of NMDAR1-AB. Here, we show that NMDAR1-AB belong to the normal autoimmune repertoire of dogs, cats, rats, mice, baboons, and rhesus macaques, and are functional in the NMDAR1 internalization assay based on human IPSC-derived cortical neurons. The age dependence of seroprevalence is lost in nonhuman primates in captivity and in human migrants, raising the intriguing possibility that chronic life stress may be related to NMDAR1-AB formation, predominantly of the IgA class. Active immunization of ApoE-/- and ApoE+/+ mice against four peptides of the extracellular NMDAR1 domain or ovalbumin (control) leads to high circulating levels of specific AB. After 4 weeks, the endogenously formed NMDAR1-AB (IgG) induce psychosis-like symptoms upon MK-801 challenge in ApoE-/- mice, characterized by an open blood-brain barrier, but not in their ApoE+/+ littermates, which are indistinguishable from ovalbumin controls. Importantly, NMDAR1-AB do not induce any sign of inflammation in the brain. Immunohistochemical staining for microglial activation markers and T lymphocytes in the hippocampus yields comparable results in ApoE-/- and ApoE+/+ mice, irrespective of immunization against NMDAR1 or ovalbumin. These data suggest that NMDAR1-AB of the IgG class shape behavioral phenotypes upon access to the brain but do not cause brain inflammation on their own.

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.

Positive allosteric modulators of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA)-type ionotropic glutamate receptors are promising compounds for treatment of neurological disorders, for example, Alzheimer's disease. Here, we report synthesis and pharmacological evaluation of a series of mono-, di-, or trialkyl-substituted 7-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides, comprising in total 16 new modulators. The trisubstituted compounds 7b, 7d, and 7e revealed potent activity (EC2× = 2.7-4.3 μM; concentration of compound responsible for a 2-fold increase of the AMPA mediated response) as AMPA receptor potentiators in an in vitro cellular fluorescence assay (FLIPR). The 4-cyclopropyl compound 7f was found to be considerably less potent (EC2× = 60 μM), in contrast to previously described 4-monoalkyl-substituted benzothiadiazine dioxides for which the cyclopropyl group constitutes the best choice of substituent. 7b was subjected to X-ray structural analysis in complex with the GluA2 ligand-binding domain. We propose an explanation of the unexpected structure-activity relationship of this new series of mono-, di-, and trialkyl-substituted 1,2,4-benzothiadiazine 1,1-dioxide compounds. The methyl substituent in the 3-position directs the binding mode of the 1,2,4-benzothiadiazine 1,1-dioxide (BTD) scaffold. When a methyl substituent is present in the 3-position of the BTD, additional methyl substituents in both the 2- and 4-positions increase potency, whereas introduction of a 4-cyclopropyl group does not enhance potency of 2,3,4-alkyl-substituted BTDs. A hydrogen bond donor in the 2-position of the BTD is not necessary for modulator potency.

Autoantibodies of the IgG class against N -methyl- d -aspartate-receptor subunit NR1 (NMDAR1) were first described in anti-NMDAR encephalitis and seen as disease indicators. Recent work on together over 5000 individuals challenged this exclusive view by showing age-dependently up to >20% NMDAR1-autoantibody seroprevalence with comparable immunoglobulin class and titer distribution across health and disease. The key question therefore is to understand the properties of these autoantibodies, also in healthy carriers, in order to assess secondary complications and possible contributions to neuropsychiatric disease. Here, we believe we provide for human NMDAR1-autoantibodies the first comprehensive analysis of their target epitopes and functionality. We selected sera of representative carriers, healthy or diagnosed with very diverse conditions, that is, schizophrenia, age-related disorders like hypertension and diabetes, or anti-NMDAR encephalitis. We show that all positive sera investigated, regardless of source (ill or healthy donor) and immunoglobulin class, provoked NMDAR1 internalization in human-induced pluripotent stem cell-derived neurons and reduction of glutamate-evoked currents in NR1-1b/NR2A-expressing Xenopus oocytes. They displayed frequently polyclonal/polyspecific epitope recognition in the extracellular or intracellular NMDAR1 domains and some additionally in NR2A. We conclude that all circulating NMDAR1-autoantibodies have pathogenic potential regarding the whole spectrum of neuronal NMDAR-mediated effects upon access to the brain in situations of increased blood–brain–barrier permeability.

Herein we describe the first structure-activity relationship study of the broad-range iGluR antagonist (2S,3R)-3-(3-carboxyphenyl)pyrrolidine-2-carboxylic acid (1) by exploring the pharmacological effect of substituents in the 4, 4', or 5' positions and the bioisosteric substitution of the distal carboxylic acid for a phosphonic acid moiety. Of particular interest is a hydroxyl group in the 4' position 2a which induced a preference in binding affinity for homomeric GluK3 over GluK1 (Ki = 0.87 and 4.8 μM, respectively). Two X-ray structures of ligand binding domains were obtained: 2e in GluA2-LBD and 2f in GluK1-LBD, both at 1.9 Å resolution. Compound 2e induces a D1-D2 domain opening in GluA2-LBD of 17.3-18.8° and 2f a domain opening in GluK1-LBD of 17.0-17.5° relative to the structures with glutamate. The pyrrolidine-2-carboxylate moiety of 2e and 2f shows a similar binding mode as kainate. The 3-carboxyphenyl ring of 2e and 2f forms contacts comparable to those of the distal carboxylate in kainate.

Ionotropic glutamate receptors (iGluRs) mediate the vast majority of excitatory neurotransmission in the central nervous system of vertebrates. In the protein family of iGluRs, kainate receptors (KARs) comprise the probably least well understood receptor class. Although KARs act as key players in the regulation of synaptic network activity, many properties and functions of these proteins remain elusive until now. Especially the precise pre-, extra-, and postsynaptic localization of KARs plays a critical role for neuronal function, as an unbalanced localization of KARs would ultimately lead to dysregulated neuronal excitability. Recently, important advances in the understanding of the regulation of surface expression, function, and agonist-dependent endocytosis of KARs have been achieved. Post-translational modifications like PKC-mediated phosphorylation and SUMOylation have been reported to critically influence surface expression and endocytosis, while newly discovered auxiliary proteins were shown to shape the functional properties of KARs.

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated cation channels that mediate excitatory signal transmission in the central nervous system (CNS) of vertebrates. The members of the iGluR subfamily of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs) mediate most of the fast excitatory signal transmission, and their abundance in the postsynaptic membrane is a major determinant of the strength of excitatory synapses. Therefore, regulation of AMPAR trafficking to the postsynaptic membrane is an important constituent of mechanisms involved in learning and memory formation, such as long-term potentiation (LTP) and long-term depression (LTD). Auxiliary subunits play a critical role in the facilitation and regulation of AMPAR trafficking and function. The currently identified auxiliary subunits of AMPARs are transmembrane AMPA receptor regulatory proteins (TARPs), suppressor of lurcher (SOL), cornichon homologues (CNIHs), synapse differentiation-induced gene I (SynDIG I), cysteine-knot AMPAR modulating proteins 44 (CKAMP44), and germ cell-specific gene 1-like (GSG1L) protein. In this review we summarize our current knowledge of the modulatory influence exerted by these important but still underappreciated proteins.

The delta receptors, GluD1 and GluD2, are regarded as a subfamily of the ionotropic glutamate receptors solely because of sequence homology. While they play important roles in cerebellar function and high-frequency hearing and appear to serve structural functions at synapses, ligand-gated ion channel function has not been observed. However, we have previously shown that GluD2 can form functional ion channels when grafted with the ligand binding domain of a kainate receptor. In this study, we characterized this chimera as well as additional rat delta receptor chimeras and point mutants in more detail. We found that the kainate receptor ligand binding domain renders GluD1 functional as well, and GluD2 becomes a functional ion channel also when provided with an AMPA receptor ligand binding domain. Point mutations indicate that the GluD2 ion pore operates similarly but not identically to that of AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and kainate receptors. GluD2 mutated at a conserved arginine within the linker region connecting the ligand binding domain to the ion pore domain displays spontaneous currents that occur in the absence of agonists and are inhibited by agonist application - a behavior reminiscent of that of the previously characterized lurcher mutant. Using our chimeric approach, we provide evidence that this inhibition of spontaneous currents by agonists may be caused by desensitization. Our results show that delta receptors have functional gating machineries and ion permeation pathways similar but not identical to those of AMPA and kainate receptors, while the key differences seem to be located within the ligand binding domain.

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate neurotransmission in animal nervous systems. Homologous proteins in plants have been implicated in root development, ion transport, and several metabolic and signaling pathways. AtGLR3.4, a plant iGluR homolog from Arabidopsis thaliana , has ion channel activity and is gated by asparagine, serine, and glycine. Using heterologous expression in Xenopus oocytes, we found that another Arabidopsis iGluR homolog, AtGLR1.4, functioned as a ligand-gated, nonselective, Ca 2+ -permeable cation channel that responded to an even broader range of amino acids, none of which are agonists of animal iGluRs. Seven of the 20 standard amino acids—mainly hydrophobic ones—acted as agonists, with methionine being most effective and most potent. Nine amino acids were antagonists, and four, including glutamate and glycine, had no effect on channel activity. We constructed a model of this previously uncharacterized ligand specificity and used knockout mutants to show that AtGLR1.4 accounts for methionine-induced membrane depolarization in Arabidopsis leaves.

Here, we present the first phylogenetic analysis of a group of species taxonomically assigned to Polinices sensu latu (Naticidae, Gastropoda) based on molecular data sets. Polinices s.l. represents a speciose group of the infaunal gastropod family Naticidae, including species that have often been assigned to subgenera of Polinices [e.g. P. (Neverita), P. (Euspira), P. (Conuber) and P. (Mammilla)] based on conchological data. The results of our molecular phylogenetic analysis confirm the validity of five genera, Conuber, Polinices, Mammilla, Euspira and Neverita, including four that have been used previously mainly as subgenera of Polinices s.l. Our results furthermore indicate a close relationship of members of the Polinicinae to Sinum—a genus traditionally placed in the naticid subfamily Sininae. We furthermore present conchological analyses to determine the validity of shell characters used traditionally in species designation in the genus Polinices. Our data reveal several characters (e.g. protoconch, operculum colour, parietal callus) to be informative, while many characters show a high degree of homoplasy (e.g. umbilicus, shell form). Among the species arranged in the genus Polinices s.s., four conchologically very similar taxa often subsumed under the common Indo-Pacific species P. mammilla are separated distinctly in phylogenetic analyses. Despite their striking conchological similarities, none of these four taxa are related directly to each other. Additional conchological analyses of available name-bearing type specimens and type figures reveal the four “mammilla”-like white Polinices species to include true P. mammilla and three additional species, which could be assigned to P. constanti (replacement name for P. dubius), P. jukesii and possibly P. tawhitirahia, based on protoconch and operculum characteristics.

The classical tango is a dance characterized by a 2/4 or 4/4 rhythm in which the partners dance in a coordinated way, allowing dynamic contact. There is a surprising similarity between the tango and how KCNE β-subunits "dance" to the fast rhythm of the cell with their partners from the Kv channel family. The five KCNE β-subunits interact with several members of the Kv channels, thereby modifying channel gating via the interaction of their single transmembrane-spanning segment, the extracellular amino terminus, and/or the intracellular carboxy terminus with the Kv α-subunit. Best studied is the molecular basis of interactions between KCNE1 and Kv7.1, which, together, supposedly form the native cardiac IKs-channel. Here we review the current knowledge about functional and molecular interactions of KCNE1 with Kv7.1 and try to summarize and interpret the tango of the KCNEs.

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.

Accessory β-subunits of the KCNE gene family modulate the function of various cation channel α-subunits by the formation of heteromultimers. Among the most dramatic changes of biophysical properties of a voltage-gated channel by KCNEs are the effects of KCNE1 on KCNQ1 channels. KCNQ1 and KCNE1 are believed to form nativeIKs channels. Here, we characterize molecular determinants of KCNE1 interaction with KCNQ1 channels by scanning mutagenesis, double mutant cycle analysis, and molecular dynamics simulations. Our findings suggest that KCNE1 binds to the outer face of the KCNQ1 channel pore domain, modifies interactions between voltage sensor, S4-S5 linker and the pore domain, leading to structural modifications of the selectivity filter and voltage sensor domain. Molecular dynamics simulations suggest a stable interaction of the KCNE1 transmembrane α-helix with the pore domain S5/S6 and part of the voltage sensor domain S4 of KCNQ1 in a putative pre-open channel state. Formation of this state may induce slow activation gating, the pivotal characteristic of native cardiac IKs channels. This new KCNQ1-KCNE1 model may become useful for dynamic modeling of disease-associated mutant IKs channels.

AMPA receptors mediate the majority of fast synaptic transmission in the central nervous system and are therefore among the most intensively studied ligand-gated ion channels over the last decades. However, the recent discovery that native AMPA receptor complexes contain auxiliary subunits classified as transmembrane AMPA receptor regulatory proteins (TARPs) was quite a surprise and dramatically changed the field of AMPA receptor research. TARPs regulate trafficking as well as synaptic localization of AMPA receptors, and alter their pharmacological and biophysical properties, generally resulting in strongly elevated receptor-mediated currents. Thus, the association of AMPA receptors with TARPs increases receptor heterogeneity and diversity of postsynaptic currents. In this regard, unravelling the mechanisms by which TARPs modulate AMPA receptor function is an intriguing challenge. Studying the functional importance of the carboxy-terminal domain (CTD) of TARPs for receptor modulation, we found that the increased trafficking mediated by the two TARPs γ2 and γ3 is attributable to their CTDs. Furthermore, we demonstrated that the CTD additionally determines the differences between TARPs regarding their modulation of AMPA receptor function. As a case in point, we showed a unique role of the CTD of γ4, suggesting that TARPs modulate AMPA receptor function via individual mechanisms.