Search

Raw data used for PMF generation and gro/xtc files used for CG and atomistic unbiased simulations.

Excitatory signaling mediated by N-methyl-D-aspartate receptor (NMDAR) is critical for brain development and function, as well as for neurological diseases and disorders. Channel blockers of NMDARs are of medical interest owing to their potential for treating depression, Alzheimer's disease, and epilepsy. However, precise mechanisms underlying binding and channel blockade have remained limited owing to challenges in obtaining high-resolution structures at the binding site within the transmembrane domains. Here, we monitor the binding of three clinically important channel blockers: phencyclidine, ketamine, and memantine in GluN1-2B NMDARs at local resolutions of 2.5-3.5 Å around the binding site using single-particle electron cryo-microscopy, molecular dynamics simulations, and electrophysiology. The channel blockers form different extents of interactions with the pore-lining residues, which control mostly off-speeds but not on-speeds. Our comparative analyses of the three unique NMDAR channel blockers provide a blueprint for developing therapeutic compounds with minimal side effects.

Under physiological conditions, N-Methyl-D-Aspartate (NMDA) receptors play a crucial role for synaptic plasticity, long-term potentiation and long-term depression. However, overactivation of NMDA receptors can result in excitotoxicity, which is associated with various neurological and neurodegenerative diseases. The physiological properties of NMDA receptors are strongly dependent on the GluN2 subunit incorporated into the heterotetrameric NMDA receptor. Therefore, subtype selective NMDA receptor modulators are of high interest. Since prototypical GluN2A-NMDA receptor antagonists TCN-201 and its MPX-analogs adopt a U-shaped conformation within the binding pocket, paracyclophanes were designed containing the phenyl rings in an already parallel orientation. Docking studies of the designed paracyclophanes show a similar binding pose as TCN-201. [2.2]Paracyclophanes with a benzoate or benzamide side chain were prepared in four-step synthesis, respectively, starting with a radical bromination in benzylic 1-position of [2.2]paracyclophane. In two-electrode voltage clamp experiments using Xenopus laevis oocytes transfected with cRNAs for the GluN1-4a and GluN2A subunits, the esters and amides (conc. 10 μM) did not show considerable inhibition of ion flux. It can be concluded that the GluN2A-NMDA receptor does not accept ligands with a paracyclophane scaffold functionalized in benzylic 1-position, although docking studies had revealed promising binding poses for benzoic acid esters and benzamides.

Neurotransmission mediated by diverse subtypes of N-methyl-D-aspartate receptors (NMDARs) is fundamental for basic brain functions and development as well as neuropsychiatric diseases and disorders. NMDARs are glycine- and glutamate-gated ion channels that exist as heterotetramers composed of obligatory GluN1 and GluN2(A-D) and/or GluN3(A-B). The GluN2C and GluN2D subunits form ion channels with distinct properties and spatio-temporal expression patterns. Here, we provide the structures of the agonist-bound human GluN1-2C NMDAR in the presence and absence of the GluN2C-selective positive allosteric potentiator (PAM), PYD-106, the agonist-bound GluN1-2A-2C tri-heteromeric NMDAR, and agonist-bound GluN1-2D NMDARs by single-particle electron cryomicroscopy. Our analysis shows unique inter-subunit and domain arrangements of the GluN2C NMDARs, which contribute to functional regulation and formation of the PAM binding pocket and is distinct from GluN2D NMDARs. Our findings here provide the fundamental blueprint to study GluN2C- and GluN2D-containing NMDARs, which are uniquely involved in neuropsychiatric disorders.

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.

N-methyl-D-aspartate receptors (NMDARs) uniquely require binding of two different neurotransmitter agonists for synaptic transmission. D-serine and glycine bind to one subunit, GluN1, while glutamate binds to the other, GluN2. These agonists bind to the receptor’s bi-lobed ligand-binding domains (LBDs), which close around the agonist during receptor activation. To better understand the unexplored mechanisms by which D-serine contributes to receptor activation, we performed multi-microsecond molecular dynamics simulations of the GluN1/GluN2A LBD dimer with free D-serine and glutamate agonists. Surprisingly, we observed D-serine binding to both GluN1 and GluN2A LBDs, suggesting that D-serine competes with glutamate for binding to GluN2A. This mechanism is confirmed by our electrophysiology experiments, which show that D-serine is indeed inhibitory at high concentrations. Although free energy calculations indicate that D-serine stabilizes the closed GluN2A LBD, its inhibitory behavior suggests that it either does not remain bound long enough or does not generate sufficient force for ion channel gating. We developed a workflow using pathway similarity analysis to identify groups of residues working together to promote binding. These conformation-dependent pathways were not significantly impacted by the presence of N-linked glycans, which act primarily by interacting with the LBD bottom lobe to stabilize the closed LBD.

Despite major advances in methodologies for membrane protein production over the last two decades, there remain challenging protein complexes that are technically difficult to yield by conventional recombinant expression methods. A large number of these proteins are multimeric membrane proteins from eukaryotic species, which are required to pass through stringent quality control mechanisms of host cells for proper folding and complex assembly. Here, we describe the development procedure to improve the production efficiency of multi-oligomeric membrane protein complexes in insect cells and recombinant baculovirus, which involves screening of promoters, enhancers, and untranslated regions for expression levels, using calcium homeostasis modulator (CALHM) and N-methyl-d-aspartate receptor (NMDAR) proteins as examples. We demonstrate that our insect cell expression strategy is effective in expression of both multi-homomeric CALHM proteins and multi-heteromeric NMDARs.

Glutamate receptors are essential ligand-gated ion channels in the central nervous system that mediate excitatory synaptic transmission in response to the release of glutamate from presynaptic terminals. The structural and biophysical basis underlying the function of these receptors has been studied for decades by a wide range of approaches. However recent structural, pharmacological, and genetic studies have provided new insight into the regions of this protein that are critical determinants of receptor function. Lack of variation in specific areas of the protein amino acid sequences in the human population has defined three regions in each receptor subunit that are under selective pressure, which has focused research efforts and driven new hypotheses. In addition, these three closely positioned elements reside near a cavity that is shown by multiple studies to be a likely site of action for allosteric modulators, one of which is currently in use as an FDA-approved anticonvulsant. These structural elements are capable of controlling gating of the pore, and appear to permit some modulators bound within the cavity to also alter permeation properties. This creates a new precedent whereby features of the channel pore can be modulated by exogenous drugs that bind outside the pore. The convergence of structural, genetic, biophysical, and pharmacological approaches is a powerful means to gain insight into the complex biological processes defined by neurotransmitter receptor function.

Hansen et al. review recent structural data that have provided insight into the function and allosteric modulation of NMDA receptors., NMDA-type glutamate receptors are ligand-gated ion channels that mediate a Ca2+-permeable component of excitatory neurotransmission in the central nervous system (CNS). They are expressed throughout the CNS and play key physiological roles in synaptic function, such as synaptic plasticity, learning, and memory. NMDA receptors are also implicated in the pathophysiology of several CNS disorders and more recently have been identified as a locus for disease-associated genomic variation. NMDA receptors exist as a diverse array of subtypes formed by variation in assembly of seven subunits (GluN1, GluN2A-D, and GluN3A-B) into tetrameric receptor complexes. These NMDA receptor subtypes show unique structural features that account for their distinct functional and pharmacological properties allowing precise tuning of their physiological roles. Here, we review the relationship between NMDA receptor structure and function with an emphasis on emerging atomic resolution structures, which begin to explain unique features of this receptor.

Excitatory neurotransmission meditated by glutamate receptors including N-methyl-D-aspartate receptors (NMDARs) is pivotal to brain development and function. NMDARs are heterotetramers composed of GluN1 and GluN2 subunits, which bind glycine and glutamate, respectively, to activate their ion channels. Despite importance in brain physiology, the precise mechanisms by which activation and inhibition occur via subunit-specific binding of agonists and antagonists remain largely unknown. Here, we show the detailed patterns of conformational changes and inter-subunit and -domain reorientation leading to agonist-gating and subunit-dependent competitive inhibition by providing multiple structures in distinct ligand states at 4 Å or better. The structures reveal that activation and competitive inhibition by both GluN1 and GluN2 antagonists occur by controlling the tension of the linker between the ligand-binding domain and the transmembrane ion channel of the GluN2 subunit. Our results provide detailed mechanistic insights into NMDAR pharmacology, activation, and inhibition, which are fundamental to the brain physiology.

AMPA and NMDA receptors are ionotropic glutamate receptors that make fundamental contributions to synaptic activity in the brain in different ways. Amin et al. show that their respective M4 segments, located on the periphery of their pore domains, contribute to their functional diversity., Ionotropic glutamate receptors (iGluRs), including AMPA receptor (AMPAR) and NMDA receptor (NMDAR) subtypes, are ligand-gated ion channels that mediate signaling at the majority of excitatory synapses in the nervous system. The iGluR pore domain is structurally and evolutionarily related to an inverted two-transmembrane K+ channel. Peripheral to the pore domain in eukaryotic iGluRs is an additional transmembrane helix, the M4 segment, which interacts with the pore domain of a neighboring subunit. In AMPARs, the integrity of the alignment of a specific face of M4 with the adjacent pore domain is essential for receptor oligomerization. In contrast to AMPARs, NMDARs are obligate heterotetramers composed of two GluN1 and typically two GluN2 subunits. Here, to address the function of the M4 segments in NMDARs, we carry out a tryptophan scan of M4 in GluN1 and GluN2A subunits. Unlike AMPARs, the M4 segments in NMDAR subunits makes only a limited contribution to their biogenesis. However, the M4 segments in both NMDAR subunits are critical for receptor activation, with mutations at some positions, most notably at the extreme extracellular end, completely halting the gating process. Furthermore, although the AMPAR M4 makes a minimal contribution to receptor desensitization, the NMDAR M4 segments have robust and subunit-specific effects on desensitization. These findings reveal that the functional roles of the M4 segments in AMPARs and NMDARs have diverged in the course of their evolution and that the M4 segments in NMDARs may act as a transduction pathway for receptor modulation at synapses.

SUMMARY The physiology of N-Methyl-D-aspartate (NMDA) receptors in mammals is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino terminal domain (ATD). Recent crystal structures of GluN1/GluN2B NMDA receptors in the presence of agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here, we combine x-ray crystallography, single-particle electron cryomicroscopy, and electrophysiology to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open-conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.

Context-dependent inhibition of N-methyl-D-aspartate (NMDA) receptors has important therapeutic implications for the treatment of neurological diseases that are associated with altered neuronal firing and signaling. This is especially true in stroke, where the proton concentration in the afflicted area can increase by an order of magnitude. A class of allosteric inhibitors, the 93-series, shows greater potency against GluN1-GluN2B NMDA receptors in such low pH environments, allowing targeted therapy only within the ischemic region. Here we map the 93-series compound binding site in the GluN1-GluN2B NMDA receptor amino terminal domain and show that the interaction of the N-alkyl group with a hydrophobic cage of the binding site is critical for pH-dependent inhibition. Mutation of residues in the hydrophobic cage alters pH-dependent potency, and remarkably, can convert inhibitors into potentiators. Our study provides a foundation for the development of highly specific neuroprotective compounds for the treatment of neurological diseases., Context-dependent inhibition of NMDA receptors has important therapeutic implications for treatment of neurological diseases. Here, the authors use structural biology and biophysics to describe the basis for pH-dependent inhibition for a class of allosteric NMDAR inhibitors, called the 93-series.

N-Methyl-d-aspartate receptors (NMDARs) are glutamate-gated ion channels, which are critically involved in brain development, learning and memory, cognition, as well as a number of neurological diseases and disorders. Structural biology of NMDARs has been challenging due to technical difficulties associated with assembling a number of different membrane protein subunits. Here, we review historical X-ray crystallographic studies on isolated extracellular domains, which are still the most effective mean to delineate compound binding modes, as well as the most recent studies using electron cryo-microscopy (cryo-EM). A number of NMDAR structures accumulated over the past 15 years provide insights into the hetero-tetrameric assembly pattern, pharmacological specificities elicited by subtypes and alternative splicing, and potential patterns of conformational dynamics; however, many more important unanswered questions remain.

Alternative gene splicing gives rise to N-methyl-D-aspartate (NMDA) receptor ion channels with defined functional properties and unique contributions to calcium signaling in a given chemical environment in the mammalian brain. Splice variants possessing the Exon 5-encoded motif at the amino terminal domain (ATD) of the GluN1 subunit are known to display robustly altered deactivation rates and pH sensitivity, however the underlying mechanism for this functional modification is largely unknown. Here, we show through electron cryo-microscopy (cryo-EM) that the presence of the Exon 5 motif in GluN1 alters the local architecture of heterotetrameric GluN1-GluN2 NMDA receptors and creates contacts with the ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, which are absent in NMDA receptors lacking the Exon 5 motif. The unique interactions established by the Exon 5 motif are essential to the stability of the ATD/LBD and LBD/LBD interfaces that are critically involved in controlling proton sensitivity and deactivation.

Two articles in this issue of Neuron (Yelshanskaya et al., 2016; Yi et al., 2016) explore the structural basis of allosteric inhibition in ionotropic glutamate receptors, providing key insights into how iGluRs function in the brain as well as how they might be pharmacologically modulated in neurological disorders and disease.

Signal transduction across cell membranes often requires assembly of heteromeric receptors with defined stoichiometry. In this issue of Structure, Morales-Perez et al. (2016) present elegant methods for the expression of heteromeric nicotinic acetylcholine receptors with a defined α4β2 stoichiometry involving controlled baculovirus-mediated transduction and subunit counting by measurement of two fluorescent signals.

Plant shoot systems derive from the shoot apical meristems (SAMs), pools of stems cells that are regulated by a feedback between the WUSCHEL (WUS) homeobox protein and CLAVATA (CLV) peptides and receptors. The maize heterotrimeric G protein α subunit COMPACT PLANT2 (CT2) functions with CLV receptors to regulate meristem development. In addition to the sole canonical Gα CT2, maize also contains three eXtra Large GTP-binding proteins (XLGs), which have a domain with homology to Gα as well as additional domains. By either forcing CT2 to be constitutively active, or by depleting XLGs using CRISPR-Cas9, here we show that both CT2 and XLGs play important roles in maize meristem regulation, and their manipulation improved agronomic traits. For example, we show that expression of a constitutively active CT2 resulted in higher spikelet density and kernel row number, larger ear inflorescence meristems (IMs) and more upright leaves, all beneficial traits selected during maize improvement. Our findings suggest that both the canonical Gα, CT2 and the non-canonical XLGs play important roles in maize meristem regulation and further demonstrate that weak alleles of plant stem cell regulatory genes have the capacity to improve agronomic traits., Author summary Maize is one of the most important cereal crops worldwide. Optimizing its yields requires fine tuning of development. Therefore, it is critical to understand the developmental signaling mechanisms to provide basic knowledge to maximize productivity. The heterotrimeric G proteins transmit signals from cell surface receptor and have been shown to regulate many biological processes, including shoot development. Here we study the role of G protein α subunits in maize development by either making the only canonical Gα constitutively active or mutating all other non-canonical Gα subunits (XLGs). We demonstrate that CT2 and XLGs have both redundant and specialized functions in regulating shoot development. Importantly, we show that a constitutively active Gα functioned as a weak allele, which introduced multiple desirable agronomic traits, such as improved kernel row number and reduced leaf angle.

Summary Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation., Highlights • The MDGA1 extracellular region has an unusual triangular multi-domain arrangement • The NL1-MDGA1 complex structure reveals how MDGA proteins block neurexin binding • MDGA1 and MDGA2 bind all NL isoforms, a process fine-tuned by alternative splicing • MDGA1 and MDGA2 suppress NL synaptogenic activity in a concentration-dependent manner, Elegheert et al. present the crystal structure of the autism-linked post-synaptic protein MDGA in complex with the synapse organizer neuroligin, providing a structural and mechanistic basis for potentially brain-wide modulation of synaptic neuroligin-neurexin signaling by MDGA proteins.

N-methyl-d-aspartate (NMDA) receptors are ligand-gated ion channels assembled from GluN1 and GluN2 subunits. We used a series of N-hydroxypyrazole-5-glycine (NHP5G) partial agonists at the GluN2 glutamate binding site as tools to study activation of GluN1/GluN2A and GluN1/GluN2D NMDA receptor subtypes. Using two-electrode voltage-clamp electrophysiology, fast-application patch-clamp, and single-channel recordings, we show that propyl- and ethyl-substituted NHP5G agonists have a broad range of agonist efficacies relative to the full agonist glutamate (

The amino terminal domain (ATD) of ionotropic glutamate receptor (iGluR) subunits resides at the extracellular region distal to the membrane. The ATD is structurally and functionally the most divergent region of the iGluR subunits. Structural studies on full-length GluA2 and the ATDs from three iGluR subfamilies have shed light on how the ATD facilitates subunit assembly, accommodates allosteric modulator compounds, and controls gating properties. Here recent developments in structural and functional studies on iGluR ATDs are reviewed.

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-free and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.

Intact NMDA receptor structure revealed For brains to develop and form memories, a signal must be transmitted from one neuron to the next. Glutamate is an important neurotransmitter that excites the receiving nerve cell by binding to an ion channel called an N -Methyl- d -Aspartate (NMDA) receptor. This activates the NMDA receptors, causing calcium ions to flood in, triggering signal transduction. Either under- or overactivation can result in a variety of neurological disorders and diseases. Karakas and Furukawa describe the crystal structure of an intact NMDA receptor composed of four separate subunits. Science , this issue p. 992

Background: Although tacrolimus-based immunosuppression has made intestinal transplantation feasible, the risk of the requisite chronic high-dose treatment has inhibited the widespread use of these procedures. We have examined our 1990–1997 experience to determine whether immunomodulatory strategies to improve outlook could be added to drug treatment. Study Design: Ninety-eight consecutive patients (59 children, 39 adults) with a panoply of indications received 104 allografts under tacrolimus-based immunosuppression: intestine only (n = 37); liver and intestine (n = 50); or multivisceral (n = 17). Of the last 42 patients, 20 received unmodified adjunct donor bone marrow cells; the other 22 were contemporaneous control patients. Results: With a mean followup of 32 ± 26 months (range, 1–86 months), 12 recipients (3 intestine only, 9 composite grafts) are alive with good nutrition beyond the 5-year milestone. Forty-seven (48%) of the total group survive bearing grafts that provide full (91%) or partial (9%) nutrition. Actuarial patient survival at 1 and 5 years (72% and 48%, respectively) was similar with isolated intestinal and composite graft recipients, but the loss rate of grafts from rejection was highest with intestine alone. The best results were in patients between 2 and 18 years of age (68% at 5 years). Adjunct bone marrow did not significantly affect the incidence of graft rejection, B-cell lymphoma, or the rate or severity of graft-versus-host disease. Conclusions: These results demonstrate that longterm rehabilitation similar to that with the other kinds of organ allografts is achievable with all three kinds of intestinal transplant procedures, that the morbidity and mortality is still too high for their widespread application, and that the liver is significantly but marginally protective of concomitantly engrafted intestine. Although none of the endpoints were markedly altered by donor leukocyte augmentation (and chimerism) with bone marrow, establishment of the safety of this adjunct procedure opens the way to further immune modulation strategies that can be added to the augmentation protocol.

Between April 1993 and May 1995, 17 adult orthotopic liver transplant recipients were found to have early hepatic artery thrombosis (HAT) after a median of 7 postoperative days (mean, 11). The HAT was diagnosed in all cases by duplex ultrasound. Thrombectomy was performed with urgent revascularization (UR), using an interposition arterial graft procured from the cadaveric liver donor, and arterial patency was verified with intraoperative angiography. In seven cases, intra-arterial urokinase was administered after the thrombectomy. Fifteen (88%) of the livers remained arterialized throughout the follow-up period (median, 15 months); the remaining two patients developed recurrent HAT after 6 and 8 months. Although there was a high rate of subsequent complications, 11 (65%) of the patients are alive without retransplantation, with a mean follow-up of 17 months. Despite having a patent hepatic artery, the remaining six patients (35%) died from infectious complications that usually were present before the UR. Thus, UR effectively restored arterial inflow in 88% of the patients with early HAT. The ultimate outcome was determined mainly by the presence of intra-abdominal complications at the time of UR. In conclusion, UR, rather than retransplantation, should be considered the prime treatment option for patients who develop early posttransplant HAT.

We have used recent structural advances in our understanding of the N-methyl-d-aspartate (NMDA) receptor amino terminal domain to explore the binding mode of multiple diaryl GluN2B-selective negative allosteric modulators at the interface between the GluN1 and GluN2B amino-terminal domains. We found that interaction of the A ring within the binding pocket seems largely invariant for a variety of structurally distinct ligands. In addition, a range of structurally diverse linkers between the two aryl rings can be accommodated by the binding site, providing a potential opportunity to tune interactions with the ligand binding pocket via changes in hydrogen bond donors, acceptors, as well as stereochemistry. The most diversity in atomic interactions between protein and ligand occur in the B ring, with functional groups that contain electron donors and acceptors providing additional atomic contacts within the pocket. A cluster of residues distant to the binding site also control ligand potency, the degree of inhibition, and show ligand-induced increases in motion during molecular dynamics simulations. Mutations at some of these residues seem to distinguish between structurally distinct ligands and raise the possibility that GluN2B-selective ligands can be divided into multiple classes. These results should help facilitate the development of well tolerated GluN2B subunit-selective antagonists.

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors that mediate a majority of excitatory synaptic transmission. NMDA receptors are comprised of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits, of which there are four subtypes (GluN2A-D) that determine many functional properties of the receptors. One unique property of the GluN1/GluN2D NMDA receptors is an unusually prolonged deactivation time course that lasts several seconds following the removal of L-glutamate. Here, we show by a combination of x-ray crystallography and electrophysiology that the deactivation time course of the GluN1/GluN2D receptors is influenced both by the conformational variability of the ligand-binding domain as well as the chemical structure and stereochemistry of the activating ligand. Of all ligands tested, L-glutamate and L-CCG-IV induce a significantly slower deactivation time course on the GluN1/GluN2D receptors than other agonists. Furthermore, crystal structures of the isolated GluN2D ligand-binding domain monomer in complex with various ligands reveal that the binding of L-glutamate induces a unique conformation at the back side of the ligand-binding site in proximity to the region where the transmembrane domain would be located in the intact receptors. These data suggest that the activity of the GluN1/GluN2D NMDA receptor is controlled distinctively by the endogenous neurotransmitter L-glutamate.

Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA (N-methyl-D-aspartate) receptors, a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function. These receptors form heteromeric ion channels and become activated after concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion-channel activity is allosterically regulated by binding of small compounds to the amino-terminal domain (ATD) in a subtype-specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1 and GluN2B NMDA receptors, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer's disease and Parkinson's disease. Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information. Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft. The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors. These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.

The mechanosensitive channel of small conductance (MscS) plays a critical role in the osmoregulation of prokaryotic cells. The crystal structure of MscS revealed a homoheptamer with three transmembrane segments and a large cytoplasmic domain. It has been suggested that the crystal structure depicts an open state, but its actual functional conformation remains controversial. In the pursuit of spectroscopical approaches to MscS gating, we determined that standard purification methods yield two forms of MscS, with a considerable amount of unfolded channel. Here, we present an improved high-yield purification method based on Escherichia coli expression and a biochemical characterization of the reconstituted channel, optimized to yield approximately 4 mg of a single monodisperse product. Upon reconstitution into lipid vesicles, MscS is unusually prone to lateral aggregation depending on the lipid composition, particularly after sample freezing. Strategies for minimizing MscS aggregation in two dimensions for spectroscopic analyses of gating have been developed.

Background The purpose of the study was to quantify changes in the quality of life of small bowel recipients before and after transplantation and of home parenteral nutrition (HPN)-dependent patients before and after therapy. We examined quality of life across multiple areas of function including physical, social, and emotional indices. Methods The Quality of Life Instrument in the form of a self-administered questionnaire was completed voluntarily by the recipients of small intestinal transplants and by a cohort of HPN-dependent patients. Results Small intestinal transplant recipients reported significant improvement in the quality of their life and function. They also rated their quality of life and function during the pretransplant, TPN-dependent period to be worse than before the development of chronic intestinal failure. Similarly, HPN recipients reported significant worsening across most areas of quality of life when they compared their premorbid period to the HPN-dependent state. Conclusions TPN dependence causes significant impairment in the quality of life in most areas of functioning. In contrast, small intestinal transplantation restores the quality of life among recipients with functioning grafts.

Purpose: A clinical trial of intestinal transplantation (ltx) under tacrolimus and prednisone immunosuppression was initiated in June 1990 in children with irreversible intestinal failure and who were dependent on total parenteral nutrition (TPN). Methods: Fifty-five patients (28 girls, 27 boys) with a median age of 3.2 years (range, 0.5 to 18 years) received 58 intestinal transplants that included isolated small bowel (SB) (n = 17), liver SB (LSB) (n = 33), and multivisceral (MV) (n = 8) allografts. Nine patients also received bone marrow infusion, and there were 20 colonic allografts. Azathioprine, cyclophosphamide, or mycophenolate mofetil were used in different phases of the series. Indications for ltx included: gastroschisis (n = 14), volvulus (n = 13), necrotizing enterocolitis (n = 6), intestinal atresia (n = 8), chronic intestinal pseudoobstruction (n = 5), Hirschsprung's disease (n = 4), microvillus inclusion disease (n = 3), multiple polyposis (n = 1), and trauma (n = 1). Results: Currently, 30 patients are alive (patient survival, 55%; graft survival, 52%). Twenty-nine children with functioning grafts are living at home and off TPN, with a mean follow-up of 962 (range, 75 to 2,424) days. Immunologic complications have included liver allograft rejection (n = 18), intestinal allograft rejection (n = 52), posttransplant lymphoproliferative disease (n = 16), cytomegalovirus (n = 16) and graft-versus-host disease (n = 4). A combination of associated complications included intestinal perforation (n = 4), biliary leak (n = 3), bile duct stenosis (n = 1), intestinal leak (n = 6), dehiscence with evisceration (n = 4), hepatic artery thrombosis (n = 3), bleeding (n = 9), portal vein stenosis (n = 1), intraabdominal abscess (n = 11), and chylous ascites (n = 4). Graft loss occurred as a result of rejection (n = 8), infection (n = 12), technical complication (n = 8), and complications of TPN after graft removal (n = 3). There were four retransplants (SB, n=1; LSB n=3). Conclusions: Intestinal transplantation is a valid therapeutic option for patients with intestinal failure suffering complications of TPN. The complex clinical and immunologic course of these patients is reflected in a higher complication rate as well as patient and graft loss than seen after heart, liver, and kidney transplantation, although better than after lung transplantation.

We are gaining experience in small intestine transplantation, however, the procedure is still experimental. Morbidity and mortality can be significant with frequent rehospitalizations. The indications for small intestine transplantation are varied though most patients have developed short gut syndrome requiring total parenteral nutrition (TPN) for nutritional support. Patients may present with a chronic illness (such as Crohn's disease), chronic pain, and psychiatric comorbidity that may need to be addressed during the perioperative period. Faced with the complicated postoperative course, transplant recipients develop a range of endogenous and organic psychiatric disorders. Psychiatric treatment may be complicated by these factors in addition to the nutritional, biochemical, and metabolic abnormalities of a transplanted small intestine.

N-methyl-D-aspartate receptors (NMDARs) belong to the family of ionotropic glutamate receptors and mediate excitatory synaptic transmission in the mammalian brain. NMDARs have been intensely studied over the past thirty years because of their critical involvement in brain development and physiology including learning and memory formation. Despite great importance, structure-function studies of NMDARs have been slow to develop compared to those of non-NMDARs. Diverse functions of NMDARs stems from the presence of pharmacological subtypes defined mainly by a combination of GluN1 subunit and four different subunits of GluN2s (GluN2A through D), which are expressed in different neuronal circuits at different developmental stages. Importantly, subtype-specific regulation of NMDAR activity is considered an effective strategy to treat neurological disorders and diseases that are caused by dysfunctional NMDARs including Alzheimer's disease, Parkinson's disease, schizophrenia, depression, and stroke. A subtype-specific compound, ifenprodil, binds to the amino terminal domain (ATD) of the GluN1/GluN2B receptor subtype and allosterically inhibits the ion channel activity. Ifenprodil and its analogues have been intensively studied over the past two decades because of their therapeutic potentials for depression, neuropathic pains, and Alzheimer's disease. However, there have been limited studies showing the mechanism of ifenprodil binding and ifenprodil-mediated allosteric inhibition. Here we show by a combination of x-ray crystallography and electrophysiology that the binding site of ifenprodil is located at the GluN1-GluN2B dimer interface and that ATD-mediated allosteric inhibition requires a conformational freedom in the GluN2B ATD. The structural and functional insights provided in this study pave the way to improve the design of therapeutic compounds.

Majority of fast excitatory synaptic transmission in the mammalian brain is mediated by a class of molecules called ionotropic glutamate receptors, which include N-methyl-D-aspartate (NMDA) receptors. NMDA receptors are hetero-tetrameric ion channels that are composed of two NR1 subunits and two NR2 (A-D) subunits or NR3 subunits. NMDA receptors play key roles in numbers of important processes including synaptic plasticity and development in normal state, whereas aberrant activity of NMDA receptors is associated with ischemic brain injury and neurodegenerative diseases including Parkinson's disease and Alzheimer's disease. Activity of NMDA receptor is tightly controlled through multiple pathways. One such mechanism is allosteric modulation through binding of small molecules to the extracellular amino terminal domain (ATD) in a subtype specific manner, i.e. polyamines and protons bind NR1, Zn2+ binds both NR2A and NR2B, and phenylethanolamine compounds bind NR2B. To understand the molecular mechanism of the ATD-dependent allosteric modulation of NMDA receptors, we have solved the structures of NR2B ATD in the zinc-bound and -free forms. The structures reveal an overall clamshell architecture with a unique domain orientation distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc binding site, ion binding sites, and the architecture of the putative phenylethanolamine binding site.