RESUMO
Glutamate transmission and activation of ionotropic glutamate receptors are the fundamental means by which neurons control their excitability and neuroplasticity1. The N-methyl-D-aspartate receptor (NMDAR) is unique among all ligand-gated channels, requiring two ligands-glutamate and glycine-for activation. These receptors function as heterotetrameric ion channels, with the channel opening dependent on the simultaneous binding of glycine and glutamate to the extracellular ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, respectively2,3. The exact molecular mechanism for channel gating by the two ligands has been unclear, particularly without structures representing the open channel and apo states. Here we show that the channel gate opening requires tension in the linker connecting the LBD and transmembrane domain (TMD) and rotation of the extracellular domain relative to the TMD. Using electron cryomicroscopy, we captured the structure of the GluN1-GluN2B (GluN1-2B) NMDAR in its open state bound to a positive allosteric modulator. This process rotates and bends the pore-forming helices in GluN1 and GluN2B, altering the symmetry of the TMD channel from pseudofourfold to twofold. Structures of GluN1-2B NMDAR in apo and single-liganded states showed that binding of either glycine or glutamate alone leads to distinct GluN1-2B dimer arrangements but insufficient tension in the LBD-TMD linker for channel opening. This mechanistic framework identifies a key determinant for channel gating and a potential pharmacological strategy for modulating NMDAR activity.
Assuntos
Ácido Glutâmico , Glicina , Ativação do Canal Iônico , Receptores de N-Metil-D-Aspartato , Animais , Ratos , Regulação Alostérica , Microscopia Crioeletrônica , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Ligantes , Modelos Moleculares , Oócitos/metabolismo , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/metabolismo , Subunidades Proteicas/química , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/ultraestrutura , Rotação , Xenopus laevisRESUMO
The chemokine co-receptors CXCR4 and CCR5 mediate HIV entry and signal transduction necessary for viral infection. However, to date only the CCR5 antagonist maraviroc is approved for treating HIV-1 infection. Given that approximately 50% of late-stage HIV patients also develop CXCR4-tropic virus, clinical anti-HIV CXCR4 antagonists are needed. Here, we describe a novel allosteric CXCR4 antagonist TIQ-15 which inhibits CXCR4-tropic HIV-1 infection of primary and transformed CD4 T cells. TIQ-15 blocks HIV entry with an IC50 of 13 nM. TIQ-15 also inhibits SDF-1α/CXCR4-mediated cAMP production, cofilin activation, and chemotactic signaling. In addition, TIQ-15 induces CXCR4 receptor internalization without affecting the levels of the CD4 receptor, suggesting that TIQ-15 may act through a novel allosteric site on CXCR4 for blocking HIV entry. Furthermore, TIQ-15 did not inhibit VSV-G pseudotyped HIV-1 infection, demonstrating its specificity in blocking CXCR4-tropic virus entry, but not CXCR4-independent endocytosis or post-entry steps. When tested against a panel of clinical isolates, TIQ-15 showed potent inhibition against CXCR4-tropic and dual-tropic viruses, and moderate inhibition against CCR5-tropic isolates. This observation was followed by a co-dosing study with maraviroc, and TIQ-15 demonstrated synergistic activity. In summary, here we describe a novel HIV-1 entry inhibitor, TIQ-15, which potently inhibits CXCR4-tropic viruses while possessing low-level synergistic activities against CCR5-tropic viruses. TIQ-15 could potentially be co-dosed with the CCR5 inhibitor maraviroc to block viruses of mixed tropisms.
Assuntos
Infecções por HIV , HIV-1 , Receptores CXCR4 , Internalização do Vírus , Humanos , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Internalização do Vírus/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Inibidores da Fusão de HIV/farmacologia , Maraviroc/farmacologia , Triazóis/farmacologia , Fármacos Anti-HIV/farmacologia , Células HEK293RESUMO
Allosteric modulators of ion channels typically alter the transitions rates between conformational states without changing the properties of the open pore. Here we describe a new class of positive allosteric modulators of N-methyl D-aspartate receptors (NMDARs) that mediate a calcium-permeable component of glutamatergic synaptic transmission and play essential roles in learning, memory and cognition, as well as neurological disease. EU1622-14 increases agonist potency and channel-open probability, slows receptor deactivation and decreases both single-channel conductance and calcium permeability. The unique functional selectivity of this chemical probe reveals a mechanism for enhancing NMDAR function while limiting excess calcium influx, and shows that allosteric modulators can act as biased modulators of ion-channel permeation.
Assuntos
Pirrolidinas/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Glicina/metabolismo , Glicina/farmacologia , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , Xenopus laevisRESUMO
NMDA receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic currents. These receptors are involved in several important brain functions, including learning and memory, and have also been implicated in neuropathological conditions and acute central nervous system injury, which has driven therapeutic interest in their modulation. The EU1794 series of positive and negative allosteric modulators of NMDA receptors has structural determinants of action near the preM1 helix that is involved in channel gating. Here, we describe the effects of the negative allosteric modulator EU1794-4 on GluN1/GluN2A channels studied in excised outside-out patches. Coapplication of EU1794-4 with a maximally effective concentration of glutamate and glycine increases the fraction of time the channel is open by nearly 1.5-fold, yet reduces single-channel conductance by increasing access of the channel to several subconductance levels, which has the net overall effect of reducing the macroscopic current. The lack of voltage-dependence of negative modulation suggests this is unrelated to a channel block mechanism. As seen with other NMDA receptor modulators that reduce channel conductance, EU1794-4 also reduces the Ca2+ permeability relative to monovalent cations of GluN1/GluN2A receptors. We conclude that EU1794-4 is a prototype for a new class of NMDA receptor negative allosteric modulators that reduce both the overall current that flows after receptor activation and the flux of Ca2+ ion relative to monovalent cations. SIGNIFICANCE STATEMENT: NMDA receptors are implicated in many neurological conditions but are challenging to target given their ubiquitous expression. Several newly identified properties of the negative allosteric modulator EU1794-4, including reducing Ca2+ flux through NMDA receptors and attenuating channel conductance, explain why this modulator reduces but does not eliminate NMDA receptor function. A modulator with these properties could have therapeutic advantages for indications in which attenuation of NMDA receptor function is beneficial, such as neurodegenerative disease and acute injury.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Cálcio/metabolismo , Permeabilidade/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Células HEK293 , Humanos , Xenopus laevisRESUMO
We describe a clinical candidate molecule from a new series of glutamate N-methyl-d-aspartate receptor subunit 2B-selective inhibitors that shows enhanced inhibition at extracellular acidic pH values relative to physiologic pH. This property should render these compounds more effective inhibitors of N-methyl-d-aspartate receptors at synapses responding to a high frequency of action potentials, since glutamate-containing vesicles are acidic within their lumen. In addition, acidification of penumbral regions around ischemic tissue should also enhance selective drug action for improved neuroprotection. The aryl piperazine we describe here shows strong neuroprotective actions with minimal side effects in preclinical studies. The clinical candidate molecule NP10679 has high oral bioavailability with good brain penetration and is suitable for both intravenous and oral dosing for therapeutic use in humans. SIGNIFICANCE STATEMENT: This study identifies a new series of glutamate N-methyl-d-aspartate (NMDA) receptor subunit 2B-selective negative allosteric modulators with properties appropriate for clinical advancement. The compounds are more potent at acidic pH, associated with ischemic tissue, and this property should increase the therapeutic safety of this class by improving efficacy in affected tissue while sparing NMDA receptor block in healthy brain.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Ácidos , Administração Oral , Animais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Feminino , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Xenopus laevisRESUMO
Nucleotide prodrugs are of great clinical interest for treating a variety of viral infections due to their ability to target tissues selectively and to deliver relatively high concentrations of the active nucleotide metabolite intracellularly. However, their clinical successes have been limited, oftentimes due to unwanted in vivo metabolic processes that reduce the quantities of nucleoside triphosphate that reach the site of action. In an attempt to circumvent this, we designed novel nucleosides that incorporate a sterically bulky group at the 5'-carbon of the phosphoester prodrug, which we reasoned would reduce the amounts of non-productive PO bond cleavage back to the corresponding nucleoside by nucleotidases. Molecular docking studies with the NS5B HCV polymerase suggested that a nucleotide containing a 5'-methyl group could be accommodated. Therefore, we synthesized mono- and diphosphate prodrugs of 2',5'-C-dimethyluridine stereoselectively and evaluated their cytotoxicity and anti-HCV activity in the HCV replicon assay. All four prodrugs exhibited anti-HCV activity with IC50 values in the single digit micromolar concentrations, with the 5'(R)-C-methyl prodrug displaying superior potency relative to its 5'(S)-C-methyl counterpart. However, when compared to the unmethylated prodrug, the potency is poorer. The poorer potency of these prodrugs may be due to unfavorable steric interactions of the 5'-C-methyl group in the active sites of the kinases that catalyze the formation of active triphosphate metabolite.
Assuntos
Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Nucleotídeos de Uracila/farmacologia , Antivirais/síntese química , Antivirais/metabolismo , Linhagem Celular , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/metabolismo , Ligação Proteica , Nucleotídeos de Uracila/síntese química , Nucleotídeos de Uracila/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
The NS5B RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is a validated target for nucleoside antiviral drug therapy. We endeavored to synthesize and test a series of 4'-thionucleosides with a monophosphate prodrug moiety for their antiviral activity against HCV and other related viruses in the Flaviviridae family. Nucleoside analogs were prepared via the stereoselective Vorbrüggen glycosylation of various nucleobases with per-acetylated 2-C-methyl-4-thio-d-ribose built in a 10-step synthetic sequence from the corresponding ribonolactone. Conjugation of the thionucleoside to a ProTide phosphoramidate allowed for evaluation of the prodrugs in the cellular HCV replicon assay with anti-HCV activities ranging from single-digit micromolar (µM) to >200 µM. The diminished anti-HCV potency of our best compound compared to its 4'-oxo congener is the subject of ongoing research in our lab and is proposed to stem from changes in sugar geometry imparted by the larger sulfur atom.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Pró-Fármacos/síntese química , Tionucleosídeos/química , Amidas/química , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Humanos , Nucleosídeos/síntese química , Fosfatos/química , Ácidos Fosfóricos/química , Pró-Fármacos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
N-methyl-d-aspartate (NMDA) receptors are ligand-gated, cation-selective channels that mediate a slow component of excitatory synaptic transmission. Subunit-selective positive allosteric modulators of NMDA receptor function have therapeutically relevant effects on multiple processes in the brain. A series of pyrrolidinones, such as PYD-106, that selectively potentiate NMDA receptors that contain the GluN2C subunit have structural determinants of activity that reside between the GluN2C amino terminal domain and the GluN2C agonist binding domain, suggesting a unique site of action. Here we use molecular biology and homology modeling to identify residues that line a candidate binding pocket for GluN2C-selective pyrrolidinones. We also show that occupancy of only one site in diheteromeric receptors is required for potentiation. Both GluN2A and GluN2B can dominate the sensitivity of triheteromeric receptors to eliminate the actions of pyrrolidinones, thus rendering this series uniquely sensitive to subunit stoichiometry. We experimentally identified NMR-derived conformers in solution, which combined with molecular modeling allows the prediction of the bioactive binding pose for this series of GluN2C-selective positive allosteric modulators of NMDA receptors. These data advance our understanding of the site and nature of the ligand-protein interaction for GluN2C-selective positive allosteric modulators for NMDA receptors.
Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Técnicas de Patch-Clamp , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Reprodutibilidade dos Testes , Estereoisomerismo , Xenopus laevisRESUMO
KEY POINTS: The kinetics of NMDA receptor (NMDAR) signalling are a critical aspect of the physiology of excitatory synaptic transmission in the brain. Here we develop a mechanistic description of NMDAR function based on the receptor tetrameric structure and the principle that each agonist-bound subunit must undergo some rate-limiting conformational change after agonist binding, prior to channel opening. By fitting this mechanism to single channel data using a new MATLAB-based software implementation of maximum likelihood fitting with correction for limited time resolution, rate constants were derived for this mechanism that reflect distinct structural changes and predict the properties of macroscopic and synaptic NMDAR currents. The principles applied here to develop a mechanistic description of the heterotetrameric NMDAR, and the software used in this analysis, can be equally applied to other heterotetrameric glutamate receptors, providing a unifying mechanistic framework to understanding the physiology of glutamate receptor signalling in the brain. ABSTRACT: NMDA receptors (NMDARs) are tetrameric complexes comprising two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Four GluN2 subunits encoded by different genes can produce up to 10 different di- and triheteromeric receptors. In addition, some neurological patients contain a de novo mutation or inherited rare variant in only one subunit. There is currently no mechanistic framework to describe tetrameric receptor function that can be extended to receptors with two different GluN1 or GluN2 subunits. Here we use the structural features of glutamate receptors to develop a mechanism describing both single channel and macroscopic NMDAR currents. We propose that each agonist-bound subunit undergoes some rate-limiting conformational change after agonist binding, prior to channel opening. We hypothesize that this conformational change occurs within a triad of interactions between a short helix preceding the M1 transmembrane helix, the highly conserved M3 motif encoded by the residues SYTANLAAF, and the linker preceding the M4 transmembrane helix of the adjacent subunit. Molecular dynamics simulations suggest that pre-M1 helix motion is uncorrelated between subunits, which we interpret to suggest independent subunit-specific conformational changes may influence these pre-gating steps. According to this interpretation, these conformational changes are the main determinants of the key kinetic properties of NMDA receptor activation following agonist binding, and so these steps sculpt their physiological role. We show that this structurally derived tetrameric model describes both single channel and macroscopic data, giving a new approach to interpreting functional properties of synaptic NMDARs that provides a logical framework to understanding receptors with non-identical subunits.
Assuntos
Ácido Glutâmico/metabolismo , Ativação do Canal Iônico , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Multimerização Proteica , Subunidades ProteicasRESUMO
N-methyl-D-aspartate (NMDA) receptors are transmembrane glutamate-binding ion channels that mediate neurotransmission in mammals. NMDA receptor subunits are tetrameric complexes of GluN1 and GluN2A-D subunits, encoded by the GRIN gene family. Of these subunits, GluN2B is suggested to be required for normal development of the central nervous system. A mutation identified in a patient with developmental delay, E413G, resides in the GluN2B ligand-binding domain and substantially reduces glutamate potency by an unknown mechanism. GluN2B Gly413, though near the agonist, is not in van der Waals contact with glutamate. Visual analysis of the GluN2B structure with the E413G mutation modeled suggests that replacement of Glu with Gly at this position increases solvent access to the ligand-binding domain. This was confirmed by molecular modeling, which showed that the ligand is more mobile in GluN2B-E413G than WT GluN2B. Evaluation of agonist occupancy using random accelerated molecular dynamics (RAMD) simulations predicts that the glutamate exits the binding-site more rapidly for GluN2B-E413G than WT receptors. This analysis was extended to other binding-site mutations, which produced qualitative agreement between experimentally determined EC50 values, deactivation time constants, and ligand motion within the binding-site. Furthermore, long sub-microsecond molecular dynamics simulations of the bi-lobed ligand-binding domain revealed that it adopted a cleft-open ligand-free state more often for GluN2B-E413G than wild-type GluN2B. This is consistent with the idea that L-glutamate binding is altered such that the ligand-binding domain occupies the open-cleft conformation associated with the closed channel.
Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Ácido Glutâmico/genética , Glicina/genética , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Mutação , Domínios Proteicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , SolventesRESUMO
HIV resistance emerging against antiretroviral drugs represents a great threat to the continued prolongation of the lifespans of HIV-infected patients. Therefore, methods capable of predicting resistance susceptibility in the development of compounds are in great need. By targeting the major reverse transcription residues Y181, K103, and L100, we used the biological activities of compounds against these enzymes and the wild-type reverse transcriptase to create Naïve Bayes Networks. Through this machine learning approach, we could predict, with high accuracy, whether a compound would be susceptible to a loss of potency due to resistance. Also, we could perfectly predict retrospectively whether compounds would be susceptible to both a K103 mutant RT and a Y181 mutant RT. In the study presented here, our method outperformed a traditional molecular mechanics approach. This method should be of broad interest beyond drug discovery efforts, and serves to expand the utility of machine learning for the prediction of physical, chemical, or biological properties using the vast information available in the literature.
Assuntos
Descoberta de Drogas/métodos , Farmacorresistência Viral , Transcriptase Reversa do HIV/genética , Aprendizado de Máquina , Mutação Puntual , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Teorema de Bayes , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Modelos BiológicosRESUMO
The HIV/AIDS epidemic, which was first reported on in 1981, progressed in just 10 years to a disease afflicting 10 million people worldwide including 1 million in the US. In 1987, AZT was approved for treating HIV/AIDS. Unfortunately, its clinical usefullness was severly limited by associated toxicities and the emergence of resistance. Three other drugs that were approved in the early 1990s suffered from similar liabilities. In 1990, the Liotta group at Emory University developed a highly diastereoselective synthesis of racemic 3'-thia-2',3'-dideoxycytidine and 3'-thia-2',3'-5-fluorodideoxycytidine and demonstrated that these compounds exhibited excellent anti-HIV activity with no apparent cytotoxicity. Subsequently, the enantiomers of these compounds were separated using enzyme-mediated kinetic resolutions and their (-)-enantiomers (3TC and FTC, respectively) were found to have exceptionally attractive preclinical profiles. In addition to their anti-HIV activity, 3TC and FTC potently inhibit the replication of hepatitis B virus. The development of FTC, which was being carried out by Burroughs Wellcome, had many remarkable starts and stops. For example, passage studies indicated that the compound rapidly selected for a single resistant mutant, M184V, and that this strain was 500-1000-fold less sensitive to FTC than was wild-type virus. Fortunately, it was found that combinations of AZT with either 3TC or FTC were synergistic. The effectiveness of AZT-3TC combination therapy was subsequently demonstrated in four independent clinical trials, and in 1997, the FDA approved Combivir, a fixed dose combination of AZT and 3TC. In phase 1 clinical trials, FTC was well tolerated by all subjects with no adverse events observed. However, the development of FTC was halted by the aquistition of Wellcome PLC by Glaxo PLC in January 1995. In 1996, Triangle Pharmaceuticals licensed FTC from Emory and initiated a series of phase I/II clinical studies that demonstrated the safety and efficacy of the drug. In August 1998, FTC was granted "Fast Track" status, based primarily on its potential for once daily dosing. While the outcomes of two subsequent phase III trials were positive, a third phase III clinical trial involving combinations of 3TC or FTC with stavudine and neviripine had to be terminated due to serious liver-related adverse events. Although analysis of the data suggested that the liver toxicity was due to neviripine, the FDA decided that the study could not be used for drug registration. Ultimately, in January 2003, Gilead Sciences acquired Triangle Pharmaceuticals and completed the development of FTC (emtricitabine), which was approved for once a day, oral administration in July 2003. A year later, Truvada, a once a day, oral, fixed dose combination of emtricitabine and tenofovir disoproxyl fumarate received FDA approval and quickly became the accepted first line therapy when used with a third antiretroviral agent. In July 2006, the FDA approved Atripla, a once a day, oral, fixed dose combination of emtricitabine, tenofovir disoproxyl fumarate, and efavirenz, which represented the culmination of two decades of research that had transformed AIDS from a death sentence to a manageable chronic disease.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , Emtricitabina/uso terapêutico , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/efeitos adversos , Ensaios Clínicos como Assunto , Descoberta de Drogas , Sinergismo Farmacológico , Quimioterapia Combinada , Emtricitabina/administração & dosagem , Emtricitabina/efeitos adversos , HIV-1/efeitos dos fármacos , Humanos , EstereoisomerismoRESUMO
N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamatergic receptors that have been implicated in learning, development, and neuropathological conditions. They are typically composed of GluN1 and GluN2A-D subunits. Whereas a great deal is known about the role of GluN2A- and GluN2B-containing NMDARs, much less is known about GluN2D-containing NMDARs. Here we explore the subunit composition of synaptic NMDARs on hippocampal interneurons. GluN2D mRNA was detected by single-cell PCR and in situ hybridization in diverse interneuron subtypes in the CA1 region of the hippocampus. The GluN2D subunit was detectable by immunoblotting and immunohistochemistry in all subfields of the hippocampus in young and adult mice. In whole-cell patch-clamp recordings from acute hippocampal slices, (+)-CIQ, the active enantiomer of the positive allosteric modulator CIQ, significantly enhanced the amplitude of the NMDAR component of miniature excitatory postsynaptic currents (mEPSCs) in CA1 interneurons but not in pyramidal cells. (+)-CIQ had no effect in slices from Grin2d-/- mice, suggesting that GluN2D-containing NMDARs participate in excitatory synaptic transmission onto hippocampal interneurons. The time course of the NMDAR component of the mEPSC was unaffected by (+)-CIQ potentiation and was not accelerated in slices from Grin2d-/- mice compared with wild-type, suggesting that GluN2D does not detectably slow the NMDAR EPSC time course at this age. (+)-CIQ increased the activity of CA1 interneurons as detected by the rate and net charge transfer of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal cells. These data provide evidence that interneurons contain synaptic NMDARs possessing a GluN2D subunit, which can influence interneuron function and signal processing.