Direct Identification of Complex Glycans via a Highly Sensitive Engineered Nanopore.

The crucial roles that glycans play in biological systems are determined by their structures. However, the analysis of glycan structures still has numerous bottlenecks due to their inherent complexities. The nanopore technology has emerged as a powerful sensor for DNA sequencing and peptide detection. This has a significant impact on the development of a related research area. Currently, nanopores are beginning to be applied for the detection of simple glycans, but the analysis of complex glycans by this technology is still challenging. Here, we designed an engineered α-hemolysin nanopore M113R/T115A to achieve the sensing of complex glycans at micromolar concentrations and under label-free conditions. By extracting characteristic features to depict a three-dimensional (3D) scatter plot, glycans with different numbers of functional groups, various chain lengths ranging from disaccharide to decasaccharide, and distinct glycosidic linkages could be distinguished. Molecular dynamics (MD) simulations show different behaviors of glycans with ß1,3- or ß1,4-glycosidic bonds in nanopores. More importantly, the designed nanopore system permitted the discrimination of each glycan isomer with different lengths in a mixture with a separation ratio of over 0.9. This work represents a proof-of-concept demonstration that complex glycans can be analyzed using nanopore sequencing technology.

The human microbiota is a beneficial reservoir for SARS-CoV-2 mutations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations are rapidly emerging. In particular, beneficial mutations in the spike (S) protein, which can either make a person more infectious or enable immunological escape, are providing a significant obstacle to the prevention and treatment of pandemics. However, how the virus acquires a high number of beneficial mutations in a short time remains a mystery. We demonstrate here that variations of concern may be mutated due in part to the influence of the human microbiome. We searched the National Center for Biotechnology Information database for homologous fragments (HFs) after finding a mutation and the six neighboring amino acids in a viral mutation fragment. Among the approximate 8,000 HFs obtained, 61 mutations in S and other outer membrane proteins were found in bacteria, accounting for 62% of all mutation sources, which is 12-fold higher than the natural variable proportion. A significant proportion of these bacterial species-roughly 70%-come from the human microbiota, are mainly found in the lung or gut, and share a composition pattern with COVID-19 patients. Importantly, SARS-CoV-2 RNA-dependent RNA polymerase replicates corresponding bacterial mRNAs harboring mutations, producing chimeric RNAs. SARS-CoV-2 may collectively pick up mutations from the human microbiota that change the original virus's binding sites or antigenic determinants. Our study clarifies the evolving mutational mechanisms of SARS-CoV-2. IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations are rapidly emerging, in particular advantageous mutations in the spike (S) protein, which either increase transmissibility or lead to immune escape and are posing a major challenge to pandemic prevention and treatment. However, how the virus acquires a high number of advantageous mutations in a short time remains a mystery. Here, we provide evidence that the human microbiota is a reservoir of advantageous mutations and aids mutational evolution and host adaptation of SARS-CoV-2. Our findings demonstrate a conceptual breakthrough on the mutational evolution mechanisms of SARS-CoV-2 for human adaptation. SARS-CoV-2 may grab advantageous mutations from the widely existing microorganisms in the host, which is undoubtedly an "efficient" manner. Our study might open a new perspective to understand the evolution of virus mutation, which has enormous implications for comprehending the trajectory of the COVID-19 pandemic.

An LQT2-related mutation in the voltage-sensing domain is involved in switching the gating polarity of hERG.

BACKGROUND: Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of hERG and HCN remains unknown. RESULTS: We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. CONCLUSIONS: Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.

Pharmacological inhibition of Kir4.1 evokes rapid-onset antidepressant responses.

Major depressive disorder, a prevalent and severe psychiatric condition, necessitates development of new and fast-acting antidepressants. Genetic suppression of astrocytic inwardly rectifying potassium channel 4.1 (Kir4.1) in the lateral habenula ameliorates depression-like phenotypes in mice. However, Kir4.1 remains an elusive drug target for depression. Here, we discovered a series of Kir4.1 inhibitors through high-throughput screening. Lys05, the most potent one thus far, effectively suppressed native Kir4.1 channels while displaying high selectivity against established targets for rapid-onset antidepressants. Cryogenic-electron microscopy structures combined with electrophysiological characterizations revealed Lys05 directly binds in the central cavity of Kir4.1. Notably, a single dose of Lys05 reversed the Kir4.1-driven depression-like phenotype and exerted rapid-onset (as early as 1 hour) antidepressant actions in multiple canonical depression rodent models with efficacy comparable to that of (S)-ketamine. Overall, we provided a proof of concept that Kir4.1 is a promising target for rapid-onset antidepressant effects.

Development of SV2A Ligands for Epilepsy Treatment: A Review of Levetiracetam, Brivaracetam, and Padsevonil.

Epilepsy is a common neurological disorder that is primarily treated with antiseizure medications (ASMs). Although dozens of ASMs are available in the clinic, approximately 30% of epileptic patients have medically refractory seizures; other limitations in most traditional ASMs include poor tolerability and drug-drug interactions. Therefore, there is an urgent need to develop alternative ASMs. Levetiracetam (LEV) is a first-line ASM that is well tolerated, has promising efficacy, and has little drug-drug interaction. Although it is widely accepted that LEV acts through a unique therapeutic target synaptic vesicle protein (SV) 2A, the molecular basis of its action remains unknown. Even so, the next-generation SV2A ligands against epilepsy based on the structure of LEV have achieved clinical success. This review highlights the research and development (R&D) process of LEV and its analogs, brivaracetam and padsevonil, to provide ideas and experience for the R&D of novel ASMs.

Ligand activation mechanisms of human KCNQ2 channel.

The human voltage-gated potassium channel KCNQ2/KCNQ3 carries the neuronal M-current, which helps to stabilize the membrane potential. KCNQ2 can be activated by analgesics and antiepileptic drugs but their activation mechanisms remain unclear. Here we report cryo-electron microscopy (cryo-EM) structures of human KCNQ2-CaM in complex with three activators, namely the antiepileptic drug cannabidiol (CBD), the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), and HN37 (pynegabine), an antiepileptic drug in the clinical trial, in an either closed or open conformation. The activator-bound structures, along with electrophysiology analyses, reveal the binding modes of two CBD, one PIP2, and two HN37 molecules in each KCNQ2 subunit, and elucidate their activation mechanisms on the KCNQ2 channel. These structures may guide the development of antiepileptic drugs and analgesics that target KCNQ2.

Mapping the Acetylamino and Carboxyl Groups on Glycans by Engineered α-Hemolysin Nanopores.

Glycan is a crucial class of biological macromolecules with important biological functions. Functional groups determine the chemical properties of glycans, which further affect their biological activities. However, the structural complexity of glycans has set a technical hurdle for their direct identification. Nanopores have emerged as highly sensitive biosensors that are capable of detecting and characterizing various analytes. Here, we identified the functional groups on glycans with a designed α-hemolysin nanopore containing arginine mutations (M113R), which is specifically sensitive to glycans with acetamido and carboxyl groups. Molecular dynamics simulations indicated that the acetamido and carboxyl groups of the glycans produce unique electrical signatures by forming polar and electrostatic interactions with the M113R nanopores. Using these electrical features as the fingerprints, we mapped the length of the glycans containing acetamido and carboxyl groups at the monosaccharide, disaccharide, and trisaccharide levels. This proof-of-concept study provides a promising foundation for developing single-molecule glycan fingerprinting libraries and demonstrates the capability of biological nanopores in glycan sequencing.

Disruption of ER ion homeostasis maintained by an ER anion channel CLCC1 contributes to ALS-like pathologies.

Although anion channel activities have been demonstrated in sarcoplasmic reticulum/endoplasmic reticulum (SR/ER), their molecular identities and functions remain unclear. Here, we link rare variants of Chloride Channel CLIC Like 1 (CLCC1) to amyotrophic lateral sclerosis (ALS)-like pathologies. We demonstrate that CLCC1 is a pore-forming component of an ER anion channel and that ALS-associated mutations impair channel conductance. CLCC1 forms homomultimers and its channel activity is inhibited by luminal Ca2+ but facilitated by phosphatidylinositol 4,5-bisphosphate (PIP2). We identified conserved residues D25 and D181 in CLCC1 N-terminus responsible for Ca2+ binding and luminal Ca2+-mediated inhibition on channel open probability and K298 in CLCC1 intraluminal loop as the critical PIP2-sensing residue. CLCC1 maintains steady-state [Cl-]ER and [K+]ER and ER morphology and regulates ER Ca2+ homeostasis, including internal Ca2+ release and steady-state [Ca2+]ER. ALS-associated mutant forms of CLCC1 increase steady-state [Cl-]ER and impair ER Ca2+ homeostasis, and animals with the ALS-associated mutations are sensitized to stress challenge-induced protein misfolding. Phenotypic comparisons of multiple Clcc1 loss-of-function alleles, including ALS-associated mutations, reveal a CLCC1 dosage dependence in the severity of disease phenotypes in vivo. Similar to CLCC1 rare variations dominant in ALS, 10% of K298A heterozygous mice developed ALS-like symptoms, pointing to a mechanism of channelopathy dominant-negatively induced by a loss-of-function mutation. Conditional knockout of Clcc1 cell-autonomously causes motor neuron loss and ER stress, misfolded protein accumulation, and characteristic ALS pathologies in the spinal cord. Thus, our findings support that disruption of ER ion homeostasis maintained by CLCC1 contributes to ALS-like pathologies.

Naphthylisoquinoline alkaloids, a new structural template inhibitor of Nav1.7 sodium channel.

Voltage-gated sodium channel 1.7 (Nav1.7) remains one of the most promising drug targets for pain relief. In the current study, we conducted a high-throughput screening of natural products in our in-house compound library to discover novel Nav1.7 inhibitors, then characterized their pharmacological properties. We identified 25 naphthylisoquinoline alkaloids (NIQs) from Ancistrocladus tectorius to be a novel type of Nav1.7 channel inhibitors. Their stereostructures including the linkage modes of the naphthalene group at the isoquinoline core were revealed by a comprehensive analysis of HRESIMS, 1D, and 2D NMR spectra as well as ECD spectra and single-crystal X-ray diffraction analysis with Cu Kα radiation. All the NIQs showed inhibitory activities against the Nav1.7 channel stably expressed in HEK293 cells, and the naphthalene ring in the C-7 position displayed a more important role in the inhibitory activity than that in the C-5 site. Among the NIQs tested, compound 2 was the most potent with an IC50 of 0.73 ± 0.03 µM. We demonstrated that compound 2 (3 µM) caused dramatical shift of steady-state slow inactivation toward the hyperpolarizing direction (V1/2 values were changed from -39.54 ± 2.77 mV to -65.53 ± 4.39 mV, which might contribute to the inhibition of compound 2 against the Nav1.7 channel. In acutely isolated dorsal root ganglion (DRG) neurons, compound 2 (10 µM) dramatically suppressed native sodium currents and action potential firing. In the formalin-induced mouse inflammatory pain model, local intraplantar administration of compound 2 (2, 20, 200 nmol) dose-dependently attenuated the nociceptive behaviors. In summary, NIQs represent a new type of Nav1.7 channel inhibitors and may act as structural templates for the following analgesic drug development.

Characterization of the role of TMEM175 in an in vitro lysosomal H+ fluxes model.

Lysosome acidification is a dynamic equilibrium of H+ influx and efflux across the membrane, which is crucial for cell physiology. The vacuolar H+ ATPase (V-ATPase) is responsible for the H+ influx or refilling of lysosomes. TMEM175 was identified as a novel H+ permeable channel on lysosomal membranes, and it plays a critical role in lysosome acidification. However, how TMEM175 participates in lysosomal acidification remains unknown. Here, we present evidence that TMEM175 regulates lysosomal H+ influx and efflux in enlarged lysosomes isolated from COS1 treated with vacuolin-1. By utilizing the whole-endolysosome patch-clamp recording technique, a series of integrated lysosomal H+ influx and efflux signals in a ten-of-second time scale under the physiological pH gradient (luminal pH 4.60, and cytosolic pH 7.20) was recorded from this in vitro system. Lysosomal H+ fluxes constitute both the lysosomal H+ refilling and releasing, and they are asymmetrical processes with distinct featured kinetics for each of the H+ fluxes. Lysosomal H+ fluxes are entirely abolished when TMEM175 losses of function in the F39V mutant and is blocked by the antagonist (2-GBI). Meanwhile, lysosomal H+ fluxes are modulated by the pH-buffering capacity of the lumen and the lysosomal glycosylated membrane proteins, lysosome-associated membrane protein 1 (LAMP1). We propose that the TMEM175-mediated lysosomal H+ fluxes model would provide novel thoughts for studying the pathology of Parkinson's disease and lysosome storage disorders.

Discovery of selective NaV1.8 inhibitors based on 5-chloro-2-(4,4-difluoroazepan-1-yl)-6-methyl nicotinamide scaffold for the treatment of pain.

The NaV1.8 channel is a genetically validated target for pain and it is mostly expressed in the peripheral nervous system. Based on the disclosed structures of NaV1.8-selective inhibitors, we designed and synthesized a series of compounds by introducing bicyclic aromatic fragments based on the nicotinamide scaffold. In this research, a systematic structure-activity relationship study was carried out. While compound 2c possessed moderate inhibitory activity (IC50 = 50.18 ± 0.04 nM) in HEK293 cells stably expressing human NaV1.8 channels, it showed potent inhibitory activity in DRG neurons and isoform selectivity (>200-fold against human NaV1.1, NaV1.5 and NaV1.7 channels). Moreover, the analgesic potency of compound 2c was identified in a post-surgical mouse model. These data demonstrate that compound 2c can be further evaluated as a non-addictive analgesic agent with reduced cardiac liabilities.

Functional characterization and in vitro pharmacological rescue of KCNQ2 pore mutations associated with epileptic encephalopathy.

Mutations in the KCNQ2 gene encoding KV7.2 subunit that mediates neuronal M-current cause a severe form of developmental and epileptic encephalopathy (DEE). Electrophysiological evaluation of KCNQ2 mutations has been proved clinically useful in improving outcome prediction and choosing rational anti-seizure medications (ASMs). In this study we described the clinical characteristics, electrophysiological phenotypes and the in vitro response to KCNQ openers of five KCNQ2 pore mutations (V250A, N258Y, H260P, A265T and G290S) from seven patients diagnosed with KCNQ2-DEE. The KCNQ2 variants were transfected into Chinese hamster ovary (CHO) cells alone, in combination with KCNQ3 (1:1) or with wild-type KCNQ2 (KCNQ2-WT) and KCNQ3 in a ratio of 1:1:2, respectively. Their expression and electrophysiological function were assessed. When transfected alone or in combination with KCNQ3, none of these mutations affected the membrane expression of KCNQ2, but most failed to induce a potassium current except A265T, in which trace currents were observed when co-transfected with KCNQ3. When co-expressed with KCNQ2-WT and KCNQ3 (1:1:2), the currents at 0 mV of these mutations were decreased by 30%-70% compared to the KCNQ2/3 channel, which could be significantly rescued by applying KCNQ openers including the approved antiepileptic drug retigabine (RTG, 10 µM), as well as two candidates subjected to clinical trials, pynegabine (HN37, 1 µM) and XEN1101 (1 µM). These newly identified pathologic variants enrich the KCNQ2-DEE mutation hotspots in the pore-forming domain. This electrophysiological study provides a rational basis for personalized therapy with KCNQ openers in DEE patients carrying loss-of-function (LOF) mutations in KCNQ2.

Extracellular vesicles mediate antibody-resistant transmission of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Antibody resistance dampens neutralizing antibody therapy and threatens current global Coronavirus (COVID-19) vaccine campaigns. In addition to the emergence of resistant SARS-CoV-2 variants, little is known about how SARS-CoV-2 evades antibodies. Here, we report a novel mechanism of extracellular vesicle (EV)-mediated cell-to-cell transmission of SARS-CoV-2, which facilitates SARS-CoV-2 to escape from neutralizing antibodies. These EVs, initially observed in SARS-CoV-2 envelope protein-expressing cells, are secreted by various SARS-CoV-2-infected cells, including Vero E6, Calu-3, and HPAEpiC cells, undergoing infection-induced pyroptosis. Various SARS-CoV-2-infected cells produce similar EVs characterized by extra-large sizes (1.6-9.5 µm in diameter, average diameter > 4.2 µm) much larger than previously reported virus-generated vesicles. Transmission electron microscopy analysis and plaque assay reveal that these SARS-CoV-2-induced EVs contain large amounts of live virus particles. In particular, the vesicle-cloaked SARS-CoV-2 virus is resistant to neutralizing antibodies and able to reinfect naïve cells independent of the reported receptors and cofactors. Consistently, the constructed 3D images show that intact EVs could be taken up by recipient cells directly, supporting vesicle-mediated cell-to-cell transmission of SARS-CoV-2. Our findings reveal a novel mechanism of receptor-independent SARS-CoV-2 infection via cell-to-cell transmission, provide new insights into antibody resistance of SARS-CoV-2 and suggest potential targets for future antiviral therapeutics.

Epilepsy phenotype and response to KCNQ openers in mice harboring the Kcnq2 R207W voltage-sensor mutation.

KCNQ2-encoded Kv7.2 subunits play a critical role in balancing neuronal excitability. Mutations in KCNQ2 are responsible for highly-heterogenous epileptic and neurodevelopmental phenotypes ranging from self-limited familial neonatal epilepsy (SeLFNE) to severe developmental and epileptic encephalopathy (DEE). Pathogenic KCNQ2 variants cluster at the voltage sensor domain (VSD), the pore domain, and the C-terminal tail. Although several knock-in mice harboring Kcnq2 pore variants have been developed, no mouse line carrying Kcnq2 voltage-sensor mutations has been described. KCNQ2-R207W is an epilepsy-causing mutation located in the VSD, mainly affecting voltage-dependent channel gating. To study the physiological consequence of Kcnq2 VSD dysfunction, we generated a Kcnq2-R207W mouse line and analyzed the pathological and pharmacological phenotypes of mutant mice. As a result, both homozygous (Kcnq2RW/RW) and heterozygous (Kcnq2RW/+) mice were viable. While Kcnq2RW/RW mice displayed a short lifespan, growth retardation, and spontaneous seizures, Kcnq2RW/+ mice survived and developed normally, although only a fraction (9/64; 14%) of them showed behavioral- and ECoG-confirmed spontaneous seizures. Kcnq2RW/+ mice displayed increased susceptibility to evoked seizures, which was dramatically ameliorated by treatment with the novel KCNQ opener pynegabine (HN37). Our results show that the Kcnq2-R207W mouse line, the first harboring a Kcnq2 voltage-sensor mutation, exhibits a unique epileptic phenotype with both spontaneous seizures and increased susceptibility to evoked seizures. In Kcnq2-R207W mice, the potent KCNQ opener HN37, currently in clinical phase I, shows strong anticonvulsant activity, suggesting it may represent a valuable option for the severe phenotypes of KCNQ2-related epilepsy.

SCN8A epileptic encephalopathy mutations display a gain-of-function phenotype and divergent sensitivity to antiepileptic drugs.

De novo missense mutations in SCN8A gene encoding voltage-gated sodium channel NaV1.6 are linked to a severe form of early infantile epileptic encephalopathy named early infantile epileptic encephalopathy type13 (EIEE13). The majority of the patients with EIEE13 does not respond favorably to the antiepileptic drugs (AEDs) in clinic and has a significantly increased risk of death. Although more than 60 EIEE13-associated mutations have been discovered, only few mutations have been functionally analyzed. In this study we investigated the functional influences of mutations N1466T and N1466K, two EIEE13-associated mutations located in the inactivation gate, on sodium channel properties. Sodium currents were recorded from CHO cells expressing the mutant and wide-type (WT) channels using the whole-cell patch-clamp technique. We found that, in comparison with WT channels, both the mutant channels exhibited increased window currents, persistent currents (INaP) and ramp currents, suggesting that N1466T and N1466K were gain-of-function (GoF) mutations. Sodium channel inhibition is one common mechanism of currently available AEDs, in which topiramate (TPM) was effective in controlling seizures of patients carrying either of the two mutations. We found that TPM (100 µM) preferentially inhibited INaP and ramp currents but did not affect transient currents (INaT) mediated by N1466T or N1466K. Among the other 6 sodium channel-inhibiting AEDs tested, phenytoin and carbamazepine displayed greater efficacy than TPM in suppressing both INaP and ramp currents. Functional characterization of mutants N1466T and N1466K is beneficial for understanding the pathogenesis of EIEE13. The divergent effects of sodium channel-inhibiting AEDs on INaP and ramp currents provide insight into the development of therapeutic strategies for the N1466T and N1466K-associated EIEE13.

Loss of MLKL ameliorates liver fibrosis by inhibiting hepatocyte necroptosis and hepatic stellate cell activation.

Background: Liver fibrosis affects millions of people worldwide without an effective treatment. Although multiple cell types in the liver contribute to the fibrogenic process, hepatocyte death is considered to be the trigger. Multiple forms of cell death, including necrosis, apoptosis, and necroptosis, have been reported to co-exist in liver diseases. Mixed lineage kinase domain-like protein (MLKL) is the terminal effector in necroptosis pathway. Although necroptosis has been reported to play an important role in a number of liver diseases, the function of MLKL in liver fibrosis has yet to be unraveled. Methods and Results: Here we report that MLKL level is positively correlated with a number of fibrotic markers in liver samples from both patients with liver fibrosis and animal models. Mlkl deletion in mice significantly reduces clinical symptoms of CCl4- and bile duct ligation (BDL) -induced liver injury and fibrosis. Further studies indicate that Mlkl-/- blocks liver fibrosis by reducing hepatocyte necroptosis and hepatic stellate cell (HSC) activation. AAV8-mediated specific knockdown of Mlkl in hepatocytes remarkably alleviates CCl4-induced liver fibrosis in both preventative and therapeutic ways. Conclusion: Our results show that MLKL-mediated signaling plays an important role in liver damage and fibrosis, and targeting MLKL might be an effective way to treat liver fibrosis.

Identification of a Subtype-Selective Allosteric Inhibitor of GluN1/GluN3 NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) are Ca2+-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca2+-permeability and lower Mg2+-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC50 value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 µM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.