RESUMEN
NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.
Asunto(s)
Receptores de N-Metil-D-Aspartato , Anticuerpos de Dominio Único , Ratas , Animales , Conejos , Receptores de N-Metil-D-Aspartato/metabolismo , Anticuerpos de Dominio Único/metabolismo , Sinapsis/fisiología , Hipocampo/metabolismo , Neuronas/metabolismo , Inmunoglobulina G/metabolismo , MamíferosRESUMEN
N-methyl-D-aspartate receptors (NMDARs) play a significant role in developing several central nervous system (CNS) disorders. Currently, memantine, used for treating Alzheimer's disease, and ketamine, known for its anesthetic and antidepressant properties, are two clinically used NMDAR open-channel blockers. However, despite extensive research into NMDAR modulators, many have shown either harmful side effects or inadequate effectiveness. For instance, dizocilpine (MK-801) is recognized for its powerful psychomimetic effects due to its high-affinity and nearly irreversible inhibition of the GluN1/GluN2 NMDAR subtypes. Unlike ketamine, memantine and MK-801 also act through a unique, low-affinity "membrane-to-channel inhibition" (MCI). We aimed to develop an open-channel blocker based on MK-801 with distinct inhibitory characteristics from memantine and MK-801. Our novel compound, K2060, demonstrated effective voltage-dependent inhibition in the micromolar range at key NMDAR subtypes, GluN1/GluN2A and GluN1/GluN2B, even in the presence of Mg2+. K2060 showed reversible inhibitory dynamics and a partially trapping open-channel blocking mechanism with a significantly stronger MCI than memantine. Using hippocampal slices, 30⯵M K2060 inhibited excitatory postsynaptic currents in CA1 hippocampal neurons by â¼51â¯%, outperforming 30⯵M memantine (â¼21â¯% inhibition). K2060 exhibited No Observed Adverse Effect Level (NOAEL) of 15â¯mg/kg upon intraperitoneal administration in mice. Administering K2060 at a 10â¯mg/kg dosage resulted in brain concentrations of approximately 2⯵M, with peak concentrations (Tmax) achieved within 15â¯minutes. Finally, applying K2060 with trimedoxime and atropine in mice exposed to tabun improved treatment outcomes. These results underscore K2060's potential as a therapeutic agent for CNS disorders linked to NMDAR dysfunction.
RESUMEN
N-methyl-D-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission in the mammalian brain, and their physiological importance is underscored by the large number of pathogenic mutations that have been identified in the receptor's GluN subunits and associated with a wide range of diseases and disorders. Here, we characterized the functional and pharmacological effects of the pathogenic N650K variant in the GluN1 subunit, which is associated with developmental delay and seizures. Our microscopy experiments showed that when expressed in HEK293 cells (from ATCC®), the GluN1-N650K subunit increases the surface expression of both GluN1/GluN2A and GluN1/GluN2B receptors, but not GluN1/GluN3A receptors, consistent with increased surface expression of the GluN1-N650K subunit expressed in hippocampal neurons (from embryonic day 18 of Wistar rats of both sexes). Using electrophysiology, we found that the GluN1-N650K variant increases the potency of GluN1/GluN2A receptors to both glutamate and glycine but decreases the receptor's conductance and open probability. In addition, the GluN1-N650K subunit does not form functional GluN1/GluN2B receptors but does form fully functional GluN1/GluN3A receptors. Moreover, in the presence of extracellular Mg2+, GluN1-N650K/GluN2A receptors have a similar and increased response to ketamine and memantine, respectively, while the effect of both drugs had markedly slower onset and offset compared to wild-type GluN1/GluN2A receptors. Finally, we found that expressing the GluN1-N650K subunit in hippocampal neurons reduces excitotoxicity, and memantine shows promising neuroprotective effects in neurons expressing either wild-type GluN1 or the GluN1-N650K subunit. This study provides the functional and pharmacological characterization of NMDARs containing the GluN1-N650K variant.
Asunto(s)
Memantina , Receptores de N-Metil-D-Aspartato , Humanos , Ratas , Femenino , Masculino , Animales , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genética , Memantina/farmacología , Células HEK293 , Ácido Glutámico , MamíferosRESUMEN
N-methyl-d-aspartate receptors (NMDARs) play an essential role in regulating glutamatergic neurotransmission. Recently, pathogenic missense mutations were identified in genes encoding NMDAR subunits; however, their effect on NMDAR activity is often poorly understood. Here, we examined whether three previously identified pathogenic mutations (M641I, A645S, and Y647S) in the M3 domain of the GluN1 subunit affect the receptor's surface delivery, agonist sensitivity, Mg2+ block, and/or inhibition by the FDA-approved NMDAR blocker memantine. When expressed in HEK293 cells, we found reduced surface expression of GluN1-M641I/GluN2A, GluN1-Y647S/GluN2A, and GluN1-Y647S/GluN2B receptors; other mutation-bearing NMDAR combinations, including GluN1/GluN3A receptors, were expressed at normal surface levels. When expressed in rat hippocampal neurons, we consistently found reduced surface expression of the GluN1-M641I and GluN1-Y647S subunits when compared with wild-type GluN1 subunit. At the functional level, we found that GluN1-M641I/GluN2 and GluN1-A645S/GluN2 receptors expressed in HEK293 cells have wild-type EC50 values for both glutamate and glycine; in contrast, GluN1-Y647S/GluN2 receptors do not produce glutamate-induced currents. In the presence of a physiological concentration of Mg2+, we found that GluN1-M641I/GluN2 receptors have a lower memantine IC50 and slower offset kinetics, whereas GluN1-A645S/GluN2 receptors have a higher memantine IC50 and faster offset kinetics when compared to wild-type receptors. Finally, we found that memantine was the most neuroprotective in hippocampal neurons expressing GluN1-M641I subunits, followed by neurons expressing wild-type GluN1 and then GluN1-A645S subunits in an NMDA-induced excitotoxicity assay. These results indicate that specific pathogenic mutations in the M3 domain of the GluN1 subunit differentially affect the trafficking and functional properties of NMDARs.
Asunto(s)
Agonistas de Aminoácidos Excitadores/administración & dosificación , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Mutación/genética , Proteínas del Tejido Nervioso/genética , Subunidades de Proteína/genética , Receptores de N-Metil-D-Aspartato/genética , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Masculino , Mutación/efectos de los fármacos , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Subunidades de Proteína/agonistas , Subunidades de Proteína/antagonistas & inhibidores , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Propiedades de Superficie/efectos de los fármacosRESUMEN
Although numerous pathogenic mutations have been identified in various subunits of N-methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC50 of both glycine and D-serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca2+ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.
Asunto(s)
Glicina/farmacología , Hipocampo/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Glicinérgicos/farmacología , Células HEK293 , Hipocampo/citología , Hipocampo/efectos de los fármacos , Humanos , Ligandos , Modelos Moleculares , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Dominios Proteicos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
N-methyl-d-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission within the mammalian central nervous system (CNS). NMDARs are heteromultimers containing GluN1, GluN2, and/or GluN3 subunits, thus giving rise to a wide variety of subunit combinations, each with unique functional and pharmacological properties. Importantly, GluN1/GluN3A and GluN1/GluN3B receptors form glycine-gated receptors. Here, we combined electrophysiology with rapid solution exchange in order to determine whether the presence of specific N-glycans and/or interactions with specific lectins regulates the functional properties of GluN1/GluN3A and GluN1/GluN3B receptors expressed in human embryonic kidney 293 (HEK293) cells. We found that removing putative N-glycosylation sites alters the functional properties of GluN1/GluN3B receptors, but has no effect on GluN1/GluN3A receptors. Moreover, we found that the functional properties of both GluN1/GluN3A and GluN1/GluN3B receptors are modulated by a variety of lectins, including Concanavalin A (ConA), Wheat Germ Agglutinin (WGA), and Aleuria Aurantia Lectin (AAL), and this effect is likely mediated by a reduction in GluN1 subunit-mediated desensitization. We also found that AAL has the most profound effect on GluN1/GluN3 receptors, and this effect is mediated partly by a single N-glycosylation site on the GluN3 subunit (specifically, N565 on GluN3A and N465 on GluN3B). Finally, we found that lectins mediate their effect only when applied to non-activated receptors and have no effect when applied in the continuous presence of glycine. These findings provide further evidence to distinguish GluN1/GluN3 receptors from the canonical GluN1/GluN2 receptors and offer insight into how GluN1/GluN3 receptors may be regulated in the mammalian CNS.
Asunto(s)
Lectinas/farmacología , Subunidades de Proteína/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Células Cultivadas , Glicina/farmacología , Glicosilación/efectos de los fármacos , Humanos , Lectinas/antagonistas & inhibidores , Potenciales de la Membrana/fisiología , Polisacáridos/metabolismo , Unión ProteicaRESUMEN
N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an essential role in mediating excitatory neurotransmission in the mammalian central nervous system (CNS). Functional NMDARs are tetramers composed of GluN1, GluN2A-D, and/or GluN3A-B subunits, giving rise to a wide variety of NMDAR subtypes with unique functional properties. Here, we examined the surface delivery and functional properties of NMDARs containing mutations in the glycine-binding sites in GluN1 and GluN3A subunits expressed in mammalian cell lines and primary rat hippocampal neurons. We found that the structural features of the glycine-binding sites in both GluN1 and GluN3A subunits are correlated with receptor forward trafficking to the cell surface. In addition, we found that a potentially clinically relevant mutation in the glycine-binding site of the human GluN3A subunit significantly reduces surface delivery of NMDARs. Taken together, these findings provide novel insight into how NMDARs are regulated by their glycine-binding sites and may provide important information regarding the role of NMDARs in both physiological and pathophysiological processes in the mammalian CNS.