N-Methyl-d-aspartate receptors (NMDARs), encoded by GRIN genes, are ionotropic glutamate receptors playing a critical role in synaptic transmission, plasticity, and synapse development. Genome sequence analyses have identified variants in GRIN genes in patients with neurodevelopmental disorders, but the underlying disease mechanisms are not well understood. Here, we have created and evaluated a transgenic mouse line carrying a missense variant Grin2bL825V , corresponding to a de novo GRIN2B variant encoding GluN2B(L825V) found in a patient with intellectual disability (ID) and autism spectrum disorder (ASD). We used HEK293T cells expressing recombinant receptors and primary hippocampal neurons prepared from heterozygous Grin2bL825V/+ (L825V/+) and wild-type (WT) Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons from L825V/+ compared with +/+ mice. The peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSCs) was unchanged, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared with +/+ neurons and were less sensitive to a GluN2B-selective antagonist ifenprodil. Together, these results suggest a decreased functional contribution of GluN2B subunits to synaptic NMDAR currents in hippocampal neurons from L825V/+ mice. The analysis of the GluN2B(L825V) subunit surface expression and synaptic localization revealed no differences compared with WT GluN2B. Behavioral testing of mice of both sexes demonstrated hypoactivity, anxiety, and impaired sensorimotor gating in the L825V/+ strain, particularly affecting males, as well as cognitive symptoms. The heterozygous L825V/+ mouse offers a clinically relevant model of GRIN2B-related ID/ASD, and our results suggest synaptic-level functional changes that may contribute to neurodevelopmental pathology.

• Keywords
• GluN2B, NMDA receptors, autism spectrum disorder, mouse model, synaptic transmission,
• MeSH
• Excitatory Postsynaptic Potentials physiology   MeSH
• HEK293 Cells MeSH
• Hippocampus metabolism   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic * MeSH
• Mice MeSH
• Neurons metabolism   MeSH
• Neurodevelopmental Disorders * genetics   physiopathology   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NR2B NMDA receptor MeSH Browser
• Receptors, N-Methyl-D-Aspartate * MeSH

N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

• Keywords
• Channelopathy, Endogenous neuroactive steroid, GRIN2 genes, Glutamate receptors, Rescue pharmacology, Surface expression,
• MeSH
• Electrophysiological Phenomena MeSH
• Humans MeSH
• Neurosteroids * MeSH
• Receptors, N-Methyl-D-Aspartate * genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• Neurosteroids * MeSH
• NR2B NMDA receptor MeSH Browser
• Receptors, N-Methyl-D-Aspartate * MeSH

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.

• MeSH
• Glycine pharmacology   MeSH
• Glycine Agents pharmacology   MeSH
• HEK293 Cells MeSH
• Hippocampus cytology   drug effects   metabolism   MeSH
• Rats MeSH
• Humans MeSH
• Ligands MeSH
• Models, Molecular MeSH
• Mutation * MeSH
• Neurons cytology   drug effects   metabolism   MeSH
• Rats, Wistar MeSH
• Protein Domains MeSH
• Nerve Tissue Proteins genetics   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glycine MeSH
• Glycine Agents MeSH
• GRIN1 protein, human MeSH Browser
• Ligands MeSH
• NMDA receptor A1 MeSH Browser
• Nerve Tissue Proteins MeSH
• Receptors, N-Methyl-D-Aspartate MeSH

N-methyl-D-aspartate receptors (NMDARs), glutamate-gated ion channels, mediate signaling at the majority of excitatory synapses in the nervous system. Recent sequencing data for neurological and psychiatric patients have indicated numerous mutations in genes encoding for NMDAR subunits. Here, we present surface expression, functional, and pharmacological analysis of 11 de novo missense mutations of the human hGluN2B subunit (P553L; V558I; W607C; N615I; V618G; S628F; E657G; G820E; G820A; M824R; L825V) located in the pre-M1, M1, M2, M3, and M4 membrane regions. These variants were identified in patients with intellectual disability, developmental delay, epileptic symptomatology, and autism spectrum disorder. Immunofluorescence microscopy indicated that the ratio of surface-to-total NMDAR expression was reduced for hGluN1/hGluN2B(S628F) receptors and increased for for hGluN1/hGluN2B(G820E) receptors. Electrophysiological recordings revealed that agonist potency was altered in hGluN1/hGluN2B(W607C; N615I; and E657G) receptors and desensitization was increased in hGluN1/hGluN2B(V558I) receptors. The probability of channel opening of hGluN1/hGluN2B (V558I; W607C; V618G; and L825V) receptors was diminished ~10-fold when compared to non-mutated receptors. Finally, the sensitivity of mutant receptors to positive allosteric modulators of the steroid origin showed that glutamate responses induced in hGluN1/hGluN2B(V558I; W607C; V618G; and G820A) receptors were potentiated by 59-96% and 406-685% when recorded in the presence of 20-oxo-pregn-5-en-3β-yl sulfate (PE-S) and androst-5-en-3β-yl hemisuccinate (AND-hSuc), respectively. Surprisingly hGluN1/hGluN2B(L825V) receptors were strongly potentiated, by 197 and 1647%, respectively, by PE-S and AND-hSuc. Synaptic-like responses induced by brief glutamate application were also potentiated and the deactivation decelerated. Further, we have used homology modeling based on the available crystal structures of GluN1/GluN2B NMDA receptor followed by molecular dynamics simulations to try to relate the functional consequences of mutations to structural changes. Overall, these data suggest that de novo missense mutations of the hGluN2B subunit located in membrane domains lead to multiple defects that manifest by the NMDAR loss of function that can be rectified by steroids. Our results provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with hypofunction of the glutamatergic system.

• Keywords
• GluN2B, NMDA receptor, de novo missense mutations, neuropsychiatric disorder, neurosteroids,
• Publication type
• Journal Article MeSH

UNLABELLED: GABAB receptors are the G-protein coupled receptors for the main inhibitory neurotransmitter in the brain, GABA. GABAB receptors were shown to associate with homo-oligomers of auxiliary KCTD8, KCTD12, KCTD12b, and KCTD16 subunits (named after their T1 K+-channel tetramerization domain) that regulate G-protein signaling of the receptor. Here we provide evidence that GABAB receptors also associate with hetero-oligomers of KCTD subunits. Coimmunoprecipitation experiments indicate that two-thirds of the KCTD16 proteins in the hippocampus of adult mice associate with KCTD12. We show that the KCTD proteins hetero-oligomerize through self-interacting T1 and H1 homology domains. Bioluminescence resonance energy transfer measurements in live cells reveal that KCTD12/KCTD16 hetero-oligomers associate with both the receptor and the G-protein. Electrophysiological experiments demonstrate that KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties on G-protein-activated Kir3 currents. During prolonged receptor activation (one min) KCTD12/KCTD16 hetero-oligomers produce moderately desensitizing fast deactivating K+ currents, whereas KCTD12 and KCTD16 homo-oligomers produce strongly desensitizing fast deactivating currents and nondesensitizing slowly deactivating currents, respectively. During short activation (2 s) KCTD12/KCTD16 hetero-oligomers produce nondesensitizing slowly deactivating currents. Electrophysiological recordings from hippocampal neurons of KCTD knock-out mice are consistent with these findings and indicate that KCTD12/KCTD16 hetero-oligomers increase the duration of slow IPSCs. In summary, our data demonstrate that simultaneous assembly of distinct KCTDs at the receptor increases the molecular and functional repertoire of native GABAB receptors and modulates physiologically induced K+ current responses in the hippocampus. SIGNIFICANCE STATEMENT: The KCTD proteins 8, 12, and 16 are auxiliary subunits of GABAB receptors that differentially regulate G-protein signaling of the receptor. The KCTD proteins are generally assumed to function as homo-oligomers. Here we show that the KCTD proteins also assemble hetero-oligomers in all possible dual combinations. Experiments in live cells demonstrate that KCTD hetero-oligomers form at least tetramers and that these tetramers directly interact with the receptor and the G-protein. KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties to GABAB receptor-induced Kir3 currents in heterologous cells. KCTD12/KCTD16 hetero-oligomers are abundant in the hippocampus, where they prolong the duration of slow IPSCs in pyramidal cells. Our data therefore support that KCTD hetero-oligomers modulate physiologically induced K+ current responses in the brain.

• Keywords
• G-protein coupled receptor, GABA-B, GPCR, KCTD12, KCTD16, Kir3,
• MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Potassium Channels genetics   metabolism   MeSH
• Electrophysiological Phenomena genetics   MeSH
• Excitatory Postsynaptic Potentials genetics   MeSH
• Kinetics MeSH
• Cricetinae MeSH
• Patch-Clamp Techniques MeSH
• Brain Chemistry genetics   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Receptors, GABA-B genetics   metabolism   MeSH
• Receptors, KIR metabolism   MeSH
• Receptors, G-Protein-Coupled metabolism   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium Channels MeSH
• Receptors, GABA-B MeSH
• Receptors, KIR MeSH
• Receptors, G-Protein-Coupled MeSH

GABAB receptors assemble from GABAB1 and GABAB2 subunits. GABAB2 additionally associates with auxiliary KCTD subunits (named after their K(+) channel tetramerization-domain). GABAB receptors couple to heterotrimeric G-proteins and activate inwardly-rectifying K(+) channels through the βγ subunits released from the G-protein. Receptor-activated K(+) currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K(+) currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast desensitization of GABAB receptor-activated K(+) currents while protein kinase-A inhibition accelerates fast desensitization. Protein kinase-A fails to regulate fast desensitization in KCTD12 knock-out mice or knock-in mice with a serine-892 to alanine mutation, thus demonstrating that serine-892 phosphorylation regulates KCTD12-induced desensitization in vivo. Fast current desensitization is accelerated in hippocampal neurons carrying the serine-892 to alanine mutation, showing that tonic serine-892 phosphorylation normally limits KCTD12-induced desensitization. Tonic serine-892 phosphorylation is in turn promoted by assembly of receptors with KCTD12. This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation.

• Keywords
• G-protein coupled receptor, GABA-B, GPCR, Kir3, PKA,
• MeSH
• Alanine genetics   metabolism   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Potassium metabolism   MeSH
• Phosphorylation MeSH
• Hippocampus cytology   metabolism   MeSH
• Cells, Cultured MeSH
• Patch-Clamp Techniques MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Neurons metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Receptors, GABA-B genetics   metabolism   MeSH
• Receptors, GABA genetics   metabolism   MeSH
• Serine genetics   metabolism   MeSH
• Amino Acid Substitution MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alanine MeSH
• Potassium MeSH
• pfetin protein, mouse MeSH Browser
• Cyclic AMP-Dependent Protein Kinases MeSH
• GTP-Binding Proteins MeSH
• Receptors, GABA-B MeSH
• Receptors, GABA MeSH
• Serine MeSH

NMDA receptors are highly expressed in the CNS and are involved in excitatory synaptic transmission and synaptic plasticity as well as excitotoxicity. They have several binding sites for allosteric modulators, including neurosteroids, endogenous compounds synthesized by the nervous tissue and expected to act locally. Whole-cell patch-clamp recording from human embryonic kidney 293 cells expressing NR1-1a/NR2B receptors revealed that neurosteroid pregnenolone sulfate (PS) (300 microm), when applied to resting NMDA receptors, potentiates the amplitude of subsequent responses to 1 mm glutamate fivefold and slows their deactivation twofold. The same concentration of PS, when applied during NMDA receptor activation by 1 mm glutamate, has only a small effect. The association and dissociation rate constants of PS binding and unbinding from resting NMDA receptors are estimated to be 3.3 +/- 2.0 mm(-1)sec(-1) and 0.12 +/- 0.02 sec(-1), respectively, corresponding to an apparent affinity K(d) of 37 microm. The results of experiments indicate that the molecular mechanism of PS potentiation of NMDA receptor responses is attributable to an increase in the peak channel open probability (P(o)). Responses to glutamate recorded in the continuous presence of PS exhibit marked time-dependent decline. Our results indicate that the decline is induced by a change of the NMDA receptor affinity for PS after receptor activation. These results suggest that the PS is a modulator of NMDA receptor P(o), the effectiveness of which is lowered by glutamate binding. This modulation may have important consequences for the neuronal excitability.

• MeSH
• Models, Biological MeSH
• Ion Channel Gating drug effects   MeSH
• Hippocampus cytology   MeSH
• Kinetics MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Glutamic Acid pharmacology   MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Neurons drug effects   metabolism   MeSH
• Animals, Newborn MeSH
• Probability MeSH
• Pregnenolone pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate agonists   drug effects   metabolism   MeSH
• Recombinant Proteins agonists   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glutamic Acid MeSH
• NR1 NMDA receptor MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Pregnenolone MeSH
• pregnenolone sulfate MeSH Browser
• Receptors, N-Methyl-D-Aspartate MeSH
• Recombinant Proteins MeSH