A steroid modulatory domain in NR2A collaborates with NR1 exon-5 to control NMDAR modulation by pregnenolone sulfate and protons.
ABSTRACT: NMDA receptor (NMDAR)-mediated excitatory synaptic transmission plays a critical role in synaptic plasticity and memory formation, whereas its dysfunction may underlie neuropsychiatric and neurodegenerative diseases. The neuroactive steroid pregnenolone sulfate (PS) acts as a cognitive enhancer in impaired animals, augments LTP in hippocampal slices by enhancing NMDAR activity, and may participate in the reduction of schizophrenia's negative symptoms by systemic pregnenolone. We report that the effects of PS on NMDAR function are diverse, varying with subunit composition and NR1 splice variant. While PS potentiates NR1-1a/NR2B receptors through a critical steroid modulatory domain in NR2B that also modulates tonic proton inhibition, potentiation of the NMDA response is not dependent upon relief of such inhibition, a finding that distinguishes it from spermine. In contrast, the presence of an NR2A subunit confers enhanced PS-potentiation at reduced pH, suggesting that it may indeed act like spermine does at NR2B-containing receptors. Additional tuning of the NMDAR response by PS comes via the N-terminal exon-5 splicing insert of NR1-1b, which regulates the magnitude of proton-dependent PS potentiation. For NR2C- and NR2D-containing receptors, negative modulation at NR2C receptors is pH-independent (like NR2B) while negative modulation at NR2D receptors is pH-dependent (like NR2A). Taken together, PS displays a rich modulatory repertoire that takes advantage of the structural diversity of NMDARs in the CNS. The differential pH sensitivity of NMDAR isoforms to PS modulation may be especially important given the emerging role of proton sensors to both learning and memory, as well as brain injury.
Project description:1. The neurosteroid pregnenolone sulphate (PS) potentiates N-methyl-D-aspartate (NMDA) receptor mediated responses in various neuronal preparations. The NR1 subunit can combine with NR2A, NR2B, NR2C, or NR2D subunits to form functional receptors. Differential NR2 subunit expression in brain and during development raises the question of how the NR2 subunit influences NMDA receptor modulation by neuroactive steroids. 2. We examined the effects of PS on the four diheteromeric NMDA receptor subtypes generated by co-expressing the NR1(100) subunit with each of the four NR2 subunits in Xenopus oocytes. Whereas PS potentiated NMDA-, glutamate-, and glycine-induced currents of NR1/NR2A and NR1/NR2B receptors, it was inhibitory at NR1/NR2C and NR1/NR2D receptors. 3. In contrast, pregnanolone sulphate (3alpha5betaS), a negative modulator of the NMDA receptor that acts at a distinct site from PS, inhibited all four subtypes, but was approximately 4 fold more potent at NR1/NR2C and NR1/NR2D than at NR1/NR2A and NR1/NR2B receptors. 4. These findings demonstrate that residues on the NR2 subunit are key determinants of modulation by PS and 3alpha5betaS. The modulatory effects of PS, but not 3alpha5betaS, on dose-response curves for NMDA, glutamate, and glycine are consistent with a two-state model in which PS either stabilizes or destabilizes the active state of the receptor, depending upon which NR2 subunit is present. 5. The selectivity of sulphated steroid modulators for NMDA receptors of specific subunit composition is consistent with a neuromodulatory role for endogenous sulphated steroids. The results indicate that it may be possible to develop therapeutic agents that target steroid modulatory sites of specific NMDA receptor subtypes.
Project description:N-methyl-D-aspartate (NMDA) receptor subunit-specific probes were used to characterize developmental changes in the distribution of excitatory amino acid receptors in the chicken's auditory brainstem nuclei. Although NR1 subunit expression does not change greatly during the development of the cochlear nuclei in the chicken (Tang and Carr  Hear. Res 191:79-89), there are significant developmental changes in NR2 subunit expression. We used in situ hybridization against NR1, NR2A, NR2B, NR2C, and NR2D to compare NR1 and NR2 expression during development. All five NMDA subunits were expressed in the auditory brainstem before embryonic day (E) 10, when electrical activity and synaptic responses appear in the nucleus magnocellularis (NM) and the nucleus laminaris (NL). At this time, the dominant form of the receptor appeared to contain NR1 and NR2B. NR2A appeared to replace NR2B by E14, a time that coincides with synaptic refinement and evoked auditory responses. NR2C did not change greatly during auditory development, whereas NR2D increased from E10 and remained at fairly high levels into adulthood. Thus changes in NMDA NR2 receptor subunits may contribute to the development of auditory brainstem responses in the chick.
Project description:N-methyl-d-aspartate (NMDA) receptors (NMDARs) are a major class of excitatory neurotransmitter receptors in the central nervous system. They form glutamate-gated ion channels that are highly permeable to calcium and mediate activity-dependent synaptic plasticity. NMDAR dysfunction is implicated in multiple brain disorders, including stroke, chronic pain and schizophrenia. NMDARs exist as multiple subtypes with distinct pharmacological and biophysical properties that are largely determined by the type of NR2 subunit (NR2A to NR2D) incorporated in the heteromeric NR1/NR2 complex. A fundamental difference between NMDAR subtypes is their channel maximal open probability (P(o)), which spans a 50-fold range from about 0.5 for NR2A-containing receptors to about 0.01 for receptors containing NR2C and NR2D; NR2B-containing receptors have an intermediate value (about 0.1). These differences in P(o) confer unique charge transfer capacities and signalling properties on each receptor subtype. The molecular basis for this profound difference in activity between NMDAR subtypes is unknown. Here we show that the subunit-specific gating of NMDARs is controlled by the region formed by the NR2 amino-terminal domain (NTD), an extracellular clamshell-like domain previously shown to bind allosteric inhibitors, and the short linker connecting the NTD to the agonist-binding domain (ABD). The subtype specificity of NMDAR P(o) largely reflects differences in the spontaneous (ligand-independent) equilibrium between open-cleft and closed-cleft conformations of the NR2-NTD. This NTD-driven gating control also affects pharmacological properties by setting the sensitivity to the endogenous inhibitors zinc and protons. Our results provide a proof of concept for a drug-based bidirectional control of NMDAR activity by using molecules acting either as NR2-NTD 'closers' or 'openers' promoting receptor inhibition or potentiation, respectively.
Project description:The four stereoisomers of azetidine-2,3-dicaroxylic acid (L-trans-ADC, L-cis-ADC, D-trans-ADC, and D-cis-ADC) were synthesized in a stereocontrolled fashion following two distinct strategies: one providing the two cis-ADC enantiomers and one giving access to the two trans-ADC enantiomers. The four azetidinic amino acids were characterized in a radioligand binding assay ([(3)H]CGP39653) at native NMDA receptors: L-trans-ADC showed the highest affinity (K(i)=10 microM) followed by the D-cis-ADC stereoisomer (21 microM). In contrast, the two analogues L-cis-ADC and D-trans-ADC were low-affinity ligands (>100 and 90 microM, respectively). Electrophysiological characterization of the ADC compounds at the four NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D expressed in Xenopus oocytes showed that L-trans-ADC displayed the highest agonist potency at NR1/NR2D (EC(50)=50 microM), which was 9.4-, 3.4-, and 1.9-fold higher than the respective potencies at NR1/NR2A-C. D-cis-ADC was shown to be a partial agonist at NR1/NR2C and NR1/NR2D with medium-range micromolar potencies (EC(50)=720 and 230 microM, respectively). A subsequent in silico ligand-protein docking study suggested an unusual binding mode for these amino acids in the agonist binding site.
Project description:N-methyl D-aspartate (NMDA) receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies have revealed reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed-onset potentiation whereby PregS approximately doubles the cell's response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS. The number of functional cell-surface NMDARs in cortical neurons increases 60-100% within 10 minutes of exposure to PregS, as shown by surface biotinylation and affinity purification. Delayed-onset potentiation is reversible and selective for expressed receptors containing the NMDAR subunit subtype 2A (NR2A) or NR2B, but not the NR2C or NR2D, subunits. Moreover, substitution of NR2B J/K helices and M4 domain with the corresponding region of NR2D ablates rapid allosteric potentiation of the NMDA response by PregS but not delayed-onset potentiation. This demonstrates that the initial phase of rapid positive allosteric modulation is not a first step in NMDAR upregulation. Delayed-onset potentiation by PregS occurs via a noncanonical, pertussis toxin-sensitive, G protein-coupled, and Ca(2+)-dependent mechanism that is independent of NMDAR ion channel activation. Further investigation into the sequelae for PregS-stimulated trafficking of NMDARs to the neuronal cell surface may uncover a new target for the pharmacological treatment of disorders in which NMDAR hypofunction has been implicated.
Project description:NMDA receptors comprised of different NR2 subunits exhibit strikingly unique biophysical and pharmacological properties. Here, we report that the extracellular amino-terminal domain (ATD) of the NR2 subunit controls pharmacological and kinetic properties of recombinant NMDA receptors, such as agonist potency, deactivation time course, open probability (P(OPEN)), and mean open/shut duration. Using ATD deletion mutants of NR2A, NR2B, NR2C, NR2D, and chimeras of NR2A and NR2D with interchanged ATD [NR2A-(2D-ATD) and NR2D-(2A-ATD)], we show that the ATD contributes to the low glutamate potency of NR2A-containing NMDA receptors and the high glutamate potency of NR2D-containing receptors. The ATD influences the deactivation time courses of NMDA receptors, as removal of the ATD from NR2A slows the deactivation rate, while removal of the ATD from NR2B, NR2C and NR2D accelerates the deactivation rate. Open probability also is influenced by the ATD. Removal of the ATD from NR2A or replacement of the NR2A-ATD with that of NR2D decreases P(OPEN) in single-channel recordings from outside-out patches of HEK 293 cells. In contrast, deletion of the ATD from NR2D or replacement of the NR2D ATD with that of NR2A increases P(OPEN) and mean open duration. These data demonstrate the modular nature of NMDA receptors, and show that the ATD of the different NR2 subunits plays an important role in fine-tuning the functional properties of the individual NMDA receptor subtypes.
Project description:In neuronal synapses, PDZ domains [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] of PSD-95 proteins interact with C termini of NMDA receptor [NMDAR (NR)] subunits, linking them to downstream neurotoxic signaling molecules. Perturbing NMDAR/PSD-95 interactions with a Tat peptide comprising the nine C-terminal residues of the NR2B subunit (Tat-NR2B9c) reduces neurons' vulnerability to excitotoxicity and ischemia. However, NR subunit C termini may bind many of >240 cellular PDZs, any of which could mediate neurotoxic signaling independently of PSD-95. Here, we performed a proteomic and biochemical analysis of the interactions of all known human PDZs with synaptic signaling proteins including NR1, NR2A-NR2D, and neuronal nitric oxide synthase (nNOS). Tat-NR2B9c, whose interactions define PDZs involved in neurotoxic signaling, was also used. NR2A-NR2D subunits and Tat-NR2B9c had similar, highly specific, PDZ protein interactions, of which the strongest were with the PSD-95 family members (PSD-95, PSD-93, SAP97, and SAP102) and Tax interaction protein 1 (TIP1). The PSD-95 PDZ2 domain bound NR2A-NR2C subunits most strongly (EC50, approximately 1 microM), and fusing the NR2B C terminus to Tat enhanced its affinity for PSD-95 PDZ2 by >100-fold (EC50, approximately 7 nM). IC50 values for Tat-NR2B9c inhibiting NR2A-NR2C/PSD-95 interactions (approximately 1-10 microM) and nNOS/PSD-95 interactions (200 nM) confirmed the feasibility of such inhibition. To determine which of the PDZ interactions of Tat-NR2B9c mediate neuroprotection, one of PSD-95, PSD-93, SAP97, SAP102, TIP1, or nNOS expression was inhibited in cortical neurons exposed to NMDA toxicity. Only neurons lacking PSD-95 or nNOS but not PSD-93, SAP97, SAP102, or TIP1 exhibited reduced excitotoxic vulnerability. Thus, despite the ubiquitousness of PDZ domain-containing proteins, PSD-95 and nNOS above any other PDZ proteins are keys in effecting NMDAR-dependent excitotoxicity. Consequently, PSD-95 inhibition may constitute a highly specific strategy for treating excitotoxic disorders.
Project description:Abnormalities in glutamate neurotransmission are thought to be among the major contributing factors to the pathophysiology of schizophrenia. Although schizophrenia has been regarded mostly as a disorder of higher cortical function, the cortex and thalamus work as a functional unit. Existing data regarding alterations of glutamate receptor subunit expression in the thalamus in schizophrenia remain equivocal. This postmortem study examined mRNA expression of ionotropic glutamate receptor (iGluR) subunits and PSD95 in 5 precisely defined and dissected thalamic subdivisions (medial and lateral sectors of the mediodorsal nucleus; and the ventral lateral posterior, ventral posterior, and centromedian nuclei) of persons with schizophrenia and matched controls using quantitative PCR with normalization to multiple endogenous controls. Among 15 genes examined (NR1 and NR2A-D subunits of the NMDA receptor; GluR1-4 subunits of the AMPA receptor; GluR5-7 and KA1-2 subunits of the kainate receptor; PSD95), all but two (GluR4 and KA1) were expressed at quantifiable levels. Differences in iGluR gene expression were seen between different thalamic nuclei but not between diagnostic groups. The relative abundance of transcripts was: NR1>>NR2A>NR2B>NR2D>NR2C for NMDA, GluR2>GluR1>GluR3 for AMPA, and KA2>GluR5>GluR7>GluR6 for kainate receptors. The expression of PSD95 correlated with the expression of NR1, NR2A, NR2B, NR2D and GluR6 in all nuclei. These results provide detailed and quantitative information on iGluR subunit expression in multiple nuclei of the human thalamus but suggest that alterations in their expression are not a prominent feature of schizophrenia.
Project description:Zinc inhibits NMDA receptor function through both voltage-dependent and voltage-independent mechanisms. In this report we have investigated the role that the NR1 subunit plays in voltage-independent Zn2+ inhibition. Our data show that inclusion of exon 5 into the NR1 subunit increases the IC50 for voltage-independent Zn2+ inhibition from 3-fold to 10-fold when full length exon 22 is also spliced into the mature NR1 transcript and the NMDA receptor complex contains the NR2A or NR2B subunits; exon 5 has little effect on Zn2+ inhibition of receptors that contain NR2C and NR2D. Mutagenesis within exon 5 indicates that the same residues that control proton inhibition, including Lys211, also control the effects of exon 5 on Zn2+ inhibition. Amino acid exchanges within the NR1 subunit but outside exon 5 (E181Q, E339Q, E342Q, N616R, N616Q, D669N, D669E, C744A, and C798A) that are known to decrease the pH sensitivity also decrease the Zn2+ sensitivity, and concentrations of spermine that relieve tonic proton inhibition also relieve Zn2+ inhibition. In summary, our results define the subunit composition of Zn2+-sensitive NMDA receptors and provide evidence for structural convergence of three allosteric regulators of receptor function: protons, polyamines, and Zn2+.
Project description:Eukaryotic ionotropic glutamate receptor subunits possess a large N-terminal domain (NTD) distinct from the neighboring agonist-binding domain. In NMDA receptors, the NTDs of NR2A and NR2B form modulatory domains binding allosteric inhibitors. Despite a high sequence homology, these two domains have been shown to bind two ligands of strikingly different chemical nature. Whereas the NTD of NR2A binds zinc with high (nanomolar) affinity, the NTD of NR2B binds the synthetic neuroprotectant ifenprodil and its derivatives. Using both NTD-mutated/deleted receptors and isolated NTDs, we now show that the NTD of NR2B, in contrast to NR2C and NR2D, also binds zinc, but with a lower affinity. Furthermore, we present evidence that zinc and ifenprodil compete for an overlapping binding site. This modulatory binding site accounts for the submicromolar zinc inhibition of NR1/NR2B receptors. Given that zinc is accumulated and released at many glutamatergic synapses in the CNS, these findings suggest that zinc is the endogenous ligand of the NTD of both NR2A and NR2B, the two major NR2 subunits. Thus, NMDA receptors contain zinc sensors capable of detecting extracellular zinc over a wide concentration range depending on their NR2 subunit composition. The coexistence of subunit-specific zinc-binding sites of high (nanomolar) and low (micromolar) affinity on NMDA receptors raises the possibility that zinc exerts both a tonic and a phasic control of membrane excitability.