Palliative care reduces emergency room visits and total hospital days among patients with metastatic HPB and GI cancers.

Palliative care services (PCS) have improved quality of life for patients across various cancer subtypes. Minimal data exists regarding PCSfor metastatic hepatopancreaticobiliary (HPB) and gastrointestinal (GI) cancers. We assessed the impact of PCS on emergency department visits, hospital admissions, and survival among these patients. Patients with metastatic HPB and GI cancer referred to outpatient PCS between 2014 and 2018 at a single institution were included. We compared the demographics, outcomes, and end-of-life indicators between those who did and did not receive PCS. The study included 183 patients, with 118 (64.5%) having received PCS. There were no significant differences in age, gender, race, marital status, or insurance. Those receiving PCS were more likely to have colorectal cancer (p = 0.0082) and receive chemotherapy (p = 0.0098). On multivariate analysis, PCS was associated with fewer ED visits (p = 0.0319), hospital admissions (p = 0.0002), and total inpatient hospital days (p < 0.0001) per 30 days of life. Overall survival was greater among patients receiving PCS (HR: 0.65 (0.46-0.92)). Outpatient PCS for patients with metastatic HPB and GI cancer is associated with fewer emergency department visits, hospital admissions, and inpatient hospital days, and improved overall survival.

A structurally derived model of subunit-dependent NMDA receptor function.

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.

An Atypical Case of Bartonella henselae Osteomyelitis and Hepatic Disease.

Bartonella henselae is a Gram-negative bacterium and the causative agent of cat scratch disease (CSD). Atypical presentations of B. henselae that involve the musculoskeletal, hepatosplenic, cardiac, or neurologic systems are rare. In this case report, we describe a case of B. henselae osteomyelitis involving bilateral iliac bones complicated by hepatic lesions in a 12-year-old immunocompetent female patient. Although B. henselae is a rare cause of osteomyelitis, it should be considered when patients who present with fever, pain, and lymphadenopathy do not respond to routine osteomyelitis therapy.

A Novel Negative Allosteric Modulator Selective for GluN2C/2D-Containing NMDA Receptors Inhibits Synaptic Transmission in Hippocampal Interneurons.

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in numerous neurological disorders. NMDARs typically comprise two GluN1 and two GluN2 subunits. The four GluN2 subtypes (GluN2A-GluN2D) have distinct functional properties and gene expression patterns, which contribute to diverse functional roles for NMDARs in the brain. Here, we present a series of GluN2C/2D-selective negative allosteric modulators built around a N-aryl benzamide (NAB) core. The prototypical compound, NAB-14, is >800-fold selective for recombinant GluN2C/GluN2D over GluN2A/GluN2B in Xenopus oocytes and has an IC50 value of 580 nM at recombinant GluN2D-containing receptors expressed in mammalian cells. NAB-14 inhibits triheteromeric (GluN1/GluN2A/GluN2C) NMDARs with modestly reduced potency and efficacy compared to diheteromeric (GluN1/GluN2C/GluN2C) receptors. Site-directed mutagenesis suggests that structural determinants for NAB-14 inhibition reside in the GluN2D M1 transmembrane helix. NAB-14 inhibits GluN2D-mediated synaptic currents in rat subthalamic neurons and mouse hippocampal interneurons, but has no effect on synaptic transmission in hippocampal pyramidal neurons, which do not express GluN2C or GluN2D. This series possesses some druglike physical properties and modest brain permeability in rat and mouse. Altogether, this work identifies a new series of negative allosteric modulators that are valuable tools for studying GluN2C- and GluN2D-containing NMDAR function in brain circuits, and suggests that the series has the potential to be developed into therapies for selectively modulating brain circuits involving the GluN2C and GluN2D subunits.

NMDA Receptors Containing the GluN2D Subunit Control Neuronal Function in the Subthalamic Nucleus.

The GluN2D subunit of the NMDA receptor is prominently expressed in the basal ganglia and associated brainstem nuclei, including the subthalamic nucleus (STN), globus pallidus, striatum, and substantia nigra. However, little is known about how GluN2D-containing NMDA receptors contribute to synaptic activity in these regions. Using Western blotting of STN tissue punches, we demonstrated that GluN2D is expressed in the rat STN throughout development [age postnatal day 7 (P7)-P60] and in the adult (age P120). Immunoelectron microscopy of the adult rat brain showed that GluN2D is predominantly expressed in dendrites, unmyelinated axons, and axon terminals within the STN. Using subunit-selective allosteric modulators of NMDA receptors (TCN-201, ifenprodil, CIQ, and DQP-1105), we provide evidence that receptors containing the GluN2B and GluN2D subunits mediate responses to exogenously applied NMDA and glycine, as well as synaptic NMDA receptor activation in the STN of rat brain slices. EPSCs in the STN were mediated primarily by AMPA and NMDA receptors and GluN2D-containing NMDA receptors controlled the slow deactivation time course of EPSCs in the STN. In vivo recordings from the STN of anesthetized adult rats demonstrated that the spike firing rate was increased by the GluN2C/D potentiator CIQ and decreased by the GluN2C/D antagonist DQP-1105, suggesting that NMDA receptor activity can influence STN output. These data indicate that the GluN2B and GluN2D NMDA receptor subunits contribute to synaptic activity in the STN and may represent potential therapeutic targets for modulating subthalamic neuron activity in neurological disorders such as Parkinson's disease.

PAR1-activated astrocytes in the nucleus of the solitary tract stimulate adjacent neurons via NMDA receptors.

Severe autonomic dysfunction, including the loss of control of the cardiovascular, respiratory, and gastrointestinal systems, is a common comorbidity of stroke and other bleeding head injuries. Previous studies suggest that this collapse of autonomic control may be caused by thrombin acting on astrocytic protease-activated receptors (PAR1) in the hindbrain. Using calcium imaging and electrophysiological techniques, we evaluated the mechanisms by which astrocytic PAR1s modulate the activity of presynaptic vagal afferent terminals and postsynaptic neurons in the rat nucleus of the solitary tract (NST). Our calcium-imaging data show that astrocytic and neuronal calcium levels increase after brain slices are treated with the PAR1 agonist SFLLRN-NH2. This increase in activity is blocked by pretreating the slices with the glial metabolic blocker fluorocitrate. In addition, PAR1-activated astrocytes communicate directly with NST neurons by releasing glutamate. Calcium responses to SFLLRN-NH2 in the astrocytes and neurons significantly increase after bath application of the excitatory amino acid transporter blocker DL-threo-ß-benzyloxyaspartic acid (TBOA) and significantly decrease after bath application of the NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (DL-AP5). Furthermore, astrocytic glutamate activates neuronal GluN2B-containing NMDA receptors. Voltage-clamp recordings of miniature EPSCs (mEPSCs) from NST neurons show that astrocytes control presynaptic vagal afferent excitability directly under resting and activated conditions. Fluorocitrate significantly decreases mEPSC frequency and SFLLRN-NH2 significantly increases mEPSC frequency. These data show that astrocytes act within a tripartite synapse in the NST, controlling the excitability of both postsynaptic NST neurons and presynaptic vagal afferent terminals.

NMDA receptors control vagal afferent excitability in the nucleus of the solitary tract.

Previous behavioral studies have demonstrated that presynaptic N-methyl-d-aspartate (NMDA) receptors expressed on vagal afferent terminals are involved in food intake and satiety. Therefore, using in vitro live cell calcium imaging of prelabeled rat hindbrain slices, we characterized which NMDA receptor GluN2 subunits may regulate vagal afferent activity. The nonselective NMDA receptor antagonist d,l-2-amino-5-phosphonopentanoic acid (d,l-AP5) significantly inhibited vagal terminal calcium influx, while the excitatory amino acid reuptake inhibitor d,l-threo-ß-benzyloxyaspartic acid (TBOA), significantly increased terminal calcium levels following pharmacological stimulation with ATP. Subunit-specific NMDA receptor antagonists and potentiators were used to identify which GluN2 subunits mediate the NMDA receptor response on the vagal afferent terminals. The GluN2B-selective antagonist, ifenprodil, selectively reduced vagal calcium influx with stimulation compared to the time control. The GluN2A-selective antagonist, 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl] benzyl]benzenesulfonamide (TCN 201) produced smaller but not statistically significant effects. Furthermore, the GluN2A/B-selective potentiator (pregnenolone sulfate) and the GluN2C/D-selective potentiator [(3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone; (CIQ)] enhanced vagal afferent calcium influx during stimulation. These data suggest that presynaptic NMDA receptors with GluN2B, GluN2C, and GluN2D subunits may predominantly control vagal afferent excitability in the nucleus of the solitary tract.

Artemisia santolinifolia enhances glutamatergic neurotransmission in the nucleus of the solitary tract.

Artemisia extracts have been used as remedies for a variety of maladies related to metabolic and gastrointestinal control. Because the vagal afferent-nucleus of the solitary tract (NST) synapse regulates the same homeostatic functions affected by Artemisia, it is possible that these extracts may have activity at the synaptic level in the NST. Therefore, we evaluated how extracts of three common medicinal Artemisia species, Artemisia santolinifolia (SANT), Artemisia scoparia (SCO), and Artemisia dracunculus L (PMI-5011), modulate the excitability of the glutamatergic vagal afferent-NST synapse. Our in vitro live cell calcium imaging data from prelabeled vagal afferent terminals show that SANT extract is a positive modulator of vagal afferent calcium levels, as the extract significantly increased the calcium signal relative to the time control. Neither SCO nor PMI-5011 extract altered the vagal calcium signals compared to the time control. Furthermore, whole cell voltage-clamp recordings from NST neurons corroborated the vagal terminal calcium data in that SANT extract also significantly increased miniature excitatory postsynaptic current (mEPSC) frequency in NST neurons. These data suggest that SANT extract could be a pharmacologically significant mediator of glutamatergic neurotransmission within the CNS.

St. John's Wort enhances the synaptic activity of the nucleus of the solitary tract.

OBJECTIVE: St. John's Wort (SJW) extract, which is commonly used to treat depression, inhibits the reuptake of several neurotransmitters, including glutamate, serotonin, norepinephrine, and dopamine. Glutamatergic visceral vagal afferents synapse upon neurons of the solitary tract (NST); thus, the aim of this study was to evaluate whether SJW extract modulates glutamatergic neurotransmission within the NST. METHODS: We used live cell calcium imaging to evaluate whether SJW and its isolated components hypericin and hyperforin increase the excitability of prelabeled vagal afferent terminals synapsing upon the NST. We used voltage-clamp recordings of spontaneous miniature excitatory postsynaptic currents (mEPSCs) to evaluate whether SJW alters glutamate release from vagal afferents onto NST neurons. RESULTS: Our imaging data show that SJW (50 µg/mL) increased the intracellular calcium levels of stimulated vagal afferent terminals compared with the bath control. This increase in presynaptic vagal afferent calcium by the extract coincides with an increase in neurotransmitter release within the nucleus of the solitary tract, as the frequency of mEPSCs is significantly higher in the presence of the extract compared with the control. Finally, our imaging data show that hyperforin, a known component of SJW extract, also significantly increases terminal calcium levels. CONCLUSION: These data suggest that SJW extract can significantly increase the probability of glutamate release from vagal afferents onto the NST by increasing presynaptic calcium. The in vitro vagal afferent synapse with NST neurons is an ideal model system to examine the mechanism of action of botanical agents on glutamatergic neurotransmission.

Structure-activity relationships and pharmacophore model of a noncompetitive pyrazoline containing class of GluN2C/GluN2D selective antagonists.

Here we describe the synthesis and structure-activity relationship for a class of pyrazoline-containing dihydroquinolone negative allosteric modulators of the NMDA receptor that show strong subunit selectivity for GluN2C- and GluN2D-containing receptors over GluN2A- and GluN2B-containing receptors. Several members of this class inhibit NMDA receptor responses in the nanomolar range and are more than 50-fold selective over GluN1/GluN2A and GluN1/GluN2B NMDA receptors, as well as AMPA, kainate, GABA, glycine, nicotinic, serotonin, and purinergic receptors. Analysis of the purified enantiomers of one of the more potent and selective compounds shows that the S-enantiomer is both more potent and more selective than the R-enantiomer. The S-enantiomer had an IC50 of 0.17-0.22 µM at GluN2D- and GluN2C-containing receptors, respectively, and showed over 70-fold selectivity over other NMDA receptor subunits. The subunit selectivity of this class of compounds should be useful in defining the role of GluN2C- and GluN2D-containing receptors in specific brain circuits in both physiological and pathophysiological conditions.

Modal gating of GluN1/GluN2D NMDA receptors.

GluN2D-containing NMDA receptors are characterized by an unusually low open probability (0.023), even in the presence of saturating glutamate and glycine. Here, we show that recombinant GluN1/GluN2D NMDA receptors can enter brief periods with exceptionally high open probability (0.65) in excised outside-out and cell-attached single channel recordings. GluN1/GluN2D channels during the enhanced gating mode have similar open durations as occurs outside of the high open probability burst of activity. However, the periods in the high gating mode only exhibit 4 brief closed duration exponential components similar to the briefest observed for openings outside the burst. GluN1/GluN2D receptors also open to a more prominent subconductance level compared to activity outside the high open probability burst. Evaluation of a five-state NMDA receptor gating model suggests that both the opening and closing rate constants differ for the periods of higher open probability compared to the high open probability arm of a gating model previously published for GluN1/GluN2D fit to a representative full length single channel recording. These data demonstrate that GluN2D-containing NMDA receptors can enter a conformation or mode that allows the pore to gate with high probability.

Structural determinants of agonist efficacy at the glutamate binding site of N-methyl-D-aspartate receptors.

N-methyl-d-aspartate (NMDA) receptors are ligand-gated ion channels assembled from GluN1 and GluN2 subunits. We used a series of N-hydroxypyrazole-5-glycine (NHP5G) partial agonists at the GluN2 glutamate binding site as tools to study activation of GluN1/GluN2A and GluN1/GluN2D NMDA receptor subtypes. Using two-electrode voltage-clamp electrophysiology, fast-application patch-clamp, and single-channel recordings, we show that propyl- and ethyl-substituted NHP5G agonists have a broad range of agonist efficacies relative to the full agonist glutamate (<1-72%). Crystal structures of the agonist binding domains (ABDs) of GluN2A and GluN2D do not reveal any differences in the overall domain conformation induced by binding of the full agonist glutamate or the partial agonist propyl-NHP5G, which is strikingly different from ABD structures of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoate (AMPA) and kainate receptors bound to full and partial agonists. Subsequent evaluation of relative NHP5G agonist efficacy at GluN2A-GluN2D chimeric subunits implicates the amino-terminal domain (ATD) as a strong determinant of agonist efficacy, suggesting that interdomain interactions between the ABD and the ATD may be a central element in controlling the manner by which agonist binding leads to channel opening. We propose that variation in the overall receptor conformation, which is strongly influenced by the nature of interdomain interactions in resting and active states, mediates differences in agonist efficacy and partial agonism at the GluN2 subunits.

GluN1 splice variant control of GluN1/GluN2D NMDA receptors.

NMDA receptors are ionotropic glutamate receptors that mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system. Recombinant GluN1-1a/GluN2D receptors are characterized by low channel open probability and prolonged deactivation time course following the removal of agonist. Here, we show that the deactivation time course, agonist potency, and single channel properties of GluN2D-containing NMDA receptors are modulated by alternative RNA splicing of GluN1. Our results demonstrate that GluN1 exon 5, which encodes a 21-amino-acid insert in the amino-terminal domain, is a key determinant of GluN1/GluN2D receptor function. GluN1-1b/GluN2D receptors, which contain the residues encoded by exon 5, deactivate with a dual exponential time course described by a τFAST of 410 ms and a τSLOW of 1100 ms. This time course is 3-fold more rapid than that for exon 5-lacking GluN1-1a/GluN2D, which deactivates with a τFAST of 1100 ms and a τSLOW of 3400 ms. Exon 5-containing NMDA receptors also have a two-fold higher open probability (0.037) than exon 5-lacking receptors (0.017). Furthermore, inclusion of exon 5-encoded residues within the GluN1-1b subunit decreases the potency for the endogenous agonist l-glutamate. Evaluation of receptor kinetics for NMDA receptors containing mutated GluN1-1b subunits and wild-type GluN2D identified residue Lys211 in GluN1-1b as a key determinant of exon 5 control of the deactivation time course and glutamate potency. Evaluation of a kinetic model of GluN1/GluN2D gating suggests that residues encoded by exon 5 influence several rate-limiting steps. These data demonstrate that the GluN1 subunit is a key determinant of the kinetic and pharmacological properties of GluN2D-containing NMDA receptors.

Mechanism for noncompetitive inhibition by novel GluN2C/D N-methyl-D-aspartate receptor subunit-selective modulators.

The compound 4-(5-(4-bromophenyl)-3-(6-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-oxobutanoic acid (DQP-1105) is a representative member of a new class of N-methyl-d-aspartate (NMDA) receptor antagonists. DQP-1105 inhibited GluN2C- and GluN2D-containing receptors with IC(50) values that were at least 50-fold lower than those for recombinant GluN2A-, GluN2B-, GluA1-, or GluK2-containing receptors. Inhibition was voltage-independent and could not be surmounted by increasing concentrations of either coagonist, glutamate or glycine, consistent with a noncompetitive mechanism of action. DQP-1105 inhibited single-channel currents in excised outside-out patches without significantly changing mean open time or single-channel conductance, suggesting that DQP inhibits a pregating step without changing the stability of the open pore conformation and thus channel closing rate. Evaluation of DQP-1105 inhibition of chimeric NMDA receptors identified two key residues in the lower lobe of the GluN2 agonist binding domain that control the selectivity of DQP-1105. These data suggest a mechanism for this new class of inhibitors and demonstrate that ligands can access, in a subunit-selective manner, a new site located in the lower, membrane-proximal portion of the agonist-binding domain.

Ligand-specific deactivation time course of GluN1/GluN2D NMDA receptors.

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors that mediate a majority of excitatory synaptic transmission. One unique property of GluN1/GluN2D NMDA receptors is an unusually prolonged deactivation time course following the removal of L-glutamate. Here we show, using x-ray crystallography and electrophysiology, that the deactivation time course of GluN1/GluN2D receptors is influenced by the conformational variability of the ligand-binding domain (LBD) as well as the structure of the activating ligand. L-glutamate and L-CCG-IV induce significantly slower deactivation time courses compared with other agonists. Crystal structures of the isolated GluN2D LBD in complex with various ligands reveal that the binding of L-glutamate induces a unique conformation at the backside of the ligand-binding site in proximity to the region at which the transmembrane domain would be located in the intact receptors. These data suggest that the activity of the GluN1/GluN2D NMDA receptor is controlled distinctively by the endogenous neurotransmitter L-glutamate.

A subunit-selective potentiator of NR2C- and NR2D-containing NMDA receptors.

NMDA receptors are tetrameric complexes of NR1 and NR2A-D subunits that mediate excitatory synaptic transmission and have a role in neurological disorders. In this article, we identify a novel subunit-selective potentiator of NMDA receptors containing the NR2C or NR2D subunit, which could allow selective modification of circuit function in regions expressing NR2C/D subunits. The substituted tetrahydroisoquinoline CIQ (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone) enhances receptor responses two-fold with an EC(50) of 3 µM by increasing channel opening frequency without altering mean open time or EC(50) values for glutamate or glycine. The actions of CIQ depend on a single residue in the M1 region (NR2D Thr592) and on the linker between the N-terminal domain and agonist binding domain. CIQ potentiates native NR2D-containing NMDA receptor currents from subthalamic neurons. Our identification of a subunit-selective NMDA receptor modulator reveals a new class of pharmacological tools with which to probe the role of NR2C- and NR2D-containing NMDA receptors in brain function and disease.

Glutamate receptor ion channels: structure, regulation, and function.

The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors.

Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.

Control of NMDA receptor function by the NR2 subunit amino-terminal domain.

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.

The serine protease plasmin cleaves the amino-terminal domain of the NR2A subunit to relieve zinc inhibition of the N-methyl-D-aspartate receptors.

Zinc is hypothesized to be co-released with glutamate at synapses of the central nervous system. Zinc binds to NR1/NR2A N-methyl-d-aspartate (NMDA) receptors with high affinity and inhibits NMDAR function in a voltage-independent manner. The serine protease plasmin can cleave a number of substrates, including protease-activated receptors, and may play an important role in several disorders of the central nervous system, including ischemia and spinal cord injury. Here, we demonstrate that plasmin can cleave the native NR2A amino-terminal domain (NR2A(ATD)), removing the functional high affinity Zn(2+) binding site. Plasmin also cleaves recombinant NR2A(ATD) at lysine 317 (Lys(317)), thereby producing a approximately 40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments observed in rat brain membrane preparations. A homology model of the NR2A(ATD) predicts that Lys(317) is near the surface of the protein and is accessible to plasmin. Recombinant expression of NR2A with an amino-terminal deletion at Lys(317) is functional and Zn(2+) insensitive. Whole cell voltage-clamp recordings show that Zn(2+) inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons is significantly reduced by plasmin treatment. Mutating the plasmin cleavage site Lys(317) on NR2A to alanine blocks the effect of plasmin on Zn(2+) inhibition. The relief of Zn(2+) inhibition by plasmin occurs in PAR1(-/-) cortical neurons and thus is independent of interaction with protease-activated receptors. These results suggest that plasmin can directly interact with NMDA receptors, and plasmin may increase NMDA receptor responses through disruption or removal of the amino-terminal domain and relief of Zn(2+) inhibition.