Publications by authors named "Jon W Johnson"

39 Publications

Interplay between Gating and Block of Ligand-Gated Ion Channels.

Brain Sci 2020 Dec 1;10(12). Epub 2020 Dec 1.

Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Drugs that inhibit ion channel function by binding in the channel and preventing current flow, known as channel blockers, can be used as powerful tools for analysis of channel properties. Channel blockers are used to probe both the sophisticated structure and basic biophysical properties of ion channels. Gating, the mechanism that controls the opening and closing of ion channels, can be profoundly influenced by channel blocking drugs. Channel block and gating are reciprocally connected; gating controls access of channel blockers to their binding sites, and channel-blocking drugs can have profound and diverse effects on the rates of gating transitions and on the stability of channel open and closed states. This review synthesizes knowledge of the inherent intertwining of block and gating of excitatory ligand-gated ion channels, with a focus on the utility of channel blockers as analytic probes of ionotropic glutamate receptor channel function.
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http://dx.doi.org/10.3390/brainsci10120928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760600PMC
December 2020

Synaptic zinc inhibition of NMDA receptors depends on the association of GluN2A with the zinc transporter ZnT1.

Sci Adv 2020 Jul 3;6(27). Epub 2020 Jul 3.

Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

The NMDA receptor (NMDAR) is inhibited by synaptically released zinc. This inhibition is thought to be the result of zinc diffusion across the synaptic cleft and subsequent binding to the extracellular domain of the NMDAR. However, this model fails to incorporate the observed association of the highly zinc-sensitive NMDAR subunit GluN2A with the postsynaptic zinc transporter ZnT1, which moves intracellular zinc to the extracellular space. Here, we report that disruption of ZnT1-GluN2A association by a cell-permeant peptide strongly reduced NMDAR inhibition by synaptic zinc in mouse dorsal cochlear nucleus synapses. Moreover, synaptic zinc inhibition of NMDARs required postsynaptic intracellular zinc, suggesting that cytoplasmic zinc is transported by ZnT1 to the extracellular space in close proximity to the NMDAR. These results challenge a decades-old dogma on how zinc inhibits synaptic NMDARs and demonstrate that presynaptic release and a postsynaptic transporter organize zinc into distinct microdomains to modulate NMDAR neurotransmission.
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http://dx.doi.org/10.1126/sciadv.abb1515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458442PMC
July 2020

Oxygen-Glucose Deprivation Differentially Affects Neocortical Pyramidal Neurons and Parvalbumin-Positive Interneurons.

Neuroscience 2019 08 30;412:72-82. Epub 2019 May 30.

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, 15260, USA; Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA, 15213, USA.

Stroke is a devastating brain disorder. The pathophysiology of stroke is associated with an impaired excitation-inhibition balance in the area that surrounds the infarct core after the insult, the peri-infarct zone. Here we exposed slices from adult mouse prefrontal cortex to oxygen-glucose deprivation and reoxygenation (OGD-RO) to study ischemia-induced changes in the activity of excitatory pyramidal neurons and inhibitory parvalbumin (PV)-positive interneurons. We found that during current-clamp recordings, PV-positive interneurons were more vulnerable to OGD-RO than pyramidal neurons as indicated by the lower percentage of recovery of PV-positive interneurons. However, neither the amplitude of OGD-induced depolarization observed in current-clamp mode nor the OGD-associated current observed in voltage-clamp mode differed between the two cell types. Large amplitude, presumably action-potential dependent, spontaneous postsynaptic inhibitory currents recorded from pyramidal neurons were less frequent after OGD-RO than in control condition. Disynaptic inhibitory postsynaptic currents (dIPSCs) in pyramidal neurons produced predominantly by PV-positive interneurons were reduced by OGD-RO. Following OGD-RO, dendrites of PV-positive interneurons exhibited more pathological beading than those of pyramidal neurons. Our data support the hypothesis that the differential vulnerability to ischemia-like conditions of excitatory and inhibitory neurons leads to the altered excitation-inhibition balance associated with stroke pathophysiology.
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http://dx.doi.org/10.1016/j.neuroscience.2019.05.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818263PMC
August 2019

Effects of Mg on recovery of NMDA receptors from inhibition by memantine and ketamine reveal properties of a second site.

Neuropharmacology 2018 07 12;137:344-358. Epub 2018 May 12.

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA; Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15213, USA. Electronic address:

Memantine and ketamine are NMDA receptor (NMDAR) open channel blockers that are thought to act via similar mechanisms at NMDARs, but exhibit divergent clinical effects. Both drugs act by entering open NMDARs and binding at a site deep within the ion channel (the deep site) at which the endogenous NMDAR channel blocker Mg also binds. Under physiological conditions, Mg increases the ICs of memantine and ketamine through competition for binding at the deep site. Memantine also can inhibit NMDARs after associating with a second site accessible in the absence of agonist, a process termed second site inhibition (SSI) that is not observed with ketamine. Here we investigated the effects of 1 mM Mg on recovery from inhibition by memantine and ketamine, and on memantine SSI, of the four main diheteromeric NMDAR subtypes. We found that: recovery from memantine inhibition depended strongly on the concentration of memantine used to inhibit the NMDAR response; Mg accelerated recovery from memantine and ketamine inhibition through distinct mechanisms and in an NMDAR subtype-dependent manner; and Mg occupation of the deep site disrupted memantine SSI in a subtype-dependent manner. Our results support the hypothesis that memantine associates with, but does not inhibit at the second site. After associating with the second site, memantine can either slowly dissociate directly to the extracellular solution, or transit to the deep site, resulting in typical channel block. Memantine's relatively slow dissociation from the second site underlies the dependence of NMDAR recovery from inhibition on both memantine concentration and on Mg.
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http://dx.doi.org/10.1016/j.neuropharm.2018.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050087PMC
July 2018

Pharmacological and Electrophysiological Characterization of Novel NMDA Receptor Antagonists.

ACS Chem Neurosci 2018 11 1;9(11):2722-2730. Epub 2018 Jun 1.

Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia i Ciències de l'Alimentació i Institut de Biomedicina (IBUB) , Universitat de Barcelona , Av. Joan XXIII, 27-31 , 08028 Barcelona , Spain.

This work reports the synthesis and pharmacological and electrophysiological evaluation of new N-methyl-d-aspartic acid receptor (NMDAR) channel blocking antagonists featuring polycyclic scaffolds. Changes in the chemical structure modulate the potency and voltage dependence of inhibition. Two of the new antagonists display properties comparable to those of memantine, a clinically approved NMDAR antagonist.
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http://dx.doi.org/10.1021/acschemneuro.8b00154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249113PMC
November 2018

New Cav2 calcium channel gating modifiers with agonist activity and therapeutic potential to treat neuromuscular disease.

Neuropharmacology 2018 03 12;131:176-189. Epub 2017 Dec 12.

Department of Neuroscience, Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, United States. Electronic address:

Voltage-gated calcium channels (VGCCs) are critical regulators of many cellular functions, including the activity-dependent release of chemical neurotransmitter from nerve terminals. At nerve terminals, the Cav2 family of VGCCs are closely positioned with neurotransmitter-containing synaptic vesicles. The relationship between calcium ions and transmitter release is such that even subtle changes in calcium flux through VGCCs have a strong influence on the magnitude of transmitter released. Therefore, modulators of the calcium influx at nerve terminals have the potential to strongly affect transmitter release at synapses. We have previously developed novel Cav2-selective VGCC gating modifiers (notably GV-58) that slow the deactivation of VGCC current, increasing total calcium ion flux. Here, we describe ten new gating modifiers based on the GV-58 structure that extend our understanding of the structure-activity relationship for this class of molecules and extend the range of modulation of channel activities. In particular, we show that one of these new compounds (MF-06) was more efficacious than GV-58, another (KK-75) acts more quickly on VGCCs than GV-58, and a third (KK-20) has a mix of increased speed and efficacy. A subset of these new VGCC agonist gating modifiers can increase transmitter release during action potentials at neuromuscular synapses, and as such, show potential as therapeutics for diseases with a presynaptic deficit that results in neuromuscular weakness. Further, several of these new compounds can be useful tool compounds for the study of VGCC gating and function.
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http://dx.doi.org/10.1016/j.neuropharm.2017.12.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820137PMC
March 2018

A versatile optical tool for studying synaptic GABA receptor trafficking.

J Cell Sci 2017 Nov 12;130(22):3933-3945. Epub 2017 Oct 12.

Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2FAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2FAP GABARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2FAP GABARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2FAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2FAP-MG dye approach reveals enhanced GABAR turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABAR trafficking.
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http://dx.doi.org/10.1242/jcs.205286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702044PMC
November 2017

Low-Density Neuronal Cultures from Human Induced Pluripotent Stem Cells.

Mol Neuropsychiatry 2017 Jul 17;3(1):28-36. Epub 2017 Jun 17.

Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented possibility to investigate defects occurring during neuronal differentiation in neuropsychiatric and neurodevelopmental disorders, but the density and intricacy of intercellular connections in neuronal cultures challenge currently available analytic methods. Low-density neuronal cultures facilitate the morphometric and functional analysis of neurons. We describe a differentiation protocol to generate low-density neuronal cultures (∼2,500 neurons/cm) from human iPSC-derived neural stem cells/early neural progenitor cells. We generated low-density cultures using cells from 3 individuals. We also evaluated the morphometric features of neurons derived from 2 of these individuals, one harboring a microdeletion on chromosome 15q11.2 and the other without the microdeletion. An approximately 7.5-fold increase in the density of dendritic filopodia was observed in the neurons with the microdeletion, consistent with previous reports. Low-density neuronal cultures enable facile and unbiased comparisons of iPSC-derived neurons from different individuals or clones.
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http://dx.doi.org/10.1159/000476034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582512PMC
July 2017

Memantine and Ketamine Differentially Alter NMDA Receptor Desensitization.

J Neurosci 2017 10 6;37(40):9686-9704. Epub 2017 Sep 6.

Department of Neuroscience and Center for Neuroscience and

Memantine and ketamine are clinically useful NMDA receptor (NMDAR) open channel blockers that inhibit NMDARs with similar potency and kinetics, but display vastly different clinical profiles. This discrepancy has been hypothesized to result from inhibition by memantine and ketamine of overlapping but distinct NMDAR subpopulations. For example, memantine but not ketamine may inhibit extrasynaptic NMDARs more effectively than synaptic NMDARs. However, the basis for preferential NMDAR inhibition depending on subcellular location has not been investigated systematically. We integrated recordings from heterologously expressed single NMDAR subtypes, kinetic modeling, and recordings of synaptically evoked NMDAR responses in acute brain slices to investigate mechanisms by which channel blockers may distinguish NMDAR subpopulations. We found that memantine and ketamine differentially alter NMDAR desensitization and that memantine stabilizes a Ca-dependent desensitized state. As a result, inhibition by memantine of GluN1/2A receptors in tsA201 cells and of native synaptic NMDARs in cortical pyramidal neurons from mice of either sex increased in conditions that enhanced intracellular Ca accumulation. Therefore, differential inhibition by memantine and ketamine based on NMDAR location is likely to result from location dependence of the intensity and duration of NMDAR activation. Modulation of Ca-dependent NMDAR desensitization is an unexplored mechanism of inhibitory action with the potential to endow drugs with NMDAR selectivity that leads to superior clinical profiles. Our results suggest that designing compounds to target specific receptor states, rather than specific receptor types, may be a viable strategy for future drug development. Memantine and ketamine are NMDA receptor (NMDAR) channel-blocking drugs with divergent clinical effects. Understanding mechanistically their differential actions may advance our understanding of nervous system disorders and suggest strategies for the design of more effective drugs. Here, we show that memantine and ketamine have contrasting effects on NMDAR desensitization. Ketamine binding decreases occupancy of desensitized states of the GluN1/2B NMDAR subtype. In contrast, memantine binding increases occupancy of GluN1/2A and native NMDAR desensitized states entered after accumulation of intracellular Ca, a novel inhibitory mechanism. These properties may contribute to inhibition of distinct NMDAR subpopulations by memantine and ketamine and help to explain their differential clinical effects. Our results suggest stabilization of Ca-dependent desensitized states as a new strategy for pharmaceutical neuroprotection.
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http://dx.doi.org/10.1523/JNEUROSCI.1173-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628409PMC
October 2017

Glial and Neuronal Glutamate Transporters Differ in the Na Requirements for Activation of the Substrate-Independent Anion Conductance.

Front Mol Neurosci 2017 29;10:150. Epub 2017 May 29.

Laboratory of Cellular and Molecular Neurobiology, National Institute of Mental Health, National Institutes of HealthBethesda, MD, United States.

Excitatory amino acid transporters (EAATs) are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na and substrate binding. The activation of the anion channel by binding of Na alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3) and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na-dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or "leak" channel activity. Choline or N-methyl-D-glucamine replacement of external Na ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na-independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system.
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http://dx.doi.org/10.3389/fnmol.2017.00150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447070PMC
May 2017

All atom NMDA receptor transmembrane domain model development and simulations in lipid bilayers and water.

PLoS One 2017 5;12(6):e0177686. Epub 2017 Jun 5.

Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

N-methyl-d-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family that mediate excitatory synaptic transmission in the central nervous system. The channels of NMDARs are permeable to Ca2+ but blocked by Mg2+, distinctive properties that underlie essential brain processes such as induction of synaptic plasticity. However, due to limited structural information about the NMDAR transmembrane ion channel forming domain, the mechanism of divalent cation permeation and block is understood poorly. In this paper we developed an atomistic model of the transmembrane domain (TMD) of NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors). The model was generated using (a) a homology model based on the structure of the NaK channel and a partially resolved structure of an AMPA receptor (AMPAR), and (b) a partially resolved X-ray structure of GluN1/2B NMDARs. Refinement and extensive Molecular Dynamics (MD) simulations of the NMDAR TMD model were performed in explicit lipid bilayer membrane and water. Targeted MD with simulated annealing was introduced to promote structure refinement. Putative positions of the Mg2+ and Ca2+ ions in the ion channel divalent cation binding site are proposed. Differences in the structural and dynamic behavior of the channel protein in the presence of Mg2+ or Ca2+ are analyzed. NMDAR protein conformational flexibility was similar with no ion bound to the divalent cation binding site and with Ca2+ bound, whereas Mg2+ binding reduced protein fluctuations. While bound at the binding site both ions retained their preferred ligand coordination numbers: 6 for Mg2+, and 7-8 for Ca2+. Four asparagine side chain oxygens, a back-bone oxygen, and a water molecule participated in binding a Mg2+ ion. The Ca2+ ion first coordination shell ligands typically included four to five side-chain oxygen atoms of the binding site asparagine residues, two water molecules and zero to two backbone oxygens of the GluN2B subunits. These results demonstrate the importance of high-resolution channel structures for elucidation of mechanisms of NMDAR permeation and block.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177686PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459333PMC
September 2017

The Role of GluN2C-Containing NMDA Receptors in Ketamine's Psychotogenic Action and in Schizophrenia Models.

J Neurosci 2016 11;36(44):11151-11157

Department of Biology, Brandeis University, Waltham, Massachusetts 02454,

The NMDA receptor (NMDAR) hypofunction hypothesis of schizophrenia is supported by multiple lines of evidence. Notably, administration of the NMDAR antagonist, ketamine, to healthy human subjects has psychotogenic action, producing both positive and negative symptoms associated with schizophrenia. NMDARs have multiple subtypes, but the subtypes through which ketamine produces its psychotogenic effects are not known. Here we address this question using quantitative data that characterize ketamine's ability to block different NMDAR subtypes. Our calculations indicate that, at a concentration that has psychotogenic action in humans, ketamine blocks a substantial fraction of GluN2C subunit-containing receptors but has less effect on GluN2A-, GluN2B-, and GluN2D-containing receptors. Thus, GluN2C-containing receptors may have preferential involvement in psychotic states produced by ketamine. A separate line of experiments also points to a special role for GluN2C. That work demonstrates the ability of NMDAR antagonists to mimic the elevation in the awake-state δ frequency EEG power that occurs in schizophrenia. Physiological experiments in rodents show that NMDAR antagonists generate δ oscillations by their action on the GluN2C-containing NMDARs that are prevalent in the thalamus. Optogenetic experiments suggest that such oscillations could contribute to symptoms of schizophrenia.
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http://dx.doi.org/10.1523/JNEUROSCI.1203-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148234PMC
November 2016

Effects of memantine on the excitation-inhibition balance in prefrontal cortex.

Neurobiol Dis 2016 Dec 18;96:75-83. Epub 2016 Aug 18.

Department of Neuroscience, and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, United States; Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15260, United States. Electronic address:

Memantine is one of the few drugs currently approved for treatment of Alzheimer's disease (AD). The clinical effects of memantine are thought to be associated with inhibition of NMDA receptors (NMDARs). Surprisingly, other open-channel NMDAR blockers have unacceptable side effects that prevent their consideration for AD treatment. One of the mechanisms proposed to explain the therapeutic benefits of memantine involves preferential decrease of excitatory drive to inhibitory neurons in the cortical circuitry and consequent changes in balance between excitation and inhibition (E/I). In this study we addressed effects of memantine on E/I balance in the prefrontal cortex (PFC). We found that a moderate concentration of memantine shifted E/I balance away from inhibition in the PFC circuitry. Indeed, memantine decreased the frequency and amplitude of spontaneous inhibitory postsynaptic currents in pyramidal neurons while leaving spontaneous excitatory postsynaptic currents unaffected. These circuitry effects of memantine were occluded by the competitive NMDAR inhibitor AP-5, and thus are associated with NMDAR inhibition. We also found that memantine decreased feed-forward disynaptic inhibitory input to pyramidal neurons, which is thought to be mediated by parvalbumin (PV)-positive interneurons. Accordingly, memantine caused a greater decrease of the amplitude of NMDAR-mediated synaptic responses in PV-positive interneurons than in pyramidal neurons. Finally, memantine reduced firing activity in PV-positive interneurons while increasing firing in pyramidal neurons. This study elucidates a novel mechanism of action of memantine associated with shifting of the E/I balance away from inhibition in neocortical circuitry, and provides important insights for AD drug development.
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http://dx.doi.org/10.1016/j.nbd.2016.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102806PMC
December 2016

Lgr5⁺ amacrine cells possess regenerative potential in the retina of adult mice.

Aging Cell 2015 Aug 20;14(4):635-43. Epub 2015 May 20.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Current knowledge indicates that the adult mammalian retina lacks regenerative capacity. Here, we show that the adult stem cell marker, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), is expressed in the retina of adult mice. Lgr5(+) cells are generated at late stages of retinal development and exhibit properties of differentiated amacrine interneurons (amacrine cells). Nevertheless, Lgr5(+) amacrine cells contribute to regeneration of new retinal cells in the adult stage. The generation of new retinal cells, including retinal neurons and Müller glia from Lgr5(+) amacrine cells, begins in early adulthood and continues as the animal ages. Together, these findings suggest that the mammalian retina is not devoid of regeneration as previously thought. It is rather dynamic, and Lgr5(+) amacrine cells function as an endogenous regenerative source. The identification of such cells in the mammalian retina may provide new insights into neuronal regeneration and point to therapeutic opportunities for age-related retinal degenerative diseases.
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http://dx.doi.org/10.1111/acel.12346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531077PMC
August 2015

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

Organogenesis 2014 ;10(4):365-77

a Department of Psychiatry ; Western Psychiatric Institute and Clinic ; University of Pittsburgh School of Medicine ; Pittsburgh , PA USA.

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.
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http://dx.doi.org/10.1080/15476278.2015.1011921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594592PMC
October 2015

Molecular bases of NMDA receptor subtype-dependent properties.

J Physiol 2015 Jan 9;593(1):83-95. Epub 2014 Sep 9.

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.

NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell survival. Several features distinguish NMDARs from other iGluRs and underlie the crucial roles NMDARs play in nervous system physiology. NMDARs display slow deactivation kinetics, are highly Ca(2+) permeable, and require depolarization to relieve channel block by external Mg(2+) ions, thereby making them effective coincidence detectors. These properties and others differ among NMDAR subtypes, which are defined by the subunits that compose the receptor. NMDARs, which are heterotetrameric, commonly are composed of two GluN1 subunits and two GluN2 subunits, of which there are four types, GluN2A-D. 'Diheteromeric' NMDARs contain two identical GluN2 subunits. Gating and ligand-binding properties (e.g. deactivation kinetics) and channel properties (e.g. channel block by Mg(2+)) depend strongly on the GluN2 subunit contained in diheteromeric NMDARs. Recent work shows that two distinct regions of GluN2 subunits control most diheteromeric NMDAR subtype-dependent properties: the N-terminal domain is responsible for most subtype dependence of gating and ligand-binding properties; a single residue difference between GluN2 subunits at a site termed the GluN2 S/L site is responsible for most subtype dependence of channel properties. Thus, two structurally and functionally distinct regions underlie the majority of subtype dependence of NMDAR properties. This topical review highlights recent studies of recombinant diheteromeric NMDARs that uncovered the involvement of the N-terminal domain and of the GluN2 S/L site in the subtype dependence of NMDAR properties.
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http://dx.doi.org/10.1113/jphysiol.2014.273763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293056PMC
January 2015

Recent insights into the mode of action of memantine and ketamine.

Curr Opin Pharmacol 2015 Feb 2;20:54-63. Epub 2014 Dec 2.

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.

The clinical benefits of the glutamate receptor antagonists memantine and ketamine have helped sustain optimism that glutamate receptors represent viable targets for development of therapeutic drugs. Both memantine and ketamine antagonize N-methyl-D-aspartate receptors (NMDARs), a glutamate receptor subfamily, by blocking the receptor-associated ion channel. Although many of the basic characteristics of NMDAR inhibition by memantine and ketamine appear similar, their effects on humans and to a lesser extent on rodents are strongly divergent. Some recent research suggests that preferential inhibition by memantine and ketamine of distinct NMDAR subpopulations may contribute to the drugs' differential clinical effects. Here we review studies that shed light on possible explanations for differences between the effects of memantine and ketamine.
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http://dx.doi.org/10.1016/j.coph.2014.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318755PMC
February 2015

Whole-cell patch-clamp analysis of recombinant NMDA receptor pharmacology using brief glutamate applications.

Methods Mol Biol 2014 ;1183:23-41

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, A210 Langley Hall, Pittsburgh, PA, 15260, USA.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that are essential for synaptic plasticity, learning and memory. Dysfunction of NMDARs has been implicated in many nervous system disorders; therefore, pharmacological modulation of NMDAR activity has great therapeutic potential. However, given the broad physiological importance of NMDARs, modulating their activity often has detrimental side effects precluding pharmaceutical use of many NMDAR modulators. One approach to possibly improve the therapeutic potential of NMDAR modulators is to identify compounds that modulate subsets of NMDARs. An obvious target for modulating NMDAR subsets is the many NMDAR subtypes produced through different combinations of NMDAR subunits. With seven identified genes that encode NMDAR subunits, there are many neuronal NMDAR subtypes with distinct properties and potentially differential pharmacological sensitivities. Study of NMDAR subtype-specific pharmacology is complicated in neurons, however, because most neurons express at least three NMDAR subtypes. Thus, use of an approach that permits study in isolation of a single receptor subtype is preferred. Additionally, the effects of drugs on agonist-activated responses typically depend on duration of agonist exposure. To evaluate drug effects on synaptic transmission, an approach should be used that allows for activation of receptor responses as brief as those observed during synaptic transmission, both in the absence and presence of drug. To address these issues, we designed a fast perfusion system capable of (1) delivering brief (~5 ms) and consistent applications of glutamate to recombinant NMDARs of known subunit composition, and (2) easily and quickly (~5 s) changing between glutamate applications in the absence and presence of drug.
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http://dx.doi.org/10.1007/978-1-4939-1096-0_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4322862PMC
February 2015

Mutant LRRK2 enhances glutamatergic synapse activity and evokes excitotoxic dendrite degeneration.

Biochim Biophys Acta 2014 Sep 27;1842(9):1596-603. Epub 2014 May 27.

Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA; Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA; The McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA; The Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA, USA; The Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA. Electronic address:

Mutations in leucine-rich repeat kinase 2 (LRRK2), which are associated with autosomal dominant Parkinson's disease, elicit progressive dendrite degeneration in neurons. We hypothesized that synaptic dysregulation contributes to mutant LRRK2-induced dendritic injury. We performed in vitro whole-cell voltage clamp studies of glutamatergic receptor agonist responses and glutamatergic synaptic activity in cultured rat cortical neurons expressing full-length wild-type and mutant forms of LRRK2. Expression of the pathogenic G2019S or R1441C LRRK2 mutants resulted in larger whole-cell current responses to direct application of AMPA and NMDA receptor agonists. In addition, mutant LRRK2-expressing neurons exhibited an increased frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in conjunction with increased excitatory synapse density as assessed by immunofluorescence for PSD95 and VGLUT1. Mutant LRRK2-expressing neurons showed enhanced vulnerability to acute synaptic glutamate stress. Furthermore, treatment with the NMDA receptor antagonist memantine significantly protected against subsequent losses in dendrite length and branching complexity. These data demonstrate an early association between mutant LRRK2 and increased excitatory synapse activity, implicating an excitotoxic contribution to mutant LRRK2 induced dendrite degeneration.
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http://dx.doi.org/10.1016/j.bbadis.2014.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144018PMC
September 2014

Comparison of behavioral effects of the NMDA receptor channel blockers memantine and ketamine in rats.

Pharmacol Biochem Behav 2013 Aug 8;109:67-76. Epub 2013 May 8.

Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Memantine and ketamine block N-methyl-D-aspartate (NMDA) receptors with similar affinity and kinetics, yet their behavioral consequences differ: e.g., memantine is used to alleviate symptoms of Alzheimer's disease, whereas ketamine reproduces symptoms of schizophrenia. The two drugs exhibit different pharmacokinetics, which may play a principal role in their differential behavioral effects. To gain insight into the drugs' behavioral consequences, we treated adult male rats acutely with varying doses (0-40 mg/kg i.p.) of memantine or ketamine and assessed exploratory behavior and spatial working memory. To examine the importance of pharmacokinetics, we assessed behavior either 15 or 45 min after drug administration. Both drugs decreased ambulation, fine movements, and rearing at the beginning of the exploratory activity test; however, at the end of the test, high doses of only memantine increased ambulation and fine movements. High doses of both drugs disrupted spontaneous alternation, a measure of working memory, but high doses of only memantine elicited perseverative behavior. Surprisingly, ketamine's effects were influenced by the delay between drug administration and testing no more frequently than were memantine's. Our findings show that, regardless of test delay, memantine and ketamine evoke similar behavioral effects at lower doses, consistent with NMDA receptors being both drugs' principal site of action, but can have divergent effects at higher doses. Our results suggest that the divergence of memantine's and ketamine's behavioral consequences is likely to result from differences in mechanisms of NMDA receptor antagonism or actions at other targets.
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http://dx.doi.org/10.1016/j.pbb.2013.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723459PMC
August 2013

Mechanistic and structural determinants of NMDA receptor voltage-dependent gating and slow Mg2+ unblock.

J Neurosci 2013 Feb;33(9):4140-50

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

NMDA receptor (NMDAR)-mediated currents depend on membrane depolarization to relieve powerful voltage-dependent NMDAR channel block by external magnesium (Mg(o)(2+)). Mg(o)(2+) unblock from native NMDARs exhibits a fast component that is consistent with rapid Mg(o)(2+) -unbinding kinetics and also a slower, millisecond time scale component (slow Mg(o)(2+) unblock). In recombinant NMDARs, slow Mg(o)(2+) unblock is prominent in GluN1/2A (an NMDAR subtype composed of GluN1 and GluN2A subunits) and GluN1/2B receptors, with slower kinetics observed for GluN1/2B receptors, but absent from GluN1/2C and GluN1/2D receptors. Slow Mg(o)(2+) unblock from GluN1/2B receptors results from inherent voltage-dependent gating, which increases channel open probability with depolarization. Here we examine the mechanisms responsible for NMDAR subtype dependence of slow Mg(o)(2+) unblock. We demonstrate that slow Mg(o)(2+) unblock from GluN1/2A receptors, like GluN1/2B receptors, results from inherent voltage-dependent gating. Surprisingly, GluN1/2A and GluN1/2B receptors exhibited equal inherent voltage dependence; faster Mg(o)(2+) unblock from GluN1/2A receptors can be explained by voltage-independent differences in gating kinetics. To investigate the absence of slow Mg(o)(2+) unblock in GluN1/2C and GluN1/2D receptors, we examined the GluN2 S/L site, a site responsible for several NMDAR subtype-dependent channel properties. Mutating the GluN2 S/L site of GluN2A subunits from serine (found in GluN2A and GluN2B subunits) to leucine (found in GluN2C and GluN2D) greatly diminished both voltage-dependent gating and slow Mg(o)(2+) unblock. Therefore, the residue at the GluN2 S/L site governs the expression of both slow Mg(o)(2+) unblock and inherent voltage dependence.
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http://dx.doi.org/10.1523/JNEUROSCI.3712-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629906PMC
February 2013

A single GluN2 subunit residue controls NMDA receptor channel properties via intersubunit interaction.

Nat Neurosci 2012 Jan 15;15(3):406-13, S1-2. Epub 2012 Jan 15.

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

NMDA receptors (NMDARs) are glutamate-gated ion channels that are present at most excitatory mammalian synapses. The four GluN2 subunits (GluN2A-D) contribute to four diheteromeric NMDAR subtypes that have divergent physiological and pathological roles. Channel properties that are fundamental to NMDAR function vary among subtypes. We investigated the amino acid residues responsible for variations in channel properties by creating and examining NMDARs containing mutant GluN2 subunits. We found that the NMDAR subtype specificity of three crucial channel properties, Mg(2+) block, selective permeability to Ca(2+) and single-channel conductance, were all controlled primarily by the residue at a single GluN2 site in the M3 transmembrane region. Mutant cycle analysis guided by molecular modeling revealed that a GluN2-GluN1 subunit interaction mediates the site's effects. We conclude that a single GluN2 subunit residue couples with the pore-forming loop of the GluN1 subunit to create naturally occurring variations in NMDAR properties that are critical to synaptic plasticity and learning.
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http://dx.doi.org/10.1038/nn.3025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288527PMC
January 2012

Tonic NMDA receptor-mediated current in prefrontal cortical pyramidal cells and fast-spiking interneurons.

J Neurophysiol 2012 Apr 11;107(8):2232-43. Epub 2012 Jan 11.

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA.

Tonically activated neuronal currents mediated by N-methyl-d-aspartate receptors (NMDARs) have been hypothesized to contribute to normal neuronal function as well as to neuronal pathology resulting from excessive activation of glutamate receptors (e.g., excitotoxicity). Whereas cortical excitatory cells are very vulnerable to excitotoxic insult, the data regarding resistance of inhibitory cells (or interneurons) are inconsistent. Types of neurons with more pronounced tonic NMDAR current potentially associated with the activation of extrasynaptic NMDARs could be expected to be more vulnerable to excessive activation by glutamate. In this study, we compared tonic activation of NMDARs in excitatory pyramidal cells and inhibitory fast-spiking interneurons in prefrontal cortical slices. We assessed tonic NMDAR current by measuring holding current shift as well as noise reduction following NMDAR blockade after removal of spontaneous glutamate release. In addition, we compared NMDAR miniature excitatory postsynaptic currents (EPSCs) in both cell types. We have demonstrated for the first time that tonic NMDAR currents are present in inhibitory fast-spiking interneurons. We found that the magnitude of tonic NMDAR current is similar in pyramidal cells and fast-spiking interneurons, and that quantal release of glutamate does not significantly impact tonic NMDAR current.
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http://dx.doi.org/10.1152/jn.01017.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3331604PMC
April 2012

HSV delivery of a ligand-regulated endogenous ion channel gene to sensory neurons results in pain control following channel activation.

Mol Ther 2011 Mar 16;19(3):500-6. Epub 2010 Nov 16.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219, USA.

Persistent pain remains a tremendous health problem due to both its prevalence and dearth of effective therapeutic interventions. To maximize pain relief while minimizing side effects, current gene therapy-based approaches have mostly exploited the expression of pain inhibitory products or interfered with pronociceptive ion channels. These methods do not enable control over the timing or duration of analgesia, nor titration to analgesic efficacy. Here, we describe a gene therapy strategy that potentially overcomes these limitations by providing exquisite control over therapy with efficacy in clinically relevant models of inflammatory pain. We utilize a herpes simplex viral (HSV) vector (vHGlyRα1) to express a ligand-regulated chloride ion channel, the glycine receptor (GlyR) in targeted sensory afferents; the subsequent exogenous addition of glycine provides the means for temporal and spatial control of afferent activity, and therefore pain. Use of an endogenous inhibitory receptor not normally present on sensory neurons both minimizes immunogenicity and maximizes therapeutic selectivity.
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http://dx.doi.org/10.1038/mt.2010.246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048176PMC
March 2011

Memantine binding to a superficial site on NMDA receptors contributes to partial trapping.

J Physiol 2009 Oct 17;587(Pt 19):4589-604. Epub 2009 Aug 17.

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Although many nervous system disorders are associated with N-methyl-D-aspartate (NMDA) receptor overactivation, pharmacological inhibition of NMDA receptors has typically demonstrated limited clinical value due to debilitating psychotomimetic side-effects. Memantine, however, induces far fewer behavioural side-effects than other NMDA receptor channel blockers such as ketamine, and slows the progressive cognitive decline associated with Alzheimer's disease. Memantine and ketamine inhibit NMDA receptors with similar affinity and kinetics. A prominent mechanistic difference between memantine and ketamine is the degree to which they are 'trapped' within the closed channel of NMDA receptors following removal of agonist: ketamine becomes trapped in nearly all NMDA receptors to which it was bound before agonist removal, whereas some bound memantine molecules dissociate after agonist removal, a phenomenon called partial trapping. Here we investigated the mechanism underlying partial trapping of memantine by recombinant NR1/2A NMDA receptors. We found that memantine dissociation from NR1/2A receptors after agonist removal (the process that results in partial trapping) followed an exponential time course with tau = 0.79 +/- 0.32 s. Neither membrane voltage depolarization nor maintained presence of memantine after agonist removal affected partial trapping, suggesting that partial trapping does not result from memantine escape through open channels. We tested the hypothesis that partial trapping results from binding of memantine to two sites, a superficial 'non-trapping' site and a deep 'trapping' site, which cannot be occupied simultaneously. This hypothesis was supported by the lack of ketamine binding to the superficial site, the voltage dependence of partial trapping, and the effect on partial trapping of a mutation near the deep site. The superficial binding site for memantine may, by causing partial trapping, contribute to memantine's unique therapeutic utility.
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http://dx.doi.org/10.1113/jphysiol.2009.176297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2768015PMC
October 2009

Mechanism of differential control of NMDA receptor activity by NR2 subunits.

Nature 2009 Jun 29;459(7247):703-7. Epub 2009 Apr 29.

Laboratoire de Neurobiologie, Ecole Normale Supérieure, CNRS, 46 rue d'Ulm, 75005 Paris, France.

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.
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http://dx.doi.org/10.1038/nature07993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711440PMC
June 2009

Mg2+ imparts NMDA receptor subtype selectivity to the Alzheimer's drug memantine.

J Neurosci 2009 Mar;29(9):2774-9

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

N-methyl-D-aspartate receptors (NMDARs) mediate interneuronal communication and are broadly involved in nervous system physiology and pathology (Dingledine et al., 1999). Memantine, a drug that blocks the ion channel formed by NMDARs, is a widely prescribed treatment of Alzheimer's disease (Schmitt, 2005; Lipton, 2006; Parsons et al., 2007). Research on memantine's mechanism of action has focused on the NMDAR subtypes most highly expressed in adult cerebral cortex, NR1/2A and NR1/2B receptors (Cull-Candy and Leszkiewicz, 2004), and has largely ignored interactions with extracellular Mg(2+) (Mg(2+)(o)). Mg(2+)(o) is an endogenous NMDAR channel blocker that binds near memantine's binding site (Kashiwagi et al., 2002; Chen and Lipton, 2005). We report that a physiological concentration (1 mM) of Mg(2+)(o) decreased memantine inhibition of NR1/2A and NR1/2B receptors nearly 20-fold at a membrane voltage near rest. In contrast, memantine inhibition of the other principal NMDAR subtypes, NR1/2C and NR1/2D receptors, was decreased only approximately 3-fold. As a result, therapeutic memantine concentrations should have negligible effects on NR1/2A or NR1/2B receptor activity but pronounced effects on NR1/2C and NR1/2D receptors. Quantitative modeling showed that the voltage dependence of memantine inhibition also is altered by 1 mM Mg(2+)(o). We report similar results with the NMDAR channel blocker ketamine, a drug used to model schizophrenia (Krystal et al., 2003). These results suggest that currently hypothesized mechanisms of memantine and ketamine action should be reconsidered and that NR1/2C and/or NR1/2D receptors play a more important role in cortical physiology and pathology than previously appreciated.
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http://dx.doi.org/10.1523/JNEUROSCI.3703-08.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679254PMC
March 2009

Voltage-dependent gating of NR1/2B NMDA receptors.

J Physiol 2008 Dec 20;586(23):5727-41. Epub 2008 Oct 20.

Department of Neuroscience and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Ligand-gated ion channels are activated by agonist binding, but may also be modulated by membrane voltage. N-Methyl-d-aspartate receptors (NMDARs) exhibit especially strong voltage dependence due to channel block by external Mg(2+) (Mg(o)(2+)). Here we demonstrate that activity of NMDARs composed of NR1 and NR2B subunits (NR1/2B receptors) is enhanced by depolarization even in 0 Mg(o)(2+), causing slow current relaxations in response to rapid voltage changes. We present a kinetic model of receptor activation that incorporates voltage-dependent gating-associated NR2B subunit conformational changes. The model accurately reproduces current relaxations during depolarizations and subsequent repolarizations in 0 Mg(o)(2+). Model simulations in physiological Mg(o)(2+) concentrations show that voltage-dependent receptor gating also underlies the slow component of Mg(o)(2+) unblock, a phenomenon that previously was shown to influence Mg(o)(2+) unblock kinetics during dendritic spikes. We propose that voltage-dependent gating of NR1/2B receptors confers enhanced voltage and time dependence on NMDAR-mediated signalling.
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http://dx.doi.org/10.1113/jphysiol.2008.160622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655412PMC
December 2008

Structural rearrangements of NR1/NR2A NMDA receptors during allosteric inhibition.

Neuron 2008 Jan;57(1):80-93

Laboratoire de Neurobiologie, Ecole Normale Supérieure, CNRS, 46 rue d'Ulm, 75005 Paris, France.

Ionotropic glutamate receptor (iGluR) subunits contain a large N-terminal domain (NTD) that precedes the agonist-binding domain (ABD) and participates in subunit oligomerization. In NMDA receptors (NMDARs), the NTDs of NR2A and NR2B subunits also form binding sites for the endogenous inhibitor Zn(2+) ion. Although these allosteric sites have been characterized in detail, the molecular mechanisms by which the NTDs communicate with the rest of the receptor to promote its inhibition remain unknown. Here, we identify the ABD dimer interface as a major structural determinant that permits coupling between the NTDs and the channel gate. The strength of this interface also controls proton inhibition, another form of allosteric modulation of NMDARs. Conformational rearrangements at the ABD dimer interface thus appear to be a key mechanism conserved in all iGluR subfamilies, but have evolved to fulfill different functions: fast desensitization at AMPA and kainate receptors, allosteric inhibition at NMDARs.
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http://dx.doi.org/10.1016/j.neuron.2007.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679256PMC
January 2008

Permeant ion effects on external Mg2+ block of NR1/2D NMDA receptors.

J Neurosci 2006 Oct;26(42):10899-910

Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

Voltage-dependent channel block by external Mg2+ (Mg2+(o)) of NMDA receptors is an essential determinant of synaptic function. The resulting Mg2+(o) inhibition of NMDA responses depends strongly on receptor subunit composition: NR1/2A and NR1/2B receptors are more strongly inhibited by Mg2+(o) than are NR1/2C or NR1/2D receptors. Previous work showed that permeant ions have profound effects on Mg2+(o) block of NMDA receptors composed of NR1, NR2A, and NR2B subunits. Whether permeant ions affect Mg2+(o) inhibition of NR1/2C or NR1/2D receptors is unknown. We investigated the effects of permeant ions on Mg2+(o) block of NR1/2D receptors by integrating results from whole-cell recordings, single-channel recordings, and kinetic modeling. Lowering internal [Cs+] caused a voltage-dependent decrease in the Mg2+(o) IC50 and in the apparent Mg2+(o) unblocking rate, and increase in the apparent Mg2+(o) blocking rate (k(+,app)) of NR1/2D receptors. Lowering external [Na+] caused modest voltage-dependent changes in the Mg2+(o) IC50 and k(+,app). These data can be explained by a kinetic model in which occupation of either of two external permeant ion binding sites prevents Mg2+(o) entry into the channel. Occupation of an internal permeant ion binding site prevents Mg2+(o) permeation and accelerates Mg2+(o) unblock to the external solution. We conclude that variations in permeant ion site properties shape the NR2 subunit dependence of Mg2+(o) block. Furthermore, the external channel entrance varies little among NMDA receptor subtypes. Differences in the Mg2+(o) blocking site, and particularly in the selectivity filter and internal channel entrance, are principally responsible for the subunit dependence of Mg2+(o) block.
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http://dx.doi.org/10.1523/JNEUROSCI.3453-06.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6674757PMC
October 2006