A Frameshift Variant of GluN2A Identified in an Epilepsy Patient Results in NMDA Receptor Mistargeting.

N-methyl-D-aspartate receptors (NMDARs) are crucial for neuronal development and synaptic plasticity. Dysfunction of NMDARs is associated with multiple neurodevelopmental disorders, including epilepsy, autism spectrum disorder, and intellectual disability. Understanding the impact of genetic variants of NMDAR subunits can shed light on the mechanisms of disease. Here, we characterized the functional implications of a de novo mutation of the GluN2A subunit (P1199Rfs*32) resulting in the truncation of the C-terminal domain. The variant was identified in a male patient with epileptic encephalopathy, multiple seizure types, severe aphasia, and neurobehavioral changes. Given the known role of the CTD in NMDAR trafficking, we examined changes in receptor localization and abundance at the postsynaptic membrane using a combination of molecular assays in heterologous cells and rat primary neuronal cultures. We observed that the GluN2A P1199Rfs*32-containing receptors traffic efficiently to the postsynaptic membrane but have increased extra-synaptic expression relative to WT GluN2A-containing NMDARs. Using in silico predictions, we hypothesized that the mutant would lose all PDZ interactions, except for the recycling protein Scribble1. Indeed, we observed impaired binding to the scaffolding protein postsynaptic protein-95 (PSD-95); however, we found the mutant interacts with Scribble1, which facilitates the recycling of both the mutant and the WT GluN2A. Finally, we found that neurons expressing GluN2A P1199Rfs*32 have fewer synapses and decreased spine density, indicating compromised synaptic transmission in these neurons. Overall, our data show that GluN2A P1199Rfs*32 is a loss-of-function variant with altered membrane localization in neurons and provide mechanistic insight into disease etiology.

Differential binding properties of [3H]dextrorphan and [3H]MK-801 in heterologously expressed NMDA receptors.

The N-methyl-D-aspartate receptor (NMDAR) antagonists: MK-801, phencyclidine and ketamine are open-channel blockers with limited clinical value due to psychotomimetic effects. Similarly, the psychotomimetic effects of the dextrorotatory opioids, dextromethorphan and its metabolite dextrorphan, derive from their NMDAR antagonist actions. Differences in the use dependency of blockade, however, suggest that the binding sites for MK-801 and dextrorphan are distinct. In the absence of exogenous glutamate and glycine, the rate of association of [3H]MK-801 with wild-type NR1-1a/NR2A receptors was considerably slower than that for [3H]dextrorphan. Glutamate individually, and in the presence of the co-agonist glycine, had substantial effects on the specific binding of [3H]MK-801, while the binding of [3H]dextrorphan was not affected. Mutation of residues N616 and A627 in the NR1 subunit had a profound effect on [3H]MK-801 binding affinity, while that of [3H]dextrorphan was unaltered. In contrast, NR1 residues, W611 and N812, were critical for specific binding of [3H]dextrorphan to NR1-1a/NR2A complexes with no corresponding influence on that of [3H]MK-801. Thus, [3H]dextrorphan and [3H]MK-801 have distinct molecular determinants for high-affinity binding. The ability of [3H]dextrorphan to bind to a closed channel, moreover, indicates that its recognition site is shallower in the ion channel domain than that of MK-801 and may be associated with the extracellular vestibule of the NMDAR.

Open probability of homomeric murine 5-HT3A serotonin receptors depends on subunit occupancy.

1. The time course of macroscopic current responses of homomeric murine serotonin 5-HT3A receptors was studied in whole cells and excised membrane patches under voltage clamp in response to rapid application of serotonin. 2. Serotonin activated whole cell currents with an EC(50) value for the peak response of 2 microM and a Hill slope of 3.0 (n = 12), suggesting that the binding of at least three agonist molecules is required to open the channel. 3. Homomeric 5-HT3A receptors in excised membrane patches had a slow activation time course (mean +/- S.E.M. 10-90 % rise time 12.5 +/- 1.6 ms; n = 9 patches) for 100 microM serotonin. The apparent activation rate was estimated by fitting an exponential function to the rising phase of responses to supramaximal serotonin to be 136 s(-1). 4. The 5-HT3A receptor response to 100 microM serotonin in outside-out patches (n = 19) and whole cells (n = 41) desensitized with a variable rate that accelerated throughout the experiment. The time course for desensitization was described by two exponential components (for patches tau(slow) 1006 +/- 139 ms, amplitude 31 %; tau(fast) 176 +/- 25 ms, amplitude 69 %). 5. Deactivation of the response following serotonin removal from excised membrane patches (n = 8) and whole cells (n = 29) was described by a dual exponential time course with time constants similar to those for desensitization (for patches tau(slow) 838 +/- 217 ms, 55 % amplitude; tau(fast) 213 +/- 44 ms, 45 % amplitude). 6. In most patches (6 of 8), the deactivation time course in response to a brief 1-5 ms pulse of serotonin was similar to or slower than desensitization. This suggests that the continued presence of agonist can induce desensitization with a similar or more rapid time course than agonist unbinding. The difference between the time course for deactivation and desensitization was voltage independent over the range -100 to -40 mV in patches (n = 4) and -100 to +50 mV in whole cells (n = 4), suggesting desensitization of these receptors in the presence of serotonin does not reflect a voltage-dependent block of the channel by agonist. 7. Simultaneously fitting the macroscopic 5-HT3A receptor responses in patches to submaximal (2 microM) and maximal (100 microM) concentrations of serotonin to a variety of state models suggests that homomeric 5-HT3A receptors require the binding of three agonists to open and possess a peak open probability greater than 0.8. Our modelling also suggests that channel open probability varies with the number of serotonin molecules bound to the receptor, with a reduced open probability for fully liganded receptors. Increasing the desensitization rate constants in this model can generate desensitization that is more rapid than deactivation, as observed in a subpopulation of our patches.

Metabotropic glutamate receptors 1 and 5 differentially regulate CA1 pyramidal cell function.

The activation of group I metabotropic glutamate receptors (mGluRs) produces a variety of actions that lead to alterations in excitability and synaptic transmission in the CA1 region of the hippocampus. The group I mGluRs, mGluR1 and mGluR5, are activated selectively by (S)-3,5-dihydroxyphenylglycine (DHPG). To identify which of these mGluR subtypes are responsible for the various actions of DHPG in area CA1, we took advantage of two novel subtype-selective antagonists. (S)-(+)-alpha-amino-a-methylbenzeneacetic acid (LY367385) is a potent competitive antagonist that is selective for mGluR1, whereas 2-methyl-6-(phenylethynyl)-pyridine (MPEP) is a potent noncompetitive antagonist that is selective for mGluR5. The use of these compounds in experiments with whole-cell patch-clamp recording and Ca(2+)-imaging techniques revealed that each group I mGluR subtype plays distinct roles in regulating the function of CA1 pyramidal neurons. The block of mGluR1 by LY367385 suppressed the DHPG-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the direct depolarization of CA1 hippocampal neurons. In addition, the increase in the frequency of spontaneous IPSCs (sIPSCs) caused by the DHPG-induced depolarization of inhibitory interneurons also was blocked by LY367385, as was the DHPG-induced inhibition of transmission at the Schaffer collateral-->CA1 synapse. On the other hand, the block of mGluR5 by MPEP antagonized the DHPG-induced suppression of the Ca(2+)-activated potassium current (I(AHP)) and potentiation of the NMDA receptor. Finally, antagonism of the DHPG-induced suppression of evoked IPSCs required the blockade of both mGluR1 and mGluR5. These data suggest that mGluR1 and mGluR5 play distinct roles in the regulation of the excitability of hippocampal CA1 pyramidal neurons.

Allosteric interaction between the amino terminal domain and the ligand binding domain of NR2A.

Fast desensitization is an important regulatory mechanism of neuronal NMDA receptor function. Only recombinant NMDA receptors composed of NR1/NR2A exhibit a fast component of desensitization similar to neuronal NMDA receptors. Here we report that the fast desensitization of NR1/NR2A receptors is caused by ambient zinc, and that a positive allosteric interaction occurs between the extracellular zinc-binding site located in the amino terminal domain and the glutamate-binding domain of NR2A. The relaxation of macroscopic currents reflects a shift to a new equilibrium due to increased zinc affinity after binding of glutamate. We also show a similar interaction between the ifenprodil binding site and the glutamate binding site of NR1/NR2B receptors. These data raise the possibility that there is an allosteric interaction between the amino terminal domain and the ligand-binding domain of other glutamate receptors. Our findings may provide insight into how zinc and other extracellular modulators regulate NMDA receptor function.

Identification of amino acid residues in GluR1 responsible for ligand binding and desensitization.

Although GluR1(o) and GluR3(o) are homologous at the amino acid level, GluR3(o) desensitizes approximately threefold faster than GluR1(o). By creating chimeras of GluR1(o) and GluR3(o) and point amino acid exchanges in their S2 regions, two residues were identified to be critical for GluR1(o) desensitization: Y716 and the R/G RNA-edited site, R757. With creation of the double-point mutant (Y716F, R757G)GluR1(o), complete exchange of the desensitization rate of GluR1(o) to that of GluR3(o) was obtained. In addition, both the potency and affinity of the subtype-selective agonist bromohomoibotenic acid were exchanged by the Y716F mutation. A model is proposed of the AMPA receptor binding site whereby a hydrogen-bonding matrix of water molecules plays an important role in determining both ligand affinity and receptor desensitization properties. Residues Y716 in GluR1 and F728 in GluR3 differentially interact with this matrix to affect the binding affinity of some ligands, providing the possibility of developing subtype-selective compounds.

Is tissue plasminogen activator a threat to neurons?

The clot-busting drug tissue plasminogen activator (tPA) is currently the only FDA-approved therapy for acute stroke. However, increasing evidence suggests that tPA can also contribute to excitotoxic neuronal damage in animal models of stroke.

Proton release as a modulator of presynaptic function.

In this issue of Neuron, DeVries (2001) describes experiments suggesting that acidification of the synaptic cleft can reduce Ca2+ channel activity and thereby act as a brake on tonic synaptic release of glutamate from cone cells. This work hints at a potentially important new facet to the regulation of synaptic transmission.

Molecular determinants of coordinated proton and zinc inhibition of N-methyl-D-aspartate NR1/NR2A receptors.

Modulation of the N-methyl-d-aspartate (NMDA)-selective glutamate receptors by extracellular protons and Zn(2+) may play important roles during ischemia in the brain and during seizures. Recombinant NR1/NR2A receptors exhibit a much higher apparent affinity for voltage-independent Zn(2+) inhibition than receptors with other subunit combinations. Here, we show that the mechanism of this apparent high-affinity, voltage-independent Zn(2+) inhibition for NR2A-containing receptors results from the enhancement of proton inhibition. We also show that the N-terminal leucine/isoleucine/valine binding protein (LIVBP)-like domain of the NR2A subunit contains critical determinants of the apparent high-affinity, voltage-independent Zn(2+) inhibition. Mutations H42A, H44G, or H128A greatly increase the Zn(2+) IC(50) (by up to approximately 700-fold) with no effect on the potencies of glutamate and glycine or on voltage-dependent block by Mg(2+). Furthermore, the amino acid residue substitution H128A, which mediates the largest effect on the apparent high-affinity Zn(2+) inhibition among all histidine substitutions we tested, is also critical to the pH-dependency of Zn(2+) inhibition. Our data revealed a unique interaction between two important extracellular modulators of NMDA receptors.

Serine proteases and brain damage - is there a link?

The protective blood-brain barrier normally allows diffusion of small molecules such as oxygen and carbon dioxide, and transport of essential nutrients, but excludes large proteins and other blood constituents from the interstitial space of the CNS. However, head trauma, stroke, status epilepticus and other pathological conditions can all compromise the integrity of this barrier, and allow blood proteins as large as albumin to gain access to the extracellular spaces that surround neurons and glia. Given their possible entry into brain tissue during cerebrovascular insult, the effects of blood-derived proteases such as thrombin, tissue plasminogen activator and plasmin in the CNS have come under increasing scrutiny. Evidence now supports a role for serine proteases in the sequence of events that can lead to glial scarring, edema, seizure and neuronal death.

Potentiation of NMDA receptor function by the serine protease thrombin.

Although serine proteases and their receptors are best known for their role in blood coagulation and fibrinolysis, the CNS expresses many components of an extracellular protease signaling system including the protease-activated receptor-1 (PAR1), for which thrombin is the most effective activator. In this report we show that activation of PAR1 potentiates hippocampal NMDA receptor responses in CA1 pyramidal cells by 2.07 +/- 0.27-fold (mean +/- SEM). Potentiation of neuronal NMDA receptor responses by thrombin can be blocked by thrombin and a protein kinase inhibitor, and the effects of thrombin can be mimicked by a peptide agonist (SFLLRN) that activates PAR1. Potentiation of the NMDA receptor by thrombin in hippocampal neurons is significantly attenuated in mice lacking PAR1. Although high concentrations of thrombin can directly cleave both native and recombinant NR1 subunits, the thrombin-induced potentiation we observe is independent of NMDA receptor cleavage. Activation of recombinant PAR1 also potentiates recombinant NR1/NR2A (1.7 +/- 0.06-fold) and NR1/NR2B (1.41 +/- 0.11-fold) receptor function but not NR1/NR2C or NR1/NR2D receptor responses. PAR1-mediated potentiation of recombinant NR1/NR2A receptors occurred after activation with as little as 300 pm thrombin. These data raise the intriguing possibility that potentiation of neuronal NMDA receptor function after entry of thrombin or other serine proteases into brain parenchyma during intracerebral hemorrhage or extravasation of plasma proteins during blood-brain barrier breakdown may exacerbate glutamate-mediated cell death and possibly participate in post-traumatic seizure. Furthermore, the ability of neuronal protease signaling to control NMDA receptor function may also have roles in normal brain development.

Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase.

Modulation of postsynaptic AMPA receptors in the brain by phosphorylation may play a role in the expression of synaptic plasticity at central excitatory synapses. It is known from biochemical studies that GluR1 AMPA receptor subunits can be phosphorylated within their C terminal by cAMP-dependent protein kinase A (PKA), which is colocalized with the phosphatase calcineurin (i.e., phosphatase 2B). We have examined the effect of PKA and calcineurin on the time course, peak open probability (P(O, PEAK)), and single-channel properties of glutamateevoked responses for neuronal AMPA receptors and homomeric GluR1(flip) receptors recorded in outside-out patches. Inclusion of purified catalytic subunit Calpha-PKA in the pipette solution increased neuronal AMPA receptor P(O,PEAK) (0.92) compared with recordings made with calcineurin included in the pipette (P(O,PEAK) 0.39). Similarly, Calpha-PKA increased P(O,PEAK) for recombinant GluR1 receptors (0. 78) compared with patches excised from cells cotransfected with a cDNA encoding the PKA peptide inhibitor PKI (P(O,PEAK) 0.50) or patches with calcineurin included in the pipette (P(O,PEAK) 0.42). Neither PKA nor calcineurin altered the amplitude of single-channel subconductance levels, weighted mean unitary current, mean channel open period, burst length, or macroscopic response waveform for recombinant GluR1 receptors. Substitution of an amino acid at the PKA phosphorylation site (S845A) on GluR1 eliminated the PKA-induced increase in P(O,PEAK), whereas the mutation of a Ca(2+), calmodulin-dependent kinase II and PKC phosphorylation site (S831A) was without effect. These results suggest that AMPA receptor peak response open probability can be increased by PKA through phosphorylation of GluR1 Ser845.

Control of voltage-independent zinc inhibition of NMDA receptors by the NR1 subunit.

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+.

Getting the most out of noise in the central nervous system.

Rather than merely a nuisance, noise in biological systems is a useful property. Before patch-clamp methods were invented, analysis of membrane current noise provided the first solid, if indirect, evidence for the existence of ion-conducting pores with discrete conductance levels. Although supplanted by single-channel recording techniques for most tasks, analysis of current membrane noise remains useful for certain problems, such as determining the properties of channels with rapid kinetics that open with a high probability and desensitize, channels localized at synapses, channels with an unusually low unitary conductance and open-channel noise. In addition, the role of noise in information processing in the CNS is increasingly being recognized. In this article, we summarize the analysis of current membrane noise with an emphasis on what the technique is still useful for, and discuss the role for noise in information processing.

Antagonist properties of a phosphono isoxazole amino acid at glutamate R1-4 (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptor subtypes.

The activity of the (R, S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor antagonist, (R,S) -2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid (ATPO), at recombinant ionotropic glutamate receptors (GluRs) was evaluated using electrophysiological techniques. Responses at homo- or heterooligomeric AMPA-preferring GluRs expressed in human embryonic kidney (HEK) 293 cells (GluR1-flip) or Xenopus laevis oocytes (GluR1-4-flop or GluR1-flop + GluR2) were potently inhibited by ATPO with apparent dissociation constants (Kb values) ranging from 3.9 to 26 microM. A Schild analysis for kainate (KA)-activated GluR1 receptors showed ATPO to have a KB of 8.2 microM and a slope of unity, indicating competitive inhibition. The antagonism by ATPO at GluR1 was of similar magnitude at holding potentials between -100 mV and +20 mV. In contrast, ATPO (<300 microM), does not inhibit responses to kainate at homomeric GluR6 or heterooligomeric GluR6/KA2 expressed in HEK 293 cells but activated GluR5 and GluR5/KA2 expressed in X. laevis oocytes. ATPO produced <15% inhibition at the maximal concentration (300 microM) of current responses through NR1A + NR2B receptors expressed in X. laevis oocytes. Thus, ATPO shows a unique pharmacological profile, being an antagonist at GluR1-4 and a weak partial agonist at GluR5 and GluR5/KA2.

Software-based correction of single compartment series resistance errors.

Resistance across a patch pipette following seal formation and attainment of the whole cell recording configuration can introduce considerable and sometimes non-intuitive errors into voltage clamp recordings. These errors can be corrected actively on most commercially available amplifiers, although decisions during the experiment often must be made concerning the degree of correction to be employed, and the decision is essentially irreversible once the data is recorded. Amplifier-based corrections assume a single compartment, and thus any degree of compensation could be applied after data collection with a delay of only a single digitization interval. This report describes computer algorithms that correct capacitative filtering that results from pipette series resistance as well as the voltage error for current responses with either linear or non linear current voltage curves. The algorithms are designed to operate on data that are recorded at a single holding potential rather than in response to voltage steps. The simplest algorithm for correction of responses with linear IV consists of about a dozen lines of C code and can easily be incorporated into data analysis programs, spreadsheets, and mathematical analysis packages. Code, sample programs, and spreadsheets that implement these algorithms are available from ftp.pharm.emory.edu.

Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition.

Activation of the tyrosine kinase Src potentiates NMDA-receptor currents, which is thought to be necessary for induction of hippocampal long-term potentiation. Although the carboxy(C)-terminal domain of the NR2A subunit contains potential tyrosine phosphorylation sites, the mechanisms by which Src modulates synaptic plasticity and NMDA receptor currents is not fully understood. Here we present evidence from NR1 mutants and splice variants that Src potentiates NMDA-receptor currents by reducing the tonic inhibition of receptors composed of NR1 and NR2A subunits by extracellular zinc. Using site-directed mutagenesis, we have identified three C-terminal tyrosine residues of NR2A that are required for Src's modulation of the zinc sensitivity of NMDA receptors. Our data link two modulatory sites of NMDA receptors that were previously thought to be independent.

Phenylethanolamines inhibit NMDA receptors by enhancing proton inhibition.

The phenylethanolamines, ifenprodil and CP-101,606, are NMDA receptor antagonists with promising neuroprotective properties. In recombinant NMDA receptors expressed in Xenopus oocytes, we found that these drugs inhibit NMDA receptors through a unique mechanism, making the receptor more sensitive to inhibition by protons, an endogenous negative modulator. These findings support a critical role for the proton sensor in gating the NMDA receptor and point the way to identifying a context-dependent NMDA receptor antagonist that is inactive at physiological pH, but is a potent inhibitor during the acidic conditions that arise during epilepsy, ischemia and brain trauma.

4-Methylhomoibotenic acid activates a novel metabotropic glutamate receptor coupled to phosphoinositide hydrolysis.

Metabotropic glutamate receptors (mGluRs) are a family of glutamate receptors that are coupled to a variety of second messenger systems through GTP-binding proteins. Of the eight subtypes cloned to date, mGluR1 and mGluR5 are coupled to phosphoinositide hydrolysis in expression systems, and both are activated by the glutamate analogue 1-aminocyclopentane-1S,3R-dicarboxylic acid. Previously, we provided evidence that in rat cortical slices, 4-bromohomoibotenic acid (BrHI) and 4-methylhomoibotenic acid (MHI) activate a 1-aminocyclopentane-1S,3R-dicarboxylic acid-insensitive phosphoinositide hydrolysis-coupled mGluR. We further examine these compounds in expression systems. In a stable cell line expressing mGluR1a, BrHI is a weak partial agonist whereas MHI has no agonist activity. In Xenopus oocytes expressing mGluR1a or mGluR5a, BrHI is a weak agonist at mGluR5a whereas MHI is without effect on either receptor. Both BrHI and MHI have weak agonist activity at mGluRs 4a and 7a expressed in stable BHK cell lines whereas neither compound had any activity on BHK cells expressing mGluR2. Finally, we found that the novel mGluR antagonist LY341495 completely blocked the activation of mGluR1 and mGluR5 and blocked the phosphoinositide hydrolysis response to DHPG in rat cortical slices. In contrast, LY341495 did not block the phosphoinositide hydrolysis response to MHI in rat cortical slices. This provides further evidence that the phosphoinositide hydrolysis response to MHI in rat cortical slices is due to activation of a novel receptor that is distinct from the previously cloned mGluRs.