1988 — 1990 |
Traynelis, Stephen F. |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Single Channel Analysis of Glutamate Receptor Subtypes |
0.966 |
1995 — 1999 |
Traynelis, Stephen F. |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Glutamate Receptor Function by the Nr1 5th Exon
Intense interest has focused on the NMDA subtype of glutamate receptor because of its roles in development, synaptic transmission, memory, and pathological situations such as ischemia and epilepsy. Although NMDA receptors are modulated by many endogenous substances, the control of receptor function by physiological concentrations of protons is particularly important, not only because the interstitial pH is highly dynamic, but also because of the extracellular acidification that accompanies ischemia and seizures. These perturbations in pH could serve as negative feedback to inhibit NMDA receptor function, and thereby limit seizure duration or glutamate-mediated damage during ischemia. My preliminary studies have shown that the 5th alternative exon of the NR1 subunit renders receptors proton-insensitive, and this effect can be mimicked by endogenous polyamines. This finding provides an opportunity to approach proton control of NMDA receptor function in structural terms, and additionally raises the possibility that the different splice variants of the NMDA receptor might be related to the selective vulnerability of central structures to ischemia-induced damage or seizure initiation. The long-term objective of this project is to determine at the structural and functional level how the 5th NR1 exon controls receptors in normal and pathological situations. The five main goals are: (1) To identify the structural determinants within the NR1 5th exon that control proton inhibition. (2) To determine at the biophysical level how the NR1 5th exon controls the receptor's proton sensitivity. (3) To identify compounds that modulate the NMDA receptor's proton sensitivity, and determine whether these compounds exert their effects in a manner similar to the 5th exon. (4) To investigate the link between the proton sensitivity of recombinant NMDA receptor splice variants and their cytotoxic potential. (5) To determine whether the 5th exon is differentially expressed in the central nervous system. These experiments will help to explain at the structural, anatomical, and functional levels how the 5th exon influences receptor function. In addition, these experiments will help to define an important regulatory site on the NMDA receptor, and identify compounds that act at that site.
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1 |
1996 — 1997 |
Traynelis, Stephen F. |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Activation of 5-Ht3 Serotonin Receptors
DESCRIPTION (Adapted from applicant's abstract): The type-3 serotonin (5-HT3) receptor is a target for several clinically useful compounds. Therefore, it is important to understand in detail the molecular composition and functional properties of this ligand-gated ion channel. This proposal focuses on two aspects of this receptor system. First, despite the isolation of a cDNA encoding the 5-HT3 receptor subunit four years ago, it is still unclear whether this subunit reproduces the full spectrum of microscopic properties displayed by the native receptor. This gap in our functional understanding of the 5-HT3 system reflects a trend, since the discovery process for cDNAs encoding neurotransmitter receptor subunits has outpaced our knowledge of the physiological properties of recombinant receptors. Moreover, the lack of a detailed understanding of recombinant ion channel physiology has blunted the promise that molecular cloning holds for linking structure to function. Second, dopamine has recently been found to activate recombinant 5-HT3 receptors, and kinetic and neurochemical arguments can be made that dopamine-activated 5-HT3 receptors might be utilized by neurons during normal function or pathological situations. Thus, an examination of the pharmacology and functional properties of dopamine-activated channels represents an important step towards assessing the role of dopamine as an endogenous agonist in the 5-HT3 receptor system. The experiments outlined in this proposal simultaneously address both of these issues by testing whether the pharmacological and biophysical properties of recombinant rat 5-HT3 ion channels isolated from rat dorsal root ganglia cDNA library match those of native receptors in sympathetic neurons. Proposed experiments explore potential differences between the two agonists and between recombinant versus native receptors in four sets of parameters: (1) desensitization properties (EC50, time course for onset/recovery, sensitivity to modulators), (2) Ca2+ permeability; (3) single channel properties (activation and open time, conductance, and probability of opening) and (4) Ki values for therapeutically useful serotonin and dopamine antagonists. These experiments constitute the first biophysical analysis of ion channel composed of only the recombinant 5-HT3 receptor. The results from both of these lines of experimentation hold implications for the treatment of neuropsychiatric disorders and chemotherapy-induced emesis.
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1 |
1998 — 2006 |
Traynelis, Stephen F. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Control of Glutamate Receptor Activation
DESCRIPTION (provided by applicant): N-methyl-D-aspartate-selective glutamate receptors (NMDA-Rs) mediate a slow component of excitatory synaptic transmission in the CNS and are involved in synaptic plasticity, learning, and memory. Activation of NMDA-Rs can contribute to the initiation and maintenance of seizures. In addition, stimulation of NMDA-Rs by extracellular glutamate that accumulates during ischemia can lead to cytotoxic levels of Ca2+ and neuronal death. Given this potential danger of NMDA-R overactivation, it is not surprising that NMDA-R function is tightly regulated by a number of endogenous extracellular ions including protons. Extracellular protons inhibit NMDA-R function completely by binding to a single binding site (Hill slope about 1) with a pKa of 7.0 (125 nM H+) or 7.4 (50 nM H+) for recombinant NRI/NR2A and NRI/NR2B receptors. Despite the potential importance of NMDA-R function, an understanding of the basic mechanisms by which NMDA-R channels open is lacking. No conceptual model exists for NMDA receptor function that explains both single channel and macroscopic receptor properties. The experiments outlined for the next period exploit our recent success obtaining excised outside-out patches that contain only one active NMDA-R channel. This single channel approach will be combined with macroscopic current recording and quantitative modeling to explore the mechanism of NMDA-R activation (spec. aims 1-3). Detailed functional information about NMDA-R gating will be required to maximize interpretation of structural information. Understanding NMDA-R gating is also a pre-requisite to understanding both the proton sensitivity of gating and the function of the therapeutically interesting compounds that regulate proton inhibition (aims 4-5). Five questions will be addressed. I. Is NMDA receptor function controlled by two independent gates? 2. Can single channel kinetics and macroscopic current response time course be reconciled by multi-gate models? 3. Do the glycine and glutamate binding subunits contribute kinetically distinct gates to the NMDA receptor pore? 4. Do protons and phenylethanolamines reduce the probability that an agonist-bound receptor will open? 5. Is the structural basis for H+ sensitivity of NMDA and G1uR6 receptors contained in the transmembrane linker regions? Together, these experiments will help define a unifying theory for NMDA receptor function that accounts for single channel and macroscopic behavior. In addition, evaluation of the hypothesis that protonation of a few key residues inhibits channel opening without changing other features of receptor function will increase our understanding of the structural nature of how glutamate receptors open and close in response to full and partial agonists. These experiments also probe the link between gating of NMDA receptors and inward rectifier K+ channels. Finally, we will determine at the single channel level the mechanisms of action of a non-competitive and therapeutically interesting class of antagonists - phenylethanolamines, such as ifenprodil.
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1 |
1998 |
Traynelis, Stephen F. |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Glutamate Receptor Function by Nr1 5th Exon |
1 |
2000 — 2007 |
Traynelis, Stephen F. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Nmda Receptor Function by Serine Protease Receptors
DESCRIPTION (provided by applicant): The thrombin receptor (protease activated receptor-1 or PAR1) is a G-protein coupled receptor that is best known for its role in coagulation and hemostasis. PAR1 is also expressed in the central nervous system (CNS) and mediates a variety of important effects. Work performed during the previous funding period demonstrated unambiguous PAR1-mediated potentiation of N-methyl-D-aspartate (NMDA) receptors and assessed the role of this effect in neuronal injury during blood brain barrier breakdown, which allows PAR1 activators direct access to brain parenchyma. We obtained strong evidence showing that PAR1 activation can exacerbate neuronal damage in vivo following 4 different CNS insults --transient ischemia, transient hypoxia, traumatic brain injury, and intra-striatal injection of NMDA. In addition, we completed cellular experiments that lead to the intriguing and unexpected working hypothesis that activation of PAR1 in astrocytes releases glutamate, which depolarizes neurons resulting in a relief of Mg2+ block of NMDA receptors. This competitive renewal proposes cellular experiments that examine whether activation of PAR1 and other astrocytic Gaq-coupled receptors triggers release of an excitatory amino acid that acts as a glial-neuronal signal. We will use mixed co-cultures of PAR1 -/- neurons on wild type glial feeder layers (and the converse) to explore the mechanism of PAR1-mediated glial-neuronal signaling. Our preliminary data suggests that astrocytic Ca2+-activated C1 channels may control release of glutamate, and we thus will explore the hypothesis that glutamate release reflects permeation through open Ca2+-dependent C1- channels. Single channel recordings will be made to examine in detail properties and permeation characteristics of Ca2+-activated C1- channels. We will address the following five experimental questions, which will expand our understanding of signaling by PAR1 and other Gaq/11-1inked receptors in the CNS as well as help explain how PAR1 could be involved in a wide range of CNS injuries. 1. Does activation of astrocytic PAR1 depolarize neurons by stimulating glutamate release? 2. Does activation of astrocytic PAR1 potentiate NMDA-Rs through depolarization-induced relief of distal Mg 2+block? 3. Does PAR1-stimulated glutamate release from astrocytes involve permeation through Ca2+ -activated C1 channels? 4. Are the effects of PAR1 activation in cultured astrocytes shared by astrocytes in brain slices? 5. Is astrocytic release of glutamate a generalized response to activation of Gaq/11-coupled receptors?
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1 |
2002 |
Traynelis, Stephen F. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Signaling by Mglur5
DESCRIPTION(Adapted from applicant's abstract): The hippocampus is a limbic cortical structure that plays an important role in learning and memory, and is a primary site of temporal lobe epilepsy and Alzheimer's disease. Glutamate is the primary neurotransmitter at excitatory synapses in the hippocampus, where it acts on both ionotropic and metabotropic glutamate receptors (mGluRs). Particular attention has been focused on the NMDA subtype of glutamate receptor because of its unique role in certain forms of learning, memory, and pathological conditions including epileptic responses and excitotoxicity. Interestingly, recent studies reveal that activation of one mGluR subtype, mGluR5, can dramatically potentiate currents through NMDA receptor channels in hippocampal neurons. Furthermore, low concentrations of NMDA potentiate responses to mGluR5 activation. This positive feedback regulation between mGluR5 and NMDA receptors could play a critical role in signal amplification and may be important for NMDA receptor function. Consistent with this, several previous studies suggest that mGluR5 plays an important role in several receptor-dependent forms of synaptic plasticity and could contribute to pathological responses to NMDA receptor activation. Previous studies reveal that mGluR5 is desensitized by activation of protein kinase C (PKC) which directly phosphorylates the receptor. We recently found that mild activation of NMDA receptors-with low concentrations of NMDA-potentiates mGluR5-mediated responses by reversing this agonist-induced desensitization. Interestingly, stronger activation of NMDA receptors reduced mGluR5-mediated responses and increased mGluR5 phosphorylation. This is especially interesting in light of recent studies that suggest that low frequency stimulation of glutamatergic afferents to hippocampal area CA1 induces preferential activation of the protein phosphatase calcineuron whereas high frequency stimulation leads to activation of PKC and a net increase in protein phosphorylation. Based on this and a number of other previous studies, we postulated that the differential effects of different concentrations of NMDA on mGluR5 are mediated by net increases and decreases in mGluR5 phosphorylation. Furthermore, we postulate that low frequency stimulation of glutamatergic afferents induces preferential dephosphorylation of mGluR5 and potentiates mGluR5-mediated responses whereas high frequency stimulation induces a net increase in mGluR5 phosphorylation and inhibition of mGluR5-mediated responses. A combination of molecular, biochemical and electrophysiological techniques will be used to directly test these hypotheses.
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1 |
2006 |
Traynelis, Stephen F. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Identification of Protease Activated Receptor-2 Modulators
DESCRIPTION (provided by applicant): Activation of G protein-coupled protease-activated receptor-2 (PAR2) in peripheral organs by the serine proteases trypsin and mast cell-derived tryptase exacerbates inflammatory processes and inflammatory disorders such as arthritis, asthma, and allergies. Neuropathological conditions such as stroke, epilepsy, and neurodegenerative diseases- involve complex neuroinflammatory processes that can exacerbate brain damage. Recent studies suggest that levels of PAR2 and its activating proteases also increase in the brain during injury and damage, suggesting that PAR2 activation may contribute to the process of inflammation in brain injury. Neuroinflammatory responses are driven largely by reactive microglia and astrocytes, and both cell types exhibit dramatic morphological changes and proliferation in response to PAR2-selective activating peptides. These observations together give rise to the hypothesis that PAR2 activation plays a pro- inflammatory role in the brain through its effects on microglial and astrocytic functions. Despite the intriguing and important roles postulated for PAR2 in both peripheral and CMS, no small molecule antagonists or modulators have been reported for this receptor in the literature. Furthermore, no small molecule antagonists or modulators are available from commercial sources. To facilitate the evaluation of the role of PAR2 in CNS injury and explore therapeutic opportunities that follow PAR2 modulation, we propose to develop a robust assay for PAR2 allosteric regulators. We propose to specifically search for small molecules that regulate the receptor in a non-competitive fashion since such molecules often bind to sites with rich and selective pharmacology, and modulators can offer advantages including increased safety and reduced side effects in the clinic. The aims of this grant are: 1. To define an assay that will allow the identification of small molecule inhibitors and potentiators. 2. To convert assay to 384 well format and design secondary assays that will allow the identification of competitive inhibitors as well as positive and negative allosteric modulators.
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1 |
2007 — 2018 |
Traynelis, Stephen F. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Control of Glutamate Receptor Function
DESCRIPTION (provided by applicant): NMDA receptors have received a great deal of attention because of their role in synaptic plasticity, seizure initiation, and ischemia-induced neuronal death. Recent crystallographic advances have provided the first structure for the agonist binding domains of NR1 and NR2A subunits, and recent functional studies have provided the first conceptual models of NR1/NR2A receptor activation that account for both single channel and macroscopic properties. These advances create an ideal opportunity to explore in detail the relationship between structure and function for NMDA receptors. We will evaluate function in the context of structure for NR1/NR2C and NR1/NR2D receptors, which remain poorly understood despite their roles in striatal and cerebellar function and the intriguing therapeutic potential of NR2C/NR2D subunit-selective modulators. The proposed experiments exploit our recent success in obtaining outside-out membrane patches that contain only one active channel to examine the function of NR1/NR2C and NR1/NR2D receptors activated by both glutamate and novel subunit-selective agonists and modulators. Single channel studies will be extended to native NR2D-containing receptors and recombinant heterotrimeric receptors containing two different NR2 subunits, which can be unambiguously isolated in one channel patches. Complementary experiments will exploit structural data by using atomic co-variance analysis of molecular dynamics simulations of the agonist binding domains for all NR1/NR2 combinations to evaluate long-range intra-protein motions. Our preliminary data reveal intra-protein motions that show domain closure, a pivot around the NR1/NR2 dimer interface, and different interdomain contacts between functionally dissimilar NR2A and NR2D. Understanding the relationship between structure and function is a pre-requisite to rational drug design, which may ultimately yield therapeutically relevant compounds. Proposed experiments will address four questions. 1. How do the NR2C and NR2D subunits control NMDA receptor activation? 2. What are the functional properties of heterotrimeric NR1/NR2A/NR2D and NR1/NR2B/NR2D receptors? 3. How does the NR2D subunit control native NMDA receptor activation? 4. What structural features of the NR1/NR2 dimer influence intra-protein motion and receptor activation?
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1 |
2008 — 2009 |
Traynelis, Stephen F. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Identification of Ligands That Bind to the Orphan Delta2 Receptor
[unreadable] DESCRIPTION (provided by applicant): The glutamate receptor family is comprised of 18 genes that can combine to form dozens of different heteromeric receptor complexes. This receptor class mediates most excitatory synaptic transmission in the mammalian brain, and plays a central role in cellular models of learning and memory as well as a wide range of neuropathologies. Of these gene products, two orphan receptor subunits (delta1 and delta2) have been designated to be in the glutamate receptor family on the basis of sequence homology even though no activating ligand has been identified. The delta2 subunit is expressed at cerebellar synapses, and a variety of electrophysiological studies suggest that delta2 plays an important role in cerebellar development and synaptic plasticity. In addition, mice that either lack the delta2 subunit or express a constitutively active mutant form of this receptor (delta2-lurcher), show distinct phenotypes with motor disturbances. Thus, a paradox exists whereby the delta2 glutamate receptor subunit is thought to play an important role in brain function yet there is a vacuum regarding what we know about the receptor. Our collaborators have recently used crystallography to determine that a small amino acid (D-serine) can bind to the delta2 ligand binding domain, making a set of atomic contacts that are consistent with glutamate binding at other glutamate receptor subunits. Interestingly, although D-serine binds to the delta2 subunit, it does not induce any current response in wild- type receptors. However, a naturally occurring point mutation within the delta2 subunit (the lurcher mutation) produces constitutively active homomeric channels, and application of D- serine to these channels inhibits the tonic current flowing through these constitutively active channels. These data suggest that binding of D-serine can lead to changes in gating, and raises the possibility that incorporation of the delta2 subunit into heteromeric receptor assemblies may endow glutamate receptors with a regulatory site for D-serine. These new data provide an unprecedented opportunity to explore delta2 receptor structure, function, and pharmacology. We propose to exploit this recent finding in 2 lines of experimentation: Aim 1: Identify new ligands that potently activate or inhibit delta2 receptors. Aim 2: Evaluate whether co-expression of delta2 with other glutamate receptor subunits adds D-serine sensitivity to the receptors. Completion of these studies will provide the first comprehensive pharmacological evaluation of this receptor class, as well as new tools with which to study its function and regulation in brain. PUBLIC HEALTH RELEVANCE: Our recent findings that D-serine can bind to the orphan glutamate receptor delta2 has provided an opportunity to significantly advance our understanding of the function of this receptor, which has been enigmatic for over a decade despite in vivo findings that suggest an important role in cerebellar development and synaptic plasticity. The goal of these studies is to identify novel and selective pharmacological agents (activators and antagonists) that could be useful research tools for understanding delta2 function in tissue. In addition, we will also test whether delta2 subunit alters the effects of D-serine on hetero-mulitmeric glutamate receptors. Completion of these studies will advance our understanding of delta2 function, which holds implications for neuronal signaling, synaptic plasticity, and neuronal development in the cerebellum. These studies may also provide insight into new therapeutic agents that target the delta2 receptor. [unreadable] [unreadable]
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1 |
2009 — 2018 |
Traynelis, Stephen F. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanism of Action of Novel Subunit-Selective Nmda Receptor Modulators
DESCRIPTION (provided by applicant): N-methyl-D-aspartate-selective glutamate receptors (NMDA receptors) are ligand-gated ion channels that mediate excitatory synaptic transmission in the brain and spinal cord. NMDA receptors are heteromultimers comprised of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four family members (NR2A,B,C,D). NMDA receptors are involved in many normal brain functions such as neuronal development, learning, memory, in addition to their roles in neuropathological conditions such as stroke, epilepsy, and neuropsychiatric disorders. Since its first description, the NR2B-selective NMDA receptor antagonist ifenprodil and its many analogues have been used in a multitude of studies exploring the role of this subunit in virtually every aspect of brain function including behavior, cognition, synaptic plasticity, neuronal development, and neuropathology. However, no highly selective antagonists have yet been described for NR2A-, NR2C-, or NR2D-containing receptors. We therefore developed an assay to identify non-competitive allosteric modulators of NR2C- and NR2D-containing NMDA receptors, and subsequently screened ~60,000 compounds for new subunit-selective antagonists and potentiators. The result was the identification of two structurally unique classes of compounds that are 100-500 fold selective inhibitors of NR1/NR2C and NR1/NR2D compared to NR2A- or NR2B-containing receptors or AMPA/kainate receptors. We also identified two structurally distinct classes of potentiators that are selective for NR2C/D-containing receptors. These new non-competitive allosteric modulators represent a breakthrough opportunity to study the role of the NR2C/D subunits in normal brain function and in neurological diseases. This proposal addresses 3 questions using these subunit-selective modulators. 1. What are the structural determinants of NR2C/D-selective inhibitors and potentiators? We will utilize site-directed mutagenesis, exploiting sequence differences between NR2A/B and NR2C/D, to identify key residues that mediate the actions of three classes of NR2C/D-selective potentiators and inhibitors. 2. What is the mechanism of action of NR2C/D selective inhibitors and potentiators? We will analyze macroscopic and single channel currents recorded under voltage clamp to define the mechanism of action of the non-competitive NR2C/NR2D inhibitors as well as NR2C/NR2D potentiators. 3. How do NR2C/D modulators alter synaptic signaling and neuronal excitability? We will evaluate the effect of inhibition and potentiation of NR2D-containing NMDA receptors at the cortical-subthalamic neuron synapse and NR2C/D-containing NMDA receptors at afferent excitatory synapses onto hippocampal interneurons in brain slices. Experiments will test the synaptic response to stimulus trains in the presence of NR2C/D modulators. We will also use these new pharmacological tools to investigate whether interneuron and subthalamic neuron excitability and spiking frequency can be altered through inhibition or potentiation of NR2C/D-containing receptors.
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1 |
2010 — 2013 |
Traynelis, Stephen |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Control of Ampa Receptor Function by Phosphorylation
DESCRIPTION (provided by applicant): The AMPA-type glutamate receptors are ligand-gated cation channels that mediate fast excitatory neurotransmission in the brain, and thus are critically involved in all aspects of brain function including cognition, movement, learning, and memory. The function and number of postsynaptic AMPA receptors are dynamically regulated to control the strength of synaptic connections, and this plasticity is a key feature of cellular models of learning and memory. Signals that trigger synaptic plasticity lead to phosphorylation of AMPA receptors by protein kinases, and this phosphorylation controls AMPA receptor trafficking and function. Phosphorylation by protein kinase C (PKC) or Ca2+/calmodulin dependent kinase II (CamKII) of an intracellular serine residue (Ser831) located on the GluR1 subunit enhances AMPA receptor function to increase synaptic strength during expression of long-term potentiation (LTP), one model of synaptic plasticity. Although previous studies observed that CamKII phosphorylation of GluR1 enhances the single channel conductance, no conceptual or structural mechanism has been described for this unique form of ion channel regulation. The goal of the experiments proposed here is to understand functionally, structurally, and conceptually how phosphorylation of GluR1 Ser831 potentiates AMPA receptor function. We will focus on Ser831 in GluR1 because of the unique mechanism of potentiation (increased unitary conductance), and will expand the study to evaluate for the first time how three nearby phosphorylation sites (Ser818, Thr840, Ser845) might functionally interact with phospho-Ser831. Furthermore, we will test whether the effects of phospho-Ser831 reflect either intra-protein interactions between the phospho-Ser831 and intracellular portions of the receptor, or inter-protein interactions between phospho-Ser831 and GluR1 binding partners. Completion of these studies will provide a comprehensive functional and structural understanding of an under-studied feature of synaptic plasticity-phosphorylation mediated changes in postsynaptic AMPA channel function. The proposed experiments address three questions: 1. What is the mechanism by which phosphorylation regulates AMPA receptor function? Single channel currents will be recorded to determine how phosphorylation of Ser831 controls GluR1 function. We will also evaluate the interactions of Ser831 with nearby phosphorylation sites, and validate our conclusions in neurons. 2. What is the structural basis for phospho-serine regulation of AMPA receptor function? We will identify intracellular GluR1 residues as phospho-Ser831 hydrogen bonding partners. We will additionally search for inter-protein interactions involving GluR1 that depend on the phosphorylation of Ser831. 3. Can models of independent subunit gating describe AMPA receptor regulation by phosphorylation? We will analyze the response of patches with one active GluR1 channel (plus stargazin) to the rapid application of a maximally effective concentration of glutamate. These data will be used to develop a novel model of subunit gating that can account for the potentiation of GluR1 channel function by phosphorylation of Ser831. PUBLIC HEALTH RELEVANCE: AMPA receptors mediate communication between neurons in the central nervous system, and thus play an important role in virtually all brain functions. The AMPA receptors are comprised of four different subunits (GluR1-4). Among these, the GluR1 subunit has been shown to play a unique role in activity-dependent synaptic plasticity. GluR1 is subject to C-terminal phosphorlyation by a variety of kinases, and this phosphorylation can influence trafficking to the membrane and AMPA receptor function. In this proposal we examine CamKII phosphorylation of GluR1-Ser831. CamKII activation has been shown to be a critical step in some forms of synaptic plasticity, presumably through phosphorylation of the GluR1 subunit. Phosphorylation of GluR1-Ser831 increases single channel conductance by an unknown mechanism. This proposal describes three series of experiments that will evaluate the underlying functional and structural mechanisms of the effects on receptor function following phosphorylation at GluR1-Ser831. Understanding how AMPA receptor function is sculpted by intracellular signaling pathways is an important step towards understanding the mechanisms of synaptic plasticity, which likely underlie higher order functions such as learning and memory.
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1 |
2012 — 2013 |
Traynelis, Stephen |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Optimization of Small Molecule Probes
DESCRIPTION (provided by applicant): N-methyl-D-aspartate (NMDA) receptors are postsynaptic ligand-gated ion channels that mediate excitatory synaptic transmission in the CNS. NMDA receptors are heteromultimers comprised of 2 glycine-binding NR1 subunits and 2 glutamate-binding NR2 subunits, of which there are four types (NR2A,B,C,D). NMDA receptors are involved in normal brain functions such as development, learning, and memory. In addition, NMDA receptor hypo-function may contribute to neuropsychiatric disorders such as schizophrenia, which has lead to the hypothesis that potentiation of NMDA receptor function could be therapeutically useful. However, no small molecule NMDA receptor potentiators exist with which to obtain proof-of-concept data. Given the lack of subunit- selective tool compounds, our objective in this proposal (in response to PAR-09-251 Optimization of small molecule probes for the nervous system, a reissue of RFA-NS-09-003) is to identify potent, subunit-selective potentiators of NMDA receptors that can be used to test specific hypotheses about NMDA receptor function in neurological diseases. To accomplish this, we developed an assay for non-competitive allosteric modulators of NMDA receptors and screened ~100,000 compounds. We identified 6 distinct but related molecules that act at a similar site to potentiate NR2C/D-containing NMDA receptor function; one scaffold appears to favor NR2C potentiation. Our working hypothesis is that these multiple scaffolds can serve as a starting point for the development of potent subunit- selective NMDA receptor potentiators. We propose to use medicinal chemistry to develop a compound with an EC50 value below 300 nM, maximal potentiation of > 2.5-fold, selectivity against other glutamate receptors > 200-fold, solubility > 10-fold EC50, lack of neurotoxicity and selectivity against 72 other CNS receptors, channels, and pumps of between 30-1000 fold. Experiments will address two questions: 1. What structural features control potency and efficacy of NR2C/D-selective NMDA receptor potentiators ? We will use parallel medicinal chemistry approaches to synthesize and test 200 analogues per year, a number supported by our preliminary data. The EC50 value for each compound will be determined at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1 receptors using 14 automated two-electrode voltage-clamp recording systems that can record/analyze concentration-effect curves for > 500 compounds/yr. Preliminary studies have identified several regions of the scaffold that control potency and subunit-selectivity with available chemical space. 2. What are the off-target liabilities and pharmacokinetic properties of NR2C/D potentiators? We will determine solubility and metabolic stability for all active compounds, and screen the best compounds at key decision points and again at the completion of the study against 72 receptors, channels, and transporters using a combination of automated multi-well assays both in the lab and at an NIMH-funded off-target screening center. This information will help to identify the best scaffold to pursue (year-1) as well as characterize the two best compounds to emerge from this study (year-2). We will evaluate plasma half-life, brain:plasma ratio, and neurotoxicity in vitro.
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0.915 |
2016 — 2019 |
Traynelis, Stephen F |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Functional Effects of Ion Channel Mutations Found Via Exome Sequencing
Genomic analysis of patients with neurological conditions is becoming increasingly more common, providing a great deal of genomic data relating changes in patient DNA to neurological disease. However, the rate at which genetic mutations are identified has not been matched by advances in understanding the functional effects of these mutations on target proteins. For most genes, known disease-associated missense mutations far outnumber mutations for which functional data exists. Thus, a large gulf exists between the mutations we know about and our understanding of how individual mutations impact protein function. This gulf is growing wider with the accelerating pace of sequencing. While computational methods exist that predict which missense mutations may be harmful, these algorithms are a poor substitute for functional data. Thus, there is a critical need for a means by which clinically-oriented laboratories can obtain functional insight into the effects of mutations. This R24 proposal seeks to fill the gap between mutations and function for the ligand-gated glutamate receptor gene family, which includes AMPA, kainate, and NMDA receptors encoded by GRIA, GRIK, and GRIN genes, respectively. The decision to focus efforts on one gene family was made in consultation with NIH, and was driven by the budget for this funding opportunity. The number of known mutations in this family of genes encoding NMDA receptor subunits is growing at a fast pace, with well over a hundred novel disease- associated mutations described in just the past 18 months, and many more known but unpublished. Thus, while we will focus on all glutamate receptor mutations, we expect most mutations identified will be in the GRIN family. Interested investigators with glutamate receptor mutations will share the amino acid change, which will be introduced into commercial cDNAs for use in heterologous expressions systems to obtain functional data describing the effects of the mutation. The Resource Center will share this functional information with the clinical lab, and work to facilitate publication of the data. This effort will fill a critical gap in the literature, advance our understanding of neurological disease, and provide new ideas about future treatment paradigms. Three activities will be supported by this R24 resource grant. First, we will contact at least 100 laboratories performing whole exome/genome sequencing to inform them of this resource. Second, for clinics or investigators who have identified GRIN mutations and would like to obtain functional analysis, we will introduce the mutation into commercially available human cDNAs and functionally evaluate the effects of the mutation on agonist potency, modulator sensitivity, response time course, and receptor surface expression. Third, we will develop a searchable database on the web to archive results from this project, and connect our results with several existing databases (ClinVar, EGI, etc.).
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1 |
2018 — 2021 |
Papa, Stella M [⬀] Traynelis, Stephen F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Regulation of Motor Function in Parkinson's Disease
Project Summary This project investigates the functional changes in striatal neurons that develop in the chronic course of Parkinson's disease (PD) and largely involve the glutamatergic signaling. In PD, the loss of DA modulation in the striatum leads to significant changes in the function of striatal projection neurons (SPNs), which then play a key pathophysiological role in motor symptoms. The dysregulation of SPNs is evidenced by a significant amount of data including major morphological and physiological changes. In particular, SPNs are markedly hyperactive in animal models and patients with PD, and this upregulation is mediated by glutamatergic input from cortex and thalamus. However, the mechanisms underlying these changes are not fully understood, and the role of particular NMDAR and AMPAR signaling is not known. Here, we will profile thoroughly the SPN changes in advanced PD and determine the impact of NMDAR and AMPAR subunit components on pathological signaling. We take a novel approach using recently developed technologies for cellular identification in primate recordings and new pharmacological tools to test glutamate mechanisms with high selectivity. The ultimate goal of this project is to uncover and validate new therapeutic targets to improve motor functionality and help patients with PD. The project includes three specific aims. In the first aim, we will determine the abnormal activity pattern of identified SPN subtypes using optogenetics in the primate model of advanced PD that reproduces the full extent of the motor phenotype of the human disease. In the second aim, we will examine the regulation of expression of glutamate receptor subunits after dopamine loss in rodent and primates to determine the potential participation of subunits in the abnormal signaling. We will use rodent models for ex-vivo physiology with comprehensive analyses of behavioral paradigms and tests of subunit-selective inhibitors. In the third aim, we will take advantage of the extensive analysis of subunit roles to pinpoint the mechanisms that may account for functional SPN changes in the primate, and challenge them with the selected inhibitors directly in the striatum for physiologic and behavioral effects. This project employs diverse experimental approaches across multiple disciplines to address an important health problem, from the use of novel viral vectors and pharmacological agents, to the ex-vivo and in-vivo evaluation of identified neurons in rodent and primate PD models, to the final evaluations of pathophysiologic mechanisms in the parkinsonian primate. The data from these translational studies will be influential in the field, advance our understanding of pathophysiologic mechanisms, and catalyze the development of new therapeutic strategies in Parkinson's disease.
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2019 — 2021 |
Traynelis, Stephen F |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Glutamate Receptors and Human Neurological Disease
This R35 Research Program proposal will use electrophysiological, molecular, and structural approaches to probe multiple aspects of excitatory synaptic function that are relevant for neurological disease. We will focus on the elucidation and modulation of the functional properties of postsynaptic glutamate receptors. We will take advantage of coincident advances in cryoEM, genetics, robotics, and receptor biology to address questions that were previously inaccessible. Four approaches will address critical gaps in our understanding of synaptic function and provide therapeutically-relevant insight into neurological disease. First, we will explore the functional and clinical implications of regional intolerance for variation in the healthy population as well as de novo disease-associated glutamate receptor mutations, most commonly found in GRIN1, GRIN2A, GRIN2B, GRIN2D, genes that are among the least tolerant in the genome. We will establish the relationship in healthy individuals between allelic frequency and functional changes, which is necessary in order to understand the potential role of SNPs in these genes as disease risk factors. Evaluation of these rare variants will also provide opportunities for precision medicine, and advance our understanding of receptor function. Second, we will develop novel compounds for proof-of-concept studies to identify new therapeutic strategies for neurological disorders. We will synthesize subunit-selective modulators of agonist potency and channel open probability to assess the roles of understudied NMDA receptor subunits (e.g. GluN2C, GluN2D) in circuits in cortex, thalamus, and striatum. We will determine the site and mechanism of action of modulators of NMDA receptors, and use medicinal chemistry to improve brain penetration, potency, and solubility in collaboration with Dennis Liotta in Chemistry at Emory. We will use the pharmacological tools that we develop to gain insight into the treatment of epilepsy, stroke, Parkinson?s disease, and Alzheimer?s disease. Third, we explore biased modulators of NMDA receptors by developing the SAR of two classes of compounds that alter ion channel selectivity. These compounds represent the first example whereby a pharmacological agent can alter channel permeation properties, demonstrating that modulators can tune distinct functions of the NMDA receptor. These compounds hold enormous potential as neuroprotectants that diminish cation flux without side effects associated with receptor blockade, which we will evaluate in vivo in models of ischemia. Fourth, we will combine information obtained through genetic analysis of regional intolerance, mechanism of allosteric modulation, and new advances in structural biology to advance our understanding of the mechanisms that convert glutamate binding to channel opening. These experiments will focus on the shared regions of the protein that control channel opening, which are the key sites of action of our allosteric modulators and common sites for disease-associated human mutations. We will collaboratively perform cryoEM and crystallographic studies to determine the binding site for modulators, as well as key features of channel structure and function.
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2021 |
Traynelis, Stephen F |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Genetic Analysis to Determine the Functional Role of Grid1
Summary The glutamate receptor gene family encodes AMPA, kainate, and NMDA receptors, which mediate excitatory synaptic transmission in the central nervous system. Two additional gene family members, GRID1 and GRID2, encode the enigmatic delta receptor GluD1 and GluD2 subunits, which can bind D-serine but do not appear to activate conventional signaling systems. There are four functional effects known for delta receptors: (1) Both delta receptors (GluD1 and GluD2) are activated when certain mutations occur in the transmembrane helices, which convert a non-gating receptor into a channel that is constitutively open, producing a tonic inward current. In addition, chimeric receptors in which the glutamate binding domain from a kainate or AMPA receptor replaces the analogous D-serine binding domain for GluD1 and GluD2 can be activated by glutamate, suggesting that the highly specialized machinery needed to convert agonist binding into pore opening is conserved in delta GluD1 receptors. (2) The binding of D-serine to GluD1 receptors harboring a TM3 mutation that renders them constitutively active can close the channel, raising that possibility that D-serine binding produces meaningful conformational changes with unknown physiological roles in WT receptors. (3) Ca2+ binding to a site at the dimer interface between two adjacent D-serine binding domains potentiates constitutive current in mutant receptors, suggesting Ca2+ could regulate GluD1 conformation and function. (4) The distal extracellular domain serves as a ligand for presynaptic Cbln2 and neurexin, which can alter synapse formation. Whereas Cbln2 and neurexin binding to GluD1 appears to be critical, it remains unclear whether channel gating, D-serine binding, or Ca2+ regulation of GluD1 play important roles in brain. A unique power of population genetics is that it can identify key functions of a protein in an unbiased manner. GRID1 is one of the least tolerant genes in the body, falling in the top 2 percentile for lacking variation, suggesting it plays essential roles. Consistent with this idea, patients with neurological conditions have been identified with missense variants in GRID1 that are absent in the general population. We will evaluate the effects of disease-associated variants in addition to well-tolerated variants commonly observed in the healthy population on three modalities associated with GRID1 function?constitutive activation, D-serine binding, Ca2+ binding. If any of these functional attributes are important, then we expect to find disease- associated variants that perturb them, while variants present in the standing population should be without effect. Three electrophysiological experiments will answer the following questions: Aim 1: Can missense variants produce active ion channels that are involved in neuropathology? Aim 2: Do missense variants alter the actions of D-serine and Ca2+ on constitutively active channels? Aim 3: Do missense variants alter the actions of glutamate on GluD1-GluK2 chimeric receptors?
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