Gabriela K. Popescu - US grants

DESCRIPTION(Provided by applicant): NMDA receptors are glutamate-gated ion channels that mediate the slow component of fast excitatory synapses.Intrinsic channel activation properties shape the post-synaptic current and contribute to critical physiological |processes in neurodevelopment and synaptic plasticity. The kinetic mechanism of NMDA receptor activation iscomplex and poorly understood. However, it represents the molecular basis of glutamate excitotoxicity underlyingneuronal cell death that occurs in stroke, epilepsy and neurodegenerative disorders. We will develop a kineticstate model describing the NMDA receptor activation and will use this model to identify structure -functionrelationships in the glutamate-binding domain. In Aim 1, single-channel currents elicited by steady-stateconcentrations of glutamate will be recorded in cell-attached patches from HEK 293 cells that express recombinantNR1/NR2A channels. We will use kinetic modeling to determine the number of closed and open states, theinterconnections between these states, and the corresponding rate constants that best describe these data. InAim 2, we will test and refine the model developed in Aim1 by using it to fit currents recorded from NR1/NR2Areceptors in excised patches, during jumps into different concentrations of glutamate. In Aim 3, we will recordsingle channel currents from NMDA receptors carrying single-substitution mutations in NR2A and NR2B subunitsand calculate rate constants for the microscopic transitions postulated by our kinetic model. This analysis willprobe the mechanism of glutamate binding, channel gating and/or desensitization. A description of the NMDAreceptor activation kinetics and these structure-function relationships will enhance our understanding of fastexcitatory synapses and will become instrumental in developing specifically targeted therapies.

microscopy; biomedical resource; lasers; clinical research;

DESCRIPTION (provided by applicant): NMDA receptors (NRs) mediate fast excitatory transmission in the brain. Their activation is critical for the normal development, maintenance and continual remodeling of excitatory synapses. Excessive NR activity is a disease mechanism in stroke, chronic pain, epilepsy, and neurodegenerative disease, whereas insufficient NR signaling has been involved with schizophrenia and cognitive disorders. Numerous endogenous and pharmacologic agents modulate NR activities and are potential candidates for therapeutic intervention. The mechanisms governing the allosteric control of NR activity are poorly understood and empirical attempts to control NR function have so far yielded disappointing results. The OBJECTIVE is to understand how allosteric modulators control NR physiologic functions in terms of molecular mechanisms, integration and consequences on synaptic physiology. The AIMS addressed in this proposal are to: i) characterize individual NR allosteric modulators in terms of their effects on NR gating dynamics and on non-stationary macroscopic behaviors;ii) establish how multiple allosteric signals are integrated to result in responses with distinct signaling profiles;and iii) investigate how allosteric modulation of NR responses impacts on synaptic physiology. First, using kinetic analysis of single-channel currents and statistical modeling we will identify and quantify the actions of individual modulators on NR elementary kinetic transitions during gating. These measured rate constants are intrinsically rich with mechanistic information;in addition they can predict modulator effects on NR macroscopic behaviors and on NR- mediated synaptic function. These predictions will be verified by measuring ensemble NR responses to patterned stimulation in excised membrane patches and by measuring evoked NR synaptic responses in brain slices. For NRs whose activation reaction is complex, this approach is entirely novel and may identify effective means to modulate specific receptor functions in separation of each other. Taken together the results will help to compile an integrated mechanistic view of how allosteric control of NR activity impacts synaptic physiology. This view should suggest new, combinatorial approaches to specifically target harmful receptor behaviors while preserving the critical functions played by NRs .

[unreadable] DESCRIPTION (provided by applicant): NMDA receptors are membrane-bound neurotransmitter receptors with critical roles in brain physiology. They are principal drug targets for Alzheimer's disease and stroke, mental retardation, and also addiction, chronic pain, schizophrenia and epilepsy. NMDA receptors are glutamate-activated, excitatory ion-channels. Their activation reaction is initiated when the neurotransmitter glutamate binds to extracellular, ligand-binding domains (LBDs) and culminates with the opening of a membrane-embedded cation-selective pore. The molecular events that make up the activation reaction of NMDA receptors remain unknown, despite their fundamental role in controlling receptor function. Kinetic studies of single-receptor activity have established that after binding agonists, NMDA receptors cycle with measurable rates between several closed-pore and open-pore conformations. Structural studies of the isolated LBDs have shown that ligands bind deep inside a crevice between two mobile lobes and have put forth the hypothesis that ligand-induced closure of the LBD is one of the required steps along the NMDA receptor activation pathway. This application proposes experiments that will directly and specifically test this hypothesis by examining the activity of NMDA receptors with restricted mobility of the LBDs. Pairs of cysteine residues will be introduced at the tips of the LBDs lobes as a means to lock these at defined distances with respect to one another. The engineered receptors will be examined by kinetic analyses of their macroscopic and single-channel behaviors in the presence and absence of oxidizing agents, and following treatment with crosslinking reagents of various lengths. The results will provide basic information regarding the identity of the molecular motions associated with NMDA receptor activation and will afford valuable insight regarding possible mechanisms of drug action at this receptor. This knowledge will form the conceptual foundation necessary to rationally control NMDA receptor activities as a therapeutic strategy for stroke, addiction, and mental illness. PUBLIC HEALTH RELEVANCE: This project will provide fundamental information regarding the mechanism by which NMDA receptors become active. This information is currently lacking and is needed for the rational design of novel therapeutic approaches for acute and chronic neurodegeneration (stroke, Alzheimer's disease), addiction, chronic pain, cognitive disorders and mental illness. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Numerous endogenous and synthetic ligands modulate NMDA receptor activities and are potential candidates for therapeutic intervention in a number of neurologic disorders. To date, empirical attempts to control en masse NMDA receptor-mediated fluxes have had only modest success in the clinic, mainly due to inadequate understanding of the mechanisms governing the allosteric control of NMDA receptor activities and of the specific roles played by these activities in brain physiology and pathology. Over the previous funding period the objective has been to delineate the mechanisms by which endogenous modulators (protons, zinc/ifenprodil, glycine) affect NMDA receptor gating dynamics and thus control the macroscopic response relevant to synaptic signaling. Over the next funding period the objective is to delineate the intracellular protein motions that constitute the NMDA receptor activation. The general approach is to capitalize on the recently solved atomic-resolution structure for a GluA2 tetrameric receptor, a NMDA receptor homologue and our growing expertise on NMDA receptor gating modulation. We will introduce mutations to perturb (increase and decrease) the relative mobility of NMDA receptor structural modules and will delineate the accompanying changes in reaction mechanism by kinetic analyses of single-molecule signals. Further, we will explore the time course of macroscopic responses obtained from receptors with restricted or enhanced internal motions to better understand how specific structural features support the unique biological functions played by NMDA receptors in brain physiology and pathology. Overall this work will provide critical information about structural correlates of NMDA receptor activation and will integrate the currently isolated structural and kinetic models of gating. Given that glutamate receptors mediate more than 90% of excitatory transmission in brain and NMDA receptors are critical to many fundamental brain functions, knowledge generated by the proposed experiments is likely to have wide impact on the fields of neurotransmission and neuromodulation.

Project Summary/Abstract In the central nervous system, proteins experience mechanical cues that vary widely across developmental stage, cell type and location, and physiological state. When acting on membrane embedded ion-channel proteins, mechanical forces can modulate the ionic flux produced by the physiological activator or can directly gate the channel. In this application, we explore the hypothesis that mechanical forces can open NMDA receptors in the absence of the endogenous neurotransmitter glutamate. NMDA receptors are glutamate-gated excitatory receptors that are widely expressed at synaptic and extrasynaptic sites in brain and spinal cord, where they play key roles in physiology and pathology of excitatory synaptic development and plasticity. These key functions rely on unique biophysical properties such as, among others, slow kinetics, large calcium permeability, voltage-dependent Mg2+ block. In this application, we propose to pursue two interrelated aims. The first will be done in recombinant receptors and will examine the type and intensity of force that can gate the channel, the biophysical properties of the force-induced current (kinetics, conductance, permeability, etc.), and how the channel senses the mechanical cue. The second aim will be done in endogenous receptors (primary cultured neurons) and will begin to explore possible roles of mechanically gated NMDA receptor currents in physiologic and pathologic conditions. In both aims, we will use electrophysiology and optical methods to monitor NMDA receptor response, total and calcium current, to experimentally-controlled mechanical perturbations. These experiments will delineate what kind of mechanical forces can activate NMDA receptors and how the signals produced by force and by glutamate compare, and will help to predict the physiological and pathological situations where mechanical forces can shape neuronal function specifically by gating NMDA receptor currents. Given that the mechanosensitivity of NMDA receptor signals is yet uncharted, the results will lay the groundwork necessary to understand how NMDA receptors contribute to the impact of mechanical forces on synaptic function and dysfunction.

? DESCRIPTION (provided by applicant): NMDA receptor activations produce substantial elevations in intracellular calcium levels, which initiate signaling cascades critical for normal ad pathological brain processes. These include normal synaptic development and plasticity, as well as psychiatric conditions such as addiction and chronic pain, schizophrenia, and neuropathologies such as stroke and Alzheimer's disease. Until recently it was assumed that the amount of calcium in the NMDA receptor current is fixed and controlled solely by gating modulators and channel blockers. This project investigates a newly reported modulatory mechanism by which intracellular signaling cascades can change in a reversible fashion the amount of calcium in the NMDA receptor current. This work seeks to identify the molecular determinants and mechanisms responsible for this new modulation. The resulting knowledge will inform novel strategies to address dysfunctions and pathologies where NMDA receptor- mediated calcium is a cause or an aggravating factor.

Project Summary  Optimal refinement of neural circuits during development is a highly controlled process that depends critically on  experience. Ample genetic evidence in mental disorders points specifically to defects in molecular targets related  to experience-­dependent developmental plasticity of excitatory synapses, and dysregulation of this fundamental  developmental process results in a variety of neuropsychiatric diseases. This project seeks to elucidate  mechanisms by which experience sculpts the functional connection of excitatory synapses during development  and how perturbations in this process can derail the normal developmental trajectory. We found that during the  critical period of their functional maturation, excitatory synapses of the mouse primary visual cortex (V1) maintain  a dynamic equilibrium in their AMPA receptor-­mediated transmission. This equilibrium requires neurogranin (Ng),  a  postsynaptic calmodulin-­binding protein  important  for  synaptic  plasticity, which is has been implicated in  schizophrenia and mental retardation. Our preliminary studies show that in addition to controlling incorporation  of  AMPA  receptors  into  AMPA  receptor-­lacking (silent) synapses and synaptic pruning, Ng levels also control  the timing of the developmental switch in NMDA receptor subunits, and change the phosphorylation profiles of  several post synaptic proteins including NMDA receptor and PSD-­93/95. This project investigates the hypothesis  that Ng levels influence the experience-­dependent reorganization of excitatory synaptic connectivity by altering  Ca/CaM-­dependent  signaling  pathways, including  PP2B  and  NMDA  receptors, using a combination of virus-­ mediated gene manipulation, synaptic physiology, channel biophysics, morphological analysis, and behavioral  interrogation. The results will elucidate the molecular pathways governing experience-­dependent refinement of  excitatory synaptic connectivity during development and will help to identify potential targets for pharmacologic  interventions in patient with neurodevelopmental disorders.     

Abstract NMDA receptors play critical roles during the normal development and function of central synapses. Mutant receptors were recently identified in patients with neurologic and psychiatric conditions and were found to be causal to the diagnosed dysfunctions. The long-term objective of this project is to correlate functional receptor states postulated by the established kinetic mechanism with structural conformations. Specifically, the two aims proposed here will integrate molecular dynamics simulations and electrophysiological measurements to delineate plausible conformations for the adult NMDA receptor isoform in closed and open conformations and for a series of rationally-targeted and naturally occurring, pathologic NMDA receptor mutants. Results will elucidate the NMDA receptor gating reaction and the mechanism by which single-residue substitutions change structure and cause dysfunction.