James Huettner, Ph.D. - US grants

Glutamate-gated ion channels play a number of important roles in the nervous system. They are known to mediate the depolarization of postsynaptic neurons at fast excitatory synapses and are thought to regulate transmitter release from presynaptic terminals of some cell types. In addition, they have been implicated in neuronal cell death associated with anoxia and hyperexcitation. Although much has been learned about glutamate receptors in the past 10 years, many aspects of their function remain poorly understood. The long term objective of the proposed research is to provide a better understanding of excitatory synaptic transmission by characterizing the operation of receptors and channels that are activated by glutamate. A second major goal is to uncover properties of the channels that may allow for clinical intervention to prevent excitotoxic cell death. Patch clamp techniques will be used to record the whole-cell and single channel currents evoked by excitatory amino acids in neurons maintained in primary cell culture and in freshly dissociated cells from peripheral sensory ganglia or from the CNS. The work involves two main projects. The first set of experiments will test the hypothesis that negative charges on the external face of the N-methyl-D-aspartate (NMDA) receptor in central neurons contribute to its unique gating and permeation properties, including regulation of the channel by glycine, Zn, polyamines, protons, Mg, and dizocilpine. The second project focuses on the activation and desensitization of non-NMDA receptors in CNS neurons and in dorsal root ganglion neurons, which express a novel form of this receptor. The specific aim is to determine the mechanism or mechanisms that underlie desensitization by studying the action of ions, drugs and proteins that are known to change the gating behavior of non-NMDA receptor channels. Another major focus of this project is to characterize the pharmacology of receptors expressed by DRG neurons with the goal of finding selective agents that can distinguish between the central and peripheral forms of the receptor. Such agents will be critical for establishing the function of the DRG receptor; they also might prove capable of controlling the sensation of pain, because the expression of this receptor in DRGs appears to be restricted to nociceptive neurons.

Glutamate-gated ion channels play a number of important roles in the nervous system. They are known to mediate the depolarization of postsynaptic neurons at fast excitatory synapses and are thought to regulate transmitter release from presynaptic terminals of some cell types. In addition, they have been implicated in neuronal cell death associated with anoxia and hyperexcitation. Although much has been learned about glutamate receptors in the past 10 years, many aspects of their function remain poorly understood. The long term objective of the proposed research is to provide a better understanding of excitatory synaptic transmission by characterizing the operation of receptors and channels that are activated by glutamate. A second major goal is to uncover properties of the channels that may allow for clinical intervention to prevent excitotoxic cell death. Patch clamp techniques will be used to record the whole-cell and single channel currents evoked by excitatory amino acids in neurons maintained in primary cell culture and in freshly dissociated cells from peripheral sensory ganglia or from the CNS. The work involves two main projects. The first set of experiments will test the hypothesis that negative charges on the external face of the N-methyl-D-aspartate (NMDA) receptor in central neurons contribute to its unique gating and permeation properties, including regulation of the channel by glycine, Zn, polyamines, protons, Mg, and dizocilpine. The second project focuses on the activation and desensitization of non-NMDA receptors in CNS neurons and in dorsal root ganglion neurons, which express a novel form of this receptor. The specific aim is to determine the mechanism or mechanisms that underlie desensitization by studying the action of ions, drugs and proteins that are known to change the gating behavior of non-NMDA receptor channels. Another major focus of this project is to characterize the pharmacology of receptors expressed by DRG neurons with the goal of finding selective agents that can distinguish between the central and peripheral forms of the receptor. Such agents will be critical for establishing the function of the DRG receptor; they also might prove capable of controlling the sensation of pain, because the expression of this receptor in DRGs appears to be restricted to nociceptive neurons.

The long term objective of my work is to provide a better understanding of synaptic transmission by studying the operation of NMDA, AMPA, and kainate receptors, which form ion channels gated by the neurotransmitter glutamate. Another major goal is to uncover properties of these receptors that may allow for clinical intervention to prevent excitotoxic cell death. The experiments outlined in this proposal focus on the functional properties of kainate receptors. This glutamate receptor subtype is thought to regulate neuronal excitability and may contribute to excitotoxicity under certain conditions; but, currently much less is known about kainate receptors than about NMDA and AMPA receptors. Patch clamp techniques will be used to record the whole-cell add single channel currents evoked by excitatory amino acids in isolated neurons that express native glutamate receptors, and in heterologous cells that express cloned glutamate receptor subunits. Kainate receptors will be characterized with respect to their agonist and antagonist pharmacology, desensitization properties, current-voltage relations, relative permeability to calcium and monovalent ions, and susceptibility to modulation. For experiments on CNS neurons, NMDA and AMRA receptors will be blocked with the non- competitive antagonists MK-801 and GYKI 53655, respectively. A second major aim of this study is to test the hypothesis that kainate receptors regulate synaptic transmission. The action of kainate initially will be studied on synapses formed by hippocampal neurons grown in microcultures. In preliminary experiments, the agonists kainate and domoate have been shown to inhibit both excitatory and inhibitory synapses. The possible mechanisms that may underlie this inhibition will be examined in detail. We will seek to establish whether this action involves pre or postsynaptic kainate receptors and whether they are identical to the receptors that mediate kainate currents in these cells. In addition, we will seek to determine the conditions under which kainate receptors may be activated by the endogenous agonist, glutamate.

9871874
Ruoff
This award provides partial support for an effort addressing the manipulation of matter on the nanoscale to form functional nanostructures. New methods and tools will be developed and used to: (i) study a new type of mechanochemistry were carbon nanotubes are mechanically strained to create very specific atom locations for chemical reaction (nanostressing stage); (ii) controllably shape surfaces, manipulate nanotubes and build structures with the AFM probe used in a new, unconventional way; (iii) dispense gas and liquid molecules (the latter in volumes down to 100 nm3) with sub-nm positional control (nanotube pipet); (iv) achieve spatiotemporal control over a new class of charged or magnetic particles using finely controlled electric fields or magnetic field gradients (designer particles).

Development of new tools and methods for nanoscale manipulation will be centered around carbon nanotubes (NT). Unique features of NT's as potential building blocks for nanotechnology include: unique size; the presence of a hollow core; unusual mechanical properties; possibilities for mechanically activated chemistry. Tools and methods which will be developed in this study will be essential for achieving one of the long term objectives of the proposed effort - turning NT's into components in functional nanostructures and as tools for building functional nanostructures.

A piezoelectric nanostressing stage will be built and used to apply mechanical stress to NT's and mechanically activate chemical reactions by straining carbon-carbon bonds. Site-specificity of such mechanically sensitized reactions, which are expected to occur in kinked regions of nanotubes, is very desirable for potential applications of NT's in nanotechnology. The nanostressing stage will also be used to elucidate the mechanical behavior of various types of NT's and of very thin graphite sheets (potentially as thin as a single atomic layer).

Tapping mode AFM, which was originally developed to minimize the interactions between the probe and the surface, will be used in a new and unconventional way to manipulate (objects on) surfaces and then image them with high resolution. Operations such as micromachining, nanohammering, and pushing objects on surfaces will be achieved by controlled increase of tip-surface interactions. High-resolution imaging after manipulation will be possible owing to our recent discovery that AFM probes can be resharpened by mechanical deposition of metal grains at the tip. New AFM methods and tools developed at Washington University and also at Zyvex will be applied to manipulation of NT's on surfaces (both at ambient conditions and under liquids) and to study the new mechanochemistry of NT's.

A nanotube pipet, capable of delivering volumes of liquids as small as several tens to a hundred nm3 (note that 1 femtoliter of water is 1 cubic micron, and that 100 nm3 contains 3000 molecules of H2O) will be built and used for fundamental studies of living cells and to construct nanostructures on surfaces. It will be capable of delivering gases, such as organometallics, which could be pyrolyzed on a surface as a means of writing nanoscale metal features, and of delivering very small volumes of biomolecules to cells.
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DESCRIPTION (provided by applicant): The long-term objective of my work is to provide a better understanding of synaptic transmission by studying the operation of NMDA, AMPA, and kainate receptors, which form ion channels gated by the neurotransmitter glutamate. Another major goal is to uncover properties of these receptors that may allow for clinical intervention to prevent excitotoxic cell death or to provide analgesia. The experiments outlined in this application focus on the regulation of kainate receptor composition and on the regulation of AMPA receptor sub-cellular distribution. In a series of studies on the electrophysiology of kainate receptors in neurons from the hippocampus, spinal cord and dorsal root ganglia, we have shown that each of these cell populations express kainate receptors with distinct pharmacological properties. The experiments in Specific Aim 1 will use molecular biology, physiology and subunit deficient mice to investigate the structural basis for these differences. Specific Aim 2 is focused on the role of specific kainate receptor subunits in spinal synaptic transmission. We have recently demonstrated three different ways in which kainate receptors participate in synaptic transmission in the dorsal horn of the spinal cord. Experiments in this aim will seek to determine whether the G1uR5 or GIuR6 subunits are required for the postsynaptic kainate receptors that mediate a component of primary afferent transmission, or for the presynaptic kainate receptors that modulate transmitter release from primary afferents and from spinal interneurons. The third major Aim of this application is to investigate the control of glutamate receptor distribution on hippocampal neurons by the low molecular weight GTPase rab 5 and other components of the endocytic trafficking pathway.

The objective of this research is to characterize the in vitro and in vivo physiology of neurons derived from embryonic stem (ES) cells. These cells represent a potentially limitless source of pluripotent, genetically normal cells for research and therapy. Mouse ES cells can differentiate in vitro into a variety of somatic cell types including neurons, astrocytes and oligodendrocytes. In addition, differentiated ES cells survive and become morphologically integrated with surrounding host tissue following transplantation into the brain or spinal cord. Based on this work with mouse ES cells, the isolation of human ES cells has raised the possibility for novel replacement therapies in which in vitro differentiated ES cells will substitute for somatic cells lost to injury or disease. Pathologies of the nervous system that might be amenable to replacement therapy include Parkinson's disease, amyotophic lateral sclerosis, stroke, Huntington's disease, and multiple sclerosis. President Bush's decision to allow federal support for research on existing human ES lines has engendered great enthusiasm to explore the promise of stem cell-derived replacement as a new way to address these previously intractable deficits; however, much basic research remains to be done before such therapies can be achieved. Our ultimate goals are to develop procedures for efficient conversion of human ES cells into specific types of neurons and to optimize the integration of ES-derived neurons into functional networks when transplanted into a host nervous system. An essential component of functional integration is the acquisition of normal physiological properties by individual stem cell derived neurons. At this point, only limited information is available about the physiology of differentiated ES cells. Thus, the goals of this proposal are: 1) To evaluate physiological differentiation of nerve cells derived from distinct ES induction protocols. 2) To characterize the physiology of ES-derived neurons after transplantation into the brain, including rigorous tests for formation of functional synaptic connections with surrounding host neurons. Human and mouse ES cells will be used in parallel to compare their developmental potentials. All research on human ES cells will use the WA01 line in the NIH human ES cell registry.

DESCRIPTION (provided by applicant): The long-term objective of my work is to provide a better understanding of synaptic transmission by studying the operation of NMDA, AMPA and kainate receptors, which form ion channels gated by the neurotransmitter glutamate. Another major goal is to uncover properties of these receptors that may allow for clinical intervention to prevent excitotoxic cell death or to provide analgesia. The experiments in this proposal arise from several interesting discoveries that we made during the current period of support. Specific Aim 1 follows up on our observation that interactions between the pore loop and adjacent transmembrane helices govern kainate receptor susceptibility to inhibition by cis-unsaturated fatty acids, such as docosahexaenoic acid (DHA). Experiments in this aim will use mutant cycle analysis to determine which residues in the channel interact with each other to control permeation, gating and modulation. Specific Aim 2 builds on our discovery that exposure to DHA appears to change the conformation of kainate receptor transmembrane helices in the open state. Reactivity of substituted cysteines, metal ion binding, and disulfide bond formation will be used to determine structural changes introduced by fatty acids. Specific Aim 3 follows up on our discovery of small molecule antagonists that prevent DHA from potentiating NMDA receptors but have little or no effect on DHA inhibition of kainate receptors. Chimeric subunits that combine domains from NMDA and kainate receptors will be used to investigate whether distinct structural interactions underlie the potentiation and inhibition of channel activity and t analyze the structural requirements for generation of functional channels. Collectively, these experiments will provide new information about the structural basis for ionotropic glutamate receptor operation and new information about how ion channels are affected by interactions with components of the lipid bilayer. A number of pathologic conditions, including brain trauma, epilepsy, and ischemia, elicit massive release of cis-unsaturated fatty acids. These compounds directly regulate many different membrane proteins including a number of ion channel subtypes. This project analyzes the molecular basis of glutamate receptor modulation by DHA, which is present at high levels in the nervous system and is known to be essential for normal brain function.