Jon W. Johnson - US grants

The class of neurotransmitter receptor molecules activated by L-glutamate appears to mediate most fast synaptic transmission in the vertebrate central nervous system. The broad objective of the project described here is to further our pharmacological and biophysical understanding of how this important class of receptors is activated and regulated. Glutamate receptors function in nearly every category of normal central nervous system activity. There is evidence for their involvement in all sensory systems, often at multiple levels from the sensory organ to cortical association areas. The presence of glutamate responses in cerebellum, striatum, and spinal cord suggest extensive involvement in motor systems. Glutamate receptors are found at high density in the cortex and appear to be essential for higher functions such as learning and memory. A group of receptors types so deeply involved in normal brain function can also play important roles in brain dysfunction; glutamate receptors have been implicated in the etiology of many brain disorders including epilepsy, Alzheimer's disease, Huntington's disease, and schizophrenia. In addition, hypoxia induced neuronal death, as a consequence, for example, of stroke, may result from excessive activation of glutamate receptors. A description of either normal or pathological brain function will require detailed understanding, of how glutamate receptors work. The research proposed here is intended to advance that understanding. Specifically, five major goals will be pursued: 1) New drugs active at glutamate receptor sites will be characterized; 2) the kinetics of receptor binding by a class of drugs that modulate one type of glutamate response will be measured; 3) the speed with which glutamate leaves one of its receptor sites will be measured; 4) the variation in glutamate receptor properties-in different parts of the brain will be studied; and, 5) the mechanisms by which glutamate responses can be regulated will be investigated. The responses of neurons and of single receptor-channel complex molecules will be studied with the electrophysiological technique of patch clamp in combination with a perfusion technique that allows rapid extracellular solution changes. Several new approaches that generalize the utility of the patch clamp technique will also be taken advantage of This research can contribute to the understanding of how synaptic transmission takes place and how its strength is regulated, and to the development of drugs that can modify synaptic transmission. The knowledge gained should help provide insight into the mechanisms that underlie the wide variety of physiological processes and brain disorders in which glutamate receptors are involved.

DESCRIPTION (Adapted from applicant's abstract): The broad objective of the proposed research is to deepen our understanding of the structure, function, and inhibition of the receptor molecules responsible for synaptic transmission. The research will focus on a subtype of glutamate receptor specifically activated by N-methyl-D-aspartate (NMDA). The family of excitatory receptors activated by the neurotransmitter glutamate is responsible for the majority of fast synaptic communications within the vertebrate nervous system. The NMDA receptor is thought to be of fundamental importance in synaptic transmission, synaptic plasticity, and development of the nervous system. It is also directly or indirectly involved in many nervous system diseases, including epilepsy, schizophrenia, ischemia, and a variety of degenerative diseases. Improved understanding of a receptor involved is such a remarkable range of processes in normal and diseased nervous systems will lead to an enhanced ability to prevent, or ameliorate the consequences of, nervous system dysfunction. Possibly because it must function in numerous neural systems, the NMDA receptor is regulated by multiple mechanisms. One mode of NMDA receptor regulation blockade by Mg2+ of the ion-conducting channel formed by the NMDA receptor. Mg2+, both from the extracellular (Mg2+e) and intracellular (Mg2+i) sides of the membrane, can enter the NMDA-activated channel and obstruct the flow of ions. Block by Mg2+i will be studied using the patch-clamp technique to measure current flow across the membranes of cultured neurons and of Chinese hamster ovary (CHO) cells that have been genetically modified to express NMDA receptors. The mechanism by which Mg2+i inhibits neuronal and CHO cell responses will be investigated by simultaneously measuring NMDA responses and the concentration of Mg2+i. Numerous drugs can block the NMDA-activated channel. The effects on humans of these drugs are diverse: some act as hallucinogens and are used as drugs of abuse; others are well tolerated clinically and are used in the treatment of neurodegenerative diseases. The mechanisms by which such drugs inhibit NMDA receptors will be examined in the proposed research. The influence of permanent ions on the interaction between blocker and channel also will be investigated. Finally, the results will be integrated into a kinetic model of the interaction of channel blockers with the NMDA receptor. By modeling and comparing block by several different compounds, basic rules of blocker-channel interactions and their dependence on blocker structure will be developed. The results will improve our understanding of channel function and how it can be influenced by drug binding, and may facilitate the design of clinically useful neuroprotective agents.

DESCRIPTION (provided by applicant): Excitatory receptors activated by the neurotransmitter L-glutamate are responsible for most fast excitatory synaptic communication in vertebrate nervous systems. In the proposed research the glutamate receptor subtypes specifically activated by N-methyl-D-aspartate (NMDA receptors) will be studied. NMDA receptors are required for many basic functions of the nervous system, including development and memory formation. Imbalanced NMDA Receptor activity has been linked to numerous nervous system disorders, including schizophrenia, Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke, and epilepsy. The unique properties of the ion channel formed by NMDA receptors are essential for its central position in nervous system function and dysfunction. The long-term objectives of the proposed research are to understand the ion channel structures and properties that are about central to NMDA receptor function. NMDA receptors are subject to many forms of physiological regulation. One of the most powerful is block of the ion channel of NMDA receptors by extracellular magnesium ions (Mg about). Block by Mg2+ in turn is strongly regulated by both extracellular and intracellular ions that can permeate the channel of NMDA receptors. To achieve he objectives of the proposed research, the following specific aims will be pursued: (1) examine how the important regulatory ion Ca2+ affects block of the channel of NMDA receptors by Mg2+ (2) determine how the effect of permeant ions on Mg2+ block varies among NMDA receptor subtypes; (3) identify amino acids within the structure of NMDA receptors that are involved in forming the site at which permeant ions bind and affect block by Mg2+ These aims will be achieved through a combination of electrophysiological recording from cells and from single NMDA receptors, computational modeling of NMDA receptor activity, and manipulation of the structure of NMDA receptors using molecular biological approaches. The results of the proposed research will extend our understanding of the structure and operation of NMDA receptors, and thus provide insight into their operation in nervous system physiology and disease.

DESCRIPTION (provided by applicant): Most excitatory synaptic excitation in the brain is mediated by glutamate receptors. NMDA receptors (NMDARs), a glutamate receptor subtype specifically activated by N-methyl-D-aspartate, are expressed on almost all mammalian central neurons. NMDARs exhibit high Ca2+ permeability and voltage-dependent channel block by Mg2+, characteristics that allow them to play central roles in synaptic plasticity and memory. NMDARs also are broadly involved in nervous system dysfunction, and have been implicated in many nervous system diseases including Alzheimer's disease (AD), Huntington's disease, schizophrenia, epilepsy, and depression. NMDARs are usually composed of two types of subunits, NR1 and NR2;there are four NR2 subunits encoded by separate genes (NR2A-NR2D), which, when combined with NR1, define four major NMDAR subtypes (NR1/2A [unreadable] NR1/2D). The function of the NR1/2A and NR1/2B NMDAR subtypes, which are heavily expressed in adult cortex, have been extensively investigated. The function of the NR1/2C and NR1/2D NMDAR subtypes, which also are expressed in adult cortex (especially NR1/2D), although at lower levels than the other NMDAR subtypes, are less well understood. Recent data suggest that the NR1/2C and/or NR1/2D NMDARs play an especially important role in the clinical utility of the widely-used AD drug memantine, which is an antagonist of NMDARs. It appears surprising that memantine, a drug that slows cognitive decline in AD patients, would act by inhibiting NMDARs, which are essential for memory. The paradoxical therapeutic effects of memantine have been proposed to result from selective inhibition of NR1/2C and NR1/2D receptor subtypes located on cortical interneurons, resulting in cortical disinhibition. The involvement of NR1/2C and/or NR1/2D NMDARs in activation of inhibitory neurons also may be of special significance to animal models of schizophrenia. The broad objectives of this application are to uncover the roles of NR1/2C and NR1/2D NMDARs in the cortex, and to improve understanding of the mechanism of action of memantine. These objectives will be accomplished by determining: the neuronal subtypes in cortex that express NR1/2C and/or NR1/2D NMDARs;their synaptic versus extrasynaptic location;whether the receptors contribute to tonically active glutamate currents;the effects of memantine on NMDAR responses of several neuronal subtypes in cortex;how genetic deletion of the NR2D subunit affects NMDAR responses in neuronal subtypes in cortex;and the influence of NR2D subunit genetic deletion on the behavioral effects of memantine. To achieve these goals we will apply, to both wild-type and mutant rodents, a powerful combination of approaches, including electrophysiological recordings from brain slices, receptor identification with new pharmacological tools, and analysis of animal behavior. The proposed research will provide fundamental information on cortical NMDARs with broad implications for nervous system function and dysfunction, and will help explain the therapeutic mechanism of a widely used AD drug.

The proposed research concerns N-methyl-D-aspartate receptors (NMDARs), brain proteins that are activated by the neurotransmitter glutamate and mediate communication between neurons. NMDARs are expressed by nearly every neuron in mammalian brains, and are required for normal brain function. NMDARs also are involved in many human disorders including Alzheimer's disease, schizophrenia, and cell death following stroke. There are several important drugs in common clinical use that act by binding to NMDARs, and there is optimism that new, more effective NMDAR-targeted drugs can be developed. Most clinically useful drugs that bind to NMDARs act as channel blockers, compounds that block current flow though the ion channel formed by NMDARs. The mechanisms by which channel blockers interact with NMDARs are not fully understood. In the proposed research we will examine a previously unknown path by which NMDAR channel blockers can access the channel: by entering the plasma membrane, and then transiting from the membrane into the ion channel. We will combine multiple approaches to uncover the characteristics and implications of channel blocker transit from membrane to channel. We will use electrophysiological approaches to record NMDAR activity from cells modified to express specific NMDAR subtypes, and will examine the properties of several channel blockers used clinically or as research tools. Similar approaches will be used to study channel block of native NMDARs in cultured neurons. We will use computational techniques in two ways: to model the mechanism by which blockers associate with NMDAR channels, and to create models of NMDAR structure to predict the likely path taken by channel blockers as they transit from membrane to channel. We then will test predictions of our structural models using molecular biological techniques to change the chemical makeup of NMDARs, followed by electrophysiological recording of resulting changes in channel blocker actions. Finally, we will use newly synthesized channel blocking compounds to help critically test our hypotheses, and to explore how the structure of channel blockers affects their interaction with NMDARs. The implications of the proposed research will be broad. We will reveal the basic properties of a newly discovered pharmacological mechanism by which an important class of inhibitors can interact with NMDARs. Understanding of this new mechanism of NMDAR inhibition is likely to provide insights into how other types of drugs interact with NMDARs, and into mechanisms that underlie modulation of voltage-gated channels. We will develop structural models of NMDARs of broad utility. We will use our structural models to predict the path by which drugs transit from membrane to channel and test our predictions experimentally. We will provide new information about the inhibitory mechanisms of both well-known and newly synthesized NMDAR channel blockers. We believe the knowledge gained from the proposed research will enable future design and synthesis of improved drugs to treat the many disorders in which NMDARs are implicated.