Vasanthi Jayaraman - US grants

9982759
Jayaraman

Communication between nerve cells serves as the basis of all brain activity. One of the fundamental steps involved in signal transmission between the nerve cells, is the conversion of a chemical signal liberated at the end of one nerve cell, into an electrical signal at the second nerve cell. This step is mediated by a class of membrane bound proteins known as neurotransmitter receptors. One member of this family is the glutamate receptor. These receptors bind to the chemical signaling molecule glutamate (ligand) and generate an electrical signal through the formation of transmembrane ion channels. In the proposed studies, Dr. Jayaraman will attempt to understand the interactions whereby the glutamate receptor protein specifically recognizes ligands such as glutamate. The ligand-protein interactions will be investigated by probing the vibrations of the ligand and the ligand-binding segment of the glutamate receptor using spectroscopic tools. Since, the vibrations of these chemical moieties are controlled by their atomic-level environment, probing the vibrations with infrared light will provide insight into the interaction between the protein and the ligand. Furthermore, extending these studies to study the changes in the protein structure upon ligand binding in a time-resolved manner, will suggest the pathway by which the protein form the ion channels upon binding the ligand. These high-resolution structural studies are required for a basic understanding of interactions that control protein-ligand recognition and will eventually allow the design of drugs that can alter the behavior of glutamate receptors. Such drugs may be useful in the treatment of traumatic head injury, and memory problems.

DESCRIPTION (provided by applicant): Ionotropic glutamate receptors are the main mediators of excitatory synaptic signals in the mammalian central nervous system. Glutamate binding to this receptor triggers the formation of transmembrane ion channels in the protein, permitting cations to flow down their electrochemical gradients across the postsynaptic membrane, depolarizing it and thereby stimulating the receiving cell. Excess glutamate, on the other hand, over activates the ionotropic glutamate receptors triggering a cascade of events resulting in neuronal death. This excitotoxic pathway is thought to be a major factor in neurodegeneration associated with a variety of acute and chronic disorders such as stroke, ALS, and Alzheimer's disease, and therefore antagonists of the ionotropic glutamate receptors have been identified as good candidates for the treatment of these diseases. A large body of initial work focused on the antagonists of the NMDA subtype of the glutamate receptors since these are Ca 2+ permeable. However, more recently it has become apparent that Ca 2+ permeable subtype of AMPA receptors may indeed be one of the primary mediators of neuronal death. For instance, selective death of motor neurons observed in ALS is thought to be mediated by the activation of the high density of Ca 2+ permeable AMPA receptors in motor neurons. The antagonists currently available for the AMPA receptors, however, are not as effective in the treatment of these diseases mainly due to their insolubility. There is hence an ongoing search for soluble antagonists of the AMPA receptor, which would be greatly facilitated by a high throughput screening assay that would be able to identify antagonists of this receptor. Traditionally, radioactive ligand binding assays have been used to screen for drugs that bind to this important protein. However, the drugs identified have to then be characterized by patch clamp methods to determine if they exhibit antagonistic properties, which is extremely labor intensive and low throughput. Here we propose to develop a fluorescence based high throughput assay that will not only allow for the screening of drugs that bind to the receptor with high specificity, but will also provide a direct readout of the functional consequences (activation or inhibition) of the drug binding, thus allowing for a selective screening of drugs with the desired functional properties.

DESCRIPTION (provided by applicant): NMDA receptor antagonists exhibit excellent neuroprotective efficacy in experimental models of stroke; however, clinical trials in stroke and traumatic brain injury with NMDA antagonists have not been successful. The failure of the currently available drugs in clinical trials can be primarily attributed to their severe side effects, which necessitate the use of significantly lower than effective doses. The key issue is to develop drugs that reduce NMDA activity without these dose-limiting side effects. This can be achieved by subtype (NR1or NR2) specific antagonists that have been clearly established to possess better side effect profiles. The currently available subtype specific compounds, however, have not been effective due to a number of problems such as activity at other receptors, insolubility, and/or inefficiency in crossing in blood brain barrier. In the proposed research we will use a promising new pharmacological approach, based on the use of RNA ligands as subtype specific antagonists for the NMDA receptors, which will address the problems with the currently available drugs. The advantage of using RNA ligands is that they can be evolved from a large pool of RNA ligands with random sequences (1015 ligands) to bind with high specificity for any target protein. Additionally, these are water soluble and have been shown to cross the blood brain barrier. The selection protocol will involve an iterative process of positive selection based on the affinity of the RNA ligand for a given subunit of the NMDA receptor followed by amplification using RT-PCR. The RNA ligands thus obtained are further selected for specificity by a negative selection step wherein those with no affinity for the other subunits of NMDA receptors are selected. The RNA ligands selected by the above steps need to be characterized to identify those with antagonistic property. This process will be greatly facilitated by the fluorescence based assay that we have developed in my laboratory. This assay provides high throughput readout of the functionality of the ligand, namely agonists or antagonist. The candidate RNA antagonists identified by the assay will be tested for antiexcitotoxic effects using in vitro and in vivo models in the Co-PI's laboratory, thus providing the basis for future preclinical and clinical trials.

DESCRIPTION (provided by applicant): The specific molecular interactions between glutamate and the glutamate receptor are central to the allosteric regulation of function in this membrane bound protein, which is main excitatory neurotransmitter receptor in the mammalian central nervous system. Recent spectroscopic investigations of the isolated ligand-binding domain of the glutamate receptor in the apo and ligand bound forms provide a first view of such interactions. What is now needed is a more detailed understanding of the role of these chemical interactions in mediating the sequence of conformational changes that ultimately regulate ion flow. We propose to address this goal using two strategies. First, using a panel of mutants that exhibit a wide range of activities we will identify changes in specific ligand protein interactions using vibrational spectroscopy and correlate these to changes in the cleft closure conformational change in the ligand binding domain using fluorescence resonance energy transfer measurements. The changes thus identified in the ligand binding domain will then be contextualized in terms of changes in receptor function that will be studied by electrophysiological measurements. Secondly, we will monitor the kinetic events in the ligand binding domain and correlate these to the functional consequences in the receptor, i.e., activation and desensitization. For such a correlation, a panel of mutants that are known to alter one or more of the kinetic steps in the ligand binding domain will be used and the specific effects on the structural changes in the ligand binding domain as well as corresponding changes in function will be investigated. These equilibrium and kinetic investigations will provide comprehensive understanding of the changes at the molecular level that drive the changes in the large conformational changes in the ligand binding domain and eventually control the functional changes of the ion channel. More importantly, such correlations will aid in bridging the gap between studies on the isolated ligand-binding domain and the behavior of the intact receptor, and provide a basis for rational design of drugs aimed at modulating the function of this important protein which is known to play an important role in diverse neuropathologies, including epilepsy and ischemia.

DESCRIPTION (provided by applicant): The specific molecular interactions between glutamate and the glutamate receptor are central to the allosteric regulation of function in this membrane bound protein, which is main excitatory neurotransmitter receptor in the mammalian central nervous system. Recent spectroscopic investigations of the isolated ligand-binding domain of the glutamate receptor in the apo and ligand bound forms provide a first view of such interactions. What is now needed is a more detailed understanding of the role of these chemical interactions in mediating the sequence of conformational changes that ultimately regulate ion flow. We propose to address this goal using two strategies. First, using a panel of mutants that exhibit a wide range of activities we will identify changes in specific ligand protein interactions using vibrational spectroscopy and correlate these to changes in the cleft closure conformational change in the ligand binding domain using fluorescence resonance energy transfer measurements. The changes thus identified in the ligand binding domain will then be contextualized in terms of changes in receptor function that will be studied by electrophysiological measurements. Secondly, we will monitor the kinetic events in the ligand binding domain and correlate these to the functional consequences in the receptor, i.e., activation and desensitization. For such a correlation, a panel of mutants that are known to alter one or more of the kinetic steps in the ligand binding domain will be used and the specific effects on the structural changes in the ligand binding domain as well as corresponding changes in function will be investigated. These equilibrium and kinetic investigations will provide comprehensive understanding of the changes at the molecular level that drive the changes in the large conformational changes in the ligand binding domain and eventually control the functional changes of the ion channel. More importantly, such correlations will aid in bridging the gap between studies on the isolated ligand-binding domain and the behavior of the intact receptor, and provide a basis for rational design of drugs aimed at modulating the function of this important protein which is known to play an important role in diverse neuropathologies, including epilepsy and ischemia.

? DESCRIPTION (provided by applicant): N-methyl D-aspartate receptors are a subtype of glutamate receptors that mediate excitatory signal transmission in the mammalian central nervous system. Their primary function involves converting the chemical signal into an electrical signal, i.e. glutamate binding to an extracellular domain in the receptor triggers the formation of cation permeable transmembrane channels in the receptor. Given the importance of these receptors in mediating a number of physiological processes and the need to modulate their function in disease states, the primary questions are how does the agonist activate the protein and how can this mechanism be modulated. The NMDA receptors are modulator in structure consisting of an amino terminal domain, agonist binding domain, channel segments and the C-terminal domains. Here we propose to study the communication between the domains and their role in dictating activation and allosteric modulation. Specifically we will investigate the role o the interactions between GluN1 agonist binding domain with the GluN2 subunit in controlling agonist binding domain dynamics and extent of activation (specific aim 1) using a combination of luminescence resonance energy transfer, smFRET, and electrophysiology. We will also investigate the pathway for allosteric modulation by establishing the conformational changes starting at the amino terminal domain through the agonist binding domain and study the changes in dynamics in the extracellular domains during the allosteric modulation (specific aim 2). The spectroscopic investigations will be complemented by electrophysiological measurements investigating the changes in function induced by alterations at the interface between the domains. These functional and structural investigations will provide a comprehensive understanding of the conformational pathway as well as role of dynamics in activation and allosteric modulation in NMDA receptor function.

? DESCRIPTION (provided by applicant): AMPA receptors mediate fast excitatory responses in the mammalian central nervous system, and ultimately control motor and cognitive functions. In neurons AMPA receptors co-assemble with auxiliary subunits. One member being transmembrane AMPA receptor regulatory proteins (TARPs), which are essential for the normal physiological functioning of these receptors. One of the dramatic effects of TARPs on AMPA receptor gating is a large increase in efficacy such that even some antagonists such as 6-cyano-7-nitroquinoxaline-2,3-dione act as partial agonists. Additionally, TARPs slow deactivation of AMPA receptor when agonist is removed and slow the rate and extent of desensitization when agonist is continually applied. These changes in properties are expected to dictate the kinetics and shape of synaptic transmission. In addition to the rapid modulations, the presence of certain subtypes of TARP's leads to resensitization on longer time scales, from the tens of milliseconds to seconds. The prolonged opening of the receptors in these long resensitization processes may play a role in glutamate mediated excitotoxicity. Here we propose to gain a fundamental understanding of the structure-dynamic changes controlling the increase in efficacy and resensitization in the AMPA receptors in the presence of TARPs using a combination of biophysical and electrophysiological methods. For the proposed study, we will use luminescence resonance energy transfer to study the conformational changes in the protein. For specific labeling of the protein with fluorescent probes we will introduce unnatural amino acids at specific sites on the protein. The conformational changes thus observed will be further verified with functional investigations of mutant proteins with mutations introduced at strategic sites that affect the observed conformational change or the stability of a specific conformational state. Finally, the position of the TARP ecto domain will be mapped to the AMPA receptor extracellular domain using mass spectrometry. To study the interaction sites between the two proteins the unnatural amino acid p-benzoyl phenylalanine will be introduced at various sites and those sites leading to crosslinking will be analyzed using high resolution tandem mass spectrometry. The proposed functional and structural investigations will provide a comprehensive understanding of the mechanism by which TARPs modulates AMPA receptor function.

PROJECT SUMMARY/ABSTRACT Glutamate receptors mediate excitatory responses in the mammalian central nervous system, and ultimately control motor and cognitive functions. Glutamate receptors are classified into three subfamilies based on affinity profiles for several synthetic and natural agonists: N-methyl-D-aspartate (NMDA), ?-amino-5-methyl-3- hydroxy-4-isoxazole propionate (AMPA) and kainate receptors. All three subtypes are broadly similar having a dimer of dimer architecture with similar topologies. However, their gating characteristics and mechanisms are unique resulting in unique roles in synaptic transmission. The work in my laboratory focuses on understanding the mechanisms underlying the fine-tuning of each of these subtypes. AMPA receptors mediate fast synaptic transmission and their gating properties are modulated by the presence of auxiliary subunits (TARPs) as well as through post-translational modifications. Work from my laboratory has provided insight into the mechanism of activation and desensitization in AMPA receptors using a combination of biophysical and biochemical methods. Here, we propose to study how these mechanisms can be translated to modulation by TARPs and post-translational modifications such as phosphorylation. For this we will use a combination of smFRET and LRET to determine the dynamics and conformational changes, and validate these structure-dynamic changes through functional characterization of changes elicited by biochemical manipulations of the receptor like cross linking and mutations. The NMDA receptors, on the other hand, mediate Ca2+ permeable long depolarizing signals and are modulated through small molecule modulators, phosphorylation and interacting partners such as calmodulin and alpha-actinin at the intracellular carboxy terminus. smFRET and LRET investigations from my laboratory as well as the X-ray and EM structures have provided significant insight into conformational changes within the ordered extracellular domains. However, the communication across domains and the role of the disordered C-terminal domain are largely unexplored. Here we propose to study the role of interactions across domains in controlling dynamics and conformational changes in the receptor, and effects of modulators and changes at the C-terminal domain on these interactions and conformational dynamics. These studies will then be correlated to functional consequences thus providing insight into the structure-dynamic pathway of activation, desensitization, and modulation in these receptors.