Susan G. Amara - US grants

gene induction /repression; site directed mutagenesis

DESCRIPTION: (adapted from Applicant's Abstract) Within the central nervous system extracellular and synaptic concentrations of most classical neurotransmitters are tightly regulated by specific, high affinity transporters that mediate the rapid reuptake into the presynaptic terminal and surrounding glial cells. Transporters for the excitatory amino acid neurotransmitters are positioned to have a major influence on both synaptic signalling and on the neurotoxic actions mediated by glutamate and aspartate, yet they are often ignored in many studies examining the role of these neurotransmitters in the CNS. The purpose of this proposal is to better define the physiological functions of excitatory amino acid transmitter transporters by 1) establishing the kinetic properties of cloned transporters that determine their capacity to take up and release glutamate, 2) identifying the structural and topological features required for transport, 3) determining their regional and cellular localization in human brain and 4) examining how signal transduction mechanisms act to modulate the reuptake process. Initial experiments will be directed at a detailed characterization of the kinetics, ion dependence, electrogenicity of three cloned excitatory amino acid transporters that have been expressed in oocytes and transfected cells. Alterations in transmembrane ion gradients, the driving forces for reuptake can have dramatic effects on the direction of transport, and thus, under a variety of conditions such as those which occur during ischemia, these changes can lead to net glutamate release through transporter reversal. The development of an electrophysiological assay for examining glutamate efflux and the ionic requirements of transport, as well as mammalian cell expression system to study the flux of radiolabeled substrates should provide insight into how the carriers function normally and how they may contribute to mechanisms of neuronal excitotoxicity. Additional goals of these studies will be to address the regional and cellular localization of carriers in the human CNS to further evaluate their potential contribution to neurodegenerative disease. Although the project is focused initially on three human carriers that have been cloned in the applicant's laboratory, it will be expanded to address the role of additional carrier subtypes and cDNAs encoding different glutamate transporters as they are identified. The importance of understanding the function, localization and regulation of different amino acid transporter subtypes is underscored by the many clinical and experimental studies which have implicated abnormal or inadequate transmitter reaccumulation in degenerative disorders such as ALS, Huntington's disease, ischemia-induced neurotoxicity, and Alzheimer's dementia.

The evolving technologies for assessing gene expression on DNA microarrays shift the scale of study from single genes to whole genomes and provide opportunities to globally examine the alterations in gene expression that occur following different drug treatments. This exploratory grant application aims to adapt gene microarray approaches to assess the adaptive changes in gene expression that take place after psychostimulant drug administration. The biogenic amine carriers have long been recognized as the primary targets for psychostimulant drugs of abuse, as well as anti-depressants, and drugs used to treat attention deficit hyperactivity disorder (ADHD). However, we know relatively little about how inhibition of transport by drugs with varying selectivities leads to such striking yet diverse behavioral consequences. The proposed studies use rat gene chip arrays to examine the impact of cocaine and eventually other monoamine transport inhibitors on gene expression profiles in key brain regions. A second aim of the proposal will examine differences in the profiles of gene expression in animals trained to self-administer cocaine. Rather than developing the DNA arrays for ourselves (a task we prefer to leave to engineers), we have chosen state-of-the-art, commercially available arrays obtained from Affymetrix/TM as the most efficient and reliable method for analyzing the expression of 10/4-10/5 different genes in parallel. The system uses microarrays of gene-specific oligonucleotides synthesized on a glass substrate which are hybridized with biotinylated probes representing the sequences expressed in the tissues of interest. Comparisons expression profiles will be accomplishes using a suite of programs available for the analysis of gene chip data. Changes in expression observed on microarrays will be confirmed using conventional approaches including Northern blotting analyses and in situ hybridization. A final aim will compare global changes in the expression profiles obtained using different classes of transport inhibitors. This latter study will assess the utility of array technology in determining "signature" gene expression profiles for different classes of drugs that act on related targets. Understanding the common and unique actions of drugs from a global, genomic perspective should enable us to characterize and understand the mechanisms that underlie their profound behavioral consequences.

DESCRIPTION (provided by applicant): Excitatory amino acid transporters (EAATs) in the CNS maintain extracellular glutamate concentrations below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. Over the previous funding period our laboratory took advantage of a highly functional cysteineless version of EAAT1, to identify the structural features required for substrate transport and ion permeation using cysteine substitutions together with sulfhydryl modifying reagents. In this competing renewal application we plan to assess proximity of different residues during the transport cycle using introduced cysteine pairs and crosslinking reagents. Studies will continue to emphasize kinetic, biochemical, pharmacological and electrophysiological analyses of EAAT function. In a second aim these approaches will be combined with experiments using computational methods to model the conformational dynamics of glutamate transporters. Gaussian network modeling (GNM) and molecular dynamic (MD) simulations are techniques ideally suited for the study of large, multifunctional structures such as ion channels and neurotransmitter transporters. To date, methods that treat such multimeric proteins have been restricted to atomic interactions or limited, sub- nanosecond time ranges, which are too localized or fast compared to the phenomena that are observable in our experiments. The use of these two complementary methods provide a robust way of identifying critical interactions, which then can be tested by structure-function experiments designed to alter the structure and mobility of the domain of interest. A third aim will explore the mechanism of action of a neuroprotective compound purified from a spider venom, which appears to enhance transport activity by altering a less studied transition step in the transport cycle, the reorientation of the unoccupied carrier to the outside. This compound, which acts selectively on the major glial carrier EAAT2, increases glutamate influx but not efflux, and provides proof of principle for the development of allosteric activators of EAATs with therapeutic potential. The importance of understanding the structure, function, and dynamics of excitatory amino acid transporters is underscored by clinical and experimental studies, which have implicated increases in extracellular glutamate concentration in degenerative disorders such as ALS, Huntington's disease, ischemia-induced neurotoxicity, and Alzheimer's dementia.