Michael W. Quick - US grants

DESCRIPTION: The Recombinant Technologies Core is envisioned as a source of reagents, equipment, and technical expertise for projects requiring the cellular transfection of DNA and other biologically active molecules. It should be emphasized that this Core is not designed as a service facility, and that experiments are to be performed by the members of the individual Center laboratories under the guidance and supervision of core personnel. The Core is characterized by its four objectives: 1) a vector reservoir for reagents, primarily viral stocks and packaging extracts to be used in transformations; 2) a particle gun supported with hoods and incubators; 3) a small cell injection facility, including an injection apparatus capable of manipulating mouse cells, mouse eggs, drosophila embryos, and cultured neurons; and 4) an oocyte injection facility, designed for the production and injection of frog oocytes. To realize these objectives, the Core will maintain a number of pieces of equipment, including three laminar flow hoods (currently operational); cell culture incubators (three in place, one to be provided by this application); a "gene gun" (already purchased); an inverted fluorescent microscope (to be purchased under this application); and a microinjection station (microscope, pipette puller, manipulator, stable-table, all to be supplied by this application). The Core will be managed on a day-to-day basis by a staff scientist supported by this application; in Years -04 and -05, once the small cell injection facility is operational, the services of a half-time research technician is also requested.

DESCRIPTION: (Applicant's Abstract) Neurotransmitter levels in brain are critical for normal brain function. Abnormal levels of neurotransmitter, resulting in inappropriate neural signaling, underlie a diverse list of brain disorders. For example, epilepsy, excitotoxic cell death, depression, and a number of conditions related to drug abuse are all related to abnormal transmitter levels in brain. Neurotransmitter transporters are proteins, located on neurons and glia, that function in part to transport transmitter from the extracellular milieu into cells. As such, they play a central role in regulating synaptic signaling. While much is being learned about the permeation properties of various neurotransmitter transporters (e.g., transport rates, substrate affinities, conducting states), little is known about how particular structural domains of the transporter participate in the permeation process. Furthermore, while it is known that transporter function can be regulated, the extent to which the regulation occurs through manipulation of the domains involved in permeation is not known. The major goal of this application is to define the role of intra-molecular and inter-molecular interactions in regulation of the GABA transporter GAT1. Specific Aim 1 is to test the hypothesis that the amino terminal cytoplasmic tail of the GABA transporter GAT1 positively regulates GABA transport through interactions with internal cytoplasmic loops of the transporter. Specific Aim 2 is to test the hypothesis that proteins that physically interact with the amino terminal tail of GAT1 negatively regulate GAT1 function by preventing the N-terminal tail from interacting with the internal cytoplasmic loops of the transporter. Specific Aim 3 is to test the hypothesis that intermolecular interactions that negatively regulate transporter function can be modulated by physiologically relevant factors. These studies are important because they will (i) define a new regulatory role in permeation for intracellular transporter domains; (ii) elucidate a novel mechanism for the cellular regulation of transporter function through protein-protein interactions; and (iii) provide data that could be useful in strategies aimed at regulating transporter function in the treatment of disorders related to abnormal transmitter levels.

Neurotransmitter levels in brain are critical for normal brain function. Abnormal levels of neurotransmitter, resulting in inappropriate neural signaling, underlie a diverse set of brain disorders. For example, epilepsy, excitotoxic cell death, depression, and a number of conditions related to drug abuse are all associated with abnormal transmitter levels in brain. Neurotransmitter transporters are proteins, located on neurons and glia, that function in part to transport transmitter from the extracellular milieu into cells. As such, they play a central role in regulating synaptic signaling. Interestingly, we know that transporters themselves are subject to regulation by a number of signal transduction cascades, in part through a subcellular redistribution of the transporter. However, the mechanisms underlying this regulation, how the signal transduction pathways interact to control transporter expression, and the physiological relevance of transporter redistribution have yet to be thoroughly examined. The major goals of this application are to determine the signalling pathways that regulate trafficking of the predominant brain GABA transporter GAT1, and to evaluate the physiological relevance of this form of regulation. Specific Aim 1 is to test the hypothesis that GAT1 redistribution occurs via the identical mechanisms that mediate the recycling of neurotransmitter- containing synaptic vesicles. Specific Aim 2 is to test the hypothesis that signal transduction cascades which regulate GAT1 redistribution do so by direct transporter phosphorylation and subsequent alterations in rates of endocytosis. Specific Aim 3 will examine the physiological relevance of the regulation of GAT1 redistribution by testing the hypothesis that alterations in GAT1 trafficking regulate GABAergic signaling in brain. These studies are important because they will (i) define the cellular machinery that participates in transporter trafficking; (ii) determine the signals that regulate transporter trafficking; (iii) determine the physiological relevance of this form of regulation; and (iv) provide data that could be useful in strategies aimed at regulating transporter function in the treatment of disorders related to abnormal transmitter levels.

DESCRIPTION (provided by applicant): This application is a request for support for a conference to be held under the auspices of the Federation of American Societies of Experimental Biology (FASEB) entitled "New Perspectives in Transporter Biology". This meeting is the third biannual conference on the subject and is scheduled to be held from July 21 to 26, 2001 at the Omni Tucson, in Tucson Arizona. It is expected to be attended by approximately 160 researchers. The conference will bring together biochemists, biophysicists, physiologists, and molecular biologists to share information on the molecular and physiological properties of the proteins responsible for mediating the transport of amino acids, peptides, neurotransmitters, and other ions across biological membranes. Recent work on the structure/function, regulation, and physiological roles of these transporters has shown them to be critical not only for normal function, but also as therapeutic targets in disease states. The explosion of new information on these molecules makes this a timely topic for interactions among the top transporter biologists. The conference sessions will be devoted to recent findings in the field of membrane transport and will include the latest information on transporters for amino acids, amines, peptides, and glucose, as well as vesicular transporters and ion exchangers. Some topics to be examined in depth include transporter physiology, structure/function, trafficking and localization, regulation, model systems, and diseases states. The conference will consist of nine oral scientific session over five days (morning and evening), and three afternoon poster sessions. Each oral session will be followed by a panel discussion of the topic. In order to promote the advancement of junior faculty, the most exciting poster submissions will be selected for short oral presentations during the regular oral sessions. This conference should provide the top transporter investigators with a forum for intimate discussions of the commonalties and differences among this important class of molecules.

DESCRIPTION (provided by applicant): Serotonin (5HT) plays a crucial role in aggression, cognition, eating, mood, motor activity, pain, and sleep. 5HT transporters (SERTs) are neuronal membrane proteins that regulate, via uptake, extracellular 5HT concentrations. SERTs are also of interest because they are targets of therapeutics (e.g., antidepressants) and drugs of abuse (e.g., cocaine, amphetamine). Interestingly, SERTs have multiple conducting states, sometimes functioning as ion channels, and other times functioning more as traditional transporters of 5HT. However, the regulatory factors that determine which state SERT occupies, and the functional roles of these conducting states in neurons that endogenously express SERT are unknown. Preliminary data show that the conducting state that SERT occupies depends on SERT's interaction with other proteins. Aim 1 tests the hypothesis that calcium shifts SERT between its states by influencing this interaction, thus providing a physiological mechanism for regulating SERT function. Aim 2 examines the signal transduction pathways by which calcium influences which state SERT occupies. Aim 3 examines state-dependent differences in 5HT uptake. Aim 4 tests the hypothesis that amphetamine dysregulates the calcium-mediated shift in SERT conductance states, and examines the mechanisms underlying this effect. Aim 5 examines the role that these states play in thalamocortical neurons that endogenously express SERT by examining state-dependent cell excitability in the presence of SERT substrates. The experiments will be performed using biochemical, pharmacological, and electrophysiological approaches in cell expression systems and in neurons that endogenously express SERT. These experiments will add to our understanding of normal SERT function and how drugs of abuse that target SERT may mediate their effects. Understanding the factors that regulate SERT may also be important for the design of strategies useful in the treatment of serotonin transporter-mediated disorders.