The aim of this project is to develop a molecular model for the mechanism of glutamate transport and through this we expect to identify novel means of pharmacologically manipulating transporter functions.
Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and activates a wide range of receptors to mediate a complex array of functions. Extracellular glutamate concentrations are tightly controlled by a family of glutamate transporters expressed in both neurons and glia. The aim of our research is to develop a structural model for how glutamate transporters work, and in this way lay the foundations for a more rational approach to the development of drugs that are both transporter-specific and subtype selective. Such compounds will help to delineate the roles of different transporter subtypes in normal brain functions and also in various neuropathological conditions, such as ischemia following a stroke, Alzheimer's disease, motor neurone disease and obsessive compulsive disorder.
The determination of a crystal structure of the bacterial glutamate transporter GltPh has created great excitement because we now have the tools to begin to understand how these important proteins work. This project will capitalize on this breakthrough by developing a structural model of how human glutamate transporters work and from this it should be possible to develop novel pharmacological tools to manipulate transporter functions. This information will be invaluable for understanding the role of glutamate and glutamate transporters in normal and disease states. The molecular basis for transport will be investigated for both the bacterial glutamate transporter, GltPh, and the human glutamate transporter, EAAT1, using a range of techniques to study the functional properties of the transporters. Recombinant GltPh protein will be purified and reconstituted into liposomes, enabling us to measure 3H-labelled substrate and ion fluxes. Human EAAT1 will be expressed in Xenopus laevis oocytes and both 3H-labelled substrate fluxes and two electrode voltage clamp techniques will be used to study function. Site-directed mutagenesis will be used to identify functionally important sites and to probe conformational changes upon binding of glutamate and the various coupled ions. Proximity relationships between regions of the transporter will be measured by cross-linking two introduced cysteine residues to gain insight into the conformational changes this protein undergoes during substrate translocation.
Glutamate transporter dysfunction has been implicated in disease states such as ischemia following a stroke, Alzheimer's disease and obsessive compulsive disorder. The expression of a neutral amino acid transporter (ASCT2) that belongs to the same gene family as the glutamate transporters is known to be upregulated in some breast and skin cancers. Through a better understanding of the mechanism of these transporters we can develop compounds that may have therapeutic benefits in these disease states.
Techniques to be used in this project will include: site-directed mutagenesis, protein purification, electrophysiology, computer modeling of protein structures, protein biochemistry. Research in the Transporter Biology Group is supported by grants from the National Health and Medical Research Council and the Australian Research Council. A top-up scholarship is available for well qualified candidates.
The opportunity ID for this research opportunity is 3