Transporters for neutral and anionic amino acids play keys roles in human physiology and are active in organs as diverse as the kidney and brain. In the central nervous system, glutamate mediates the majority of fast excitatory signaling, a form of neuron-neuron communication that is essential to the development and maintenance of the nervous system. A fundamental component of glutamate-mediated signaling is the removal of glutamate from the synaptic cleft, following an excitatory stimulus, by sodium-dependent, high affinity glutamate transporters in neurons and other cells, such as glial cells. At the present time, there is no atomic resolution structural information on a glutamate transporter, which greatly hampers our understanding of their architecture and mechanism of action. In this application I propose to determine the structure of a bacterial protein that has significant sequence identity to the eukaryotic glutamate transporters, using x-ray crystallography. Furthermore, I plan to determine the functional behavior of the bacterial homolog, and to test structure-based mechanisms of transporter function. In addition, by using the crystal structure(s) of the bacterial protein as a guide, I will create a homology model of selected eukaryotic transporters and, together with previously determined structure and function information, this will place structure and function relationships of the eukaryotic transporters in an atomic-resolution, three-dimensional context. Taken together, the proposed research will substantially further our understanding of both eukaryotic and prokaryotic glutamate transporters, and, because they are related to transporters of dicarboxylic acids and of neutral amino acids, our knowledge of these secondary transporters will be increased as well. Lastly, because glutamatergic signaling is pervasive in the human nervous system, the structure of the bacterial transporter, along with the homology models of the eukaryotic transporters, should facilitate the design of new molecules that may have therapeutic potential.
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