This project explores the molecular mechanism of two ion-coupled transporters: The (Na+ + K+)-coupled glutamate transporter GLT-1 from brain an the H+-organic cation multidrug antiporter EmrE from E. coli. These two system provide unique experimental paradigms for structure/function relationships. In GLT-1 we have already identified several amino acid residues lining the translocation pathway. Moreover, homologous bacterial transporters provide a powerful tool to apply bacterial genetics to study the system. In the case of EmrE, it is the smallest ion-coupled transporter and displays unique stability and solubility properties. This makes it amenable to structural studies. These investigations will progress towards understanding the molecular mechanism by (a) identification of residues in the translocation pathway, (b) in depth biochemical and electrophysiological analysis of the role of critical residues, (c) identification of new ones upon study of second site revertants of the bacterially expressed proteins, and (d) study of the topology, helix packing and oligomeric structure. In addition to contributing to understanding the structural basis of ion-coupled transporter function, these studies may provide important clues to the role of these transporters not only under normal physiological conditions, but also in disease.
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