Conformations of EAAT3 Measured by Transition Metal Ion FRET

The excitatory amino acid transporter 3 (EAAT3) is a secondary active glutamate transporter that is vital to proper neuronal function. Defects in the normal function and expression of EAAT3 have dire consequences including epilepsy, cell death during ischemic stroke, amyotrophic lateral sclerosis (ALS), and Alzheimers disease. Because of the clinical importance of this transporter, EAAT3 has been assiduously studied; however, there are still major sections of the transport cycle that remain incompletely characterized; Notably the details of the molecular basis of the translocation step and binding site of a potassium ion remains elusive. To study the binding and translocation of potassium through EAAT3, we propose using transition metal ion fluorescence resonance energy transfer (FRET). In conjunction with using transition metal ion FRET, we will use traditional methods of electrophysiology, such as whole cell patch clamp, molecular modeling, and APBS computations. Whole cell patch clamp techniques will provide information on charge movements and the transition metal ion FRET will provide measurements of distinct spatial movements within small areas between the transmembrane helices of EAAT3. Taken together, these data should determine the binding site of potassium on EAAT3 as well as distinguish the potassium binding site from the sodium 1 from sodium 3 binding sites. This combination of techniques will also characterize EAAT3 movements specifically associated with potassium translocation. Additionally, the novel combination of these methods will further describe the binding and translocation of the sodium ion in the well-characterized sodium 1 site. Successful use of this novel application has the potential to drive drug design and will stand as a model transition metal ion FRET system for future research in transmembrane transporters. These experiments are to be conducted at the Medical University of Vienna in the Center of Physiology and Pharmacology with Harald Sitte and Thomas Stockner. MUW has a strong reputation in the field of transporter proteins and Harald Sitte is a pioneer in the application of fluorescence spectroscopy on transmembrane transporters. The international applicant, Catherine B Zander has experience with whole cell patch clamp, mutagenesis studies, molecular modeling, and APBS computational studies, gained through her doctoral training in the laboratory of Christof Grewer. The Grewer lab is known for its expert knowledge of the EAATs and other neurologically significant secondary active transporter proteins.