Doctor of Philosophy (PHD)
Physiology and Biophysics (Medicine)
Date of Defense
First Committee Member
H. Peter Larsson
Second Committee Member
Third Committee Member
Fourth Committee Member
Fifth Committee Member
Excitatory amino acid transporters (EAATs) remove glutamate from synapses in a reuptake process driven by the Na+ gradient to prevent glutamate concentrations from reaching neurotoxic levels. Malfunction of this reuptake mechanism is implicated in stroke. However, methods to treat glutamate transporter malfunction are limited due to the lack of knowledge of the detailed transport mechanism of these transporters under physiological and pathological conditions. To date no crystal structure of human glutamate transporter is available. All crystal structures of glutamate transporter are from the archaeal glutamate transporter homologue Gltph. Inspection of the available Gltph crystal structures in different states gave rise to two crucial questions that are addressed in our study. Aim 1: To identify the third Na+ binding site in glutamate transporter and the role of this Na+ ion in the transport mechanism. In Glpth crystal structure, only two cation binding sites are visible, representing two Na+ binding sites. However, three Na+ ions are cotransported with one substrate in each transport cycle. This raises the question of where the third Na+ ion binds and what role this Na+ plays in the transport mechanism. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical monte carlo and free energy simulations performed on different iso-forms of Gltph as well on a homology model of EAAT3, we locate the third sodium binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in GltPh) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs. Results for Aim 1 are presented in Chapter 2. Aim 2: To study the conformational changes of the human glutamate transporter during the transport cycle in a physiological environment. Crystal structures of the inward and outward facing states of Gltph suggest that there are large rearrangements between the two conformations. In contrast, our previous fluorescence resonance energy transfer (FRET) measurements in human glutamate transporter EAAT3 suggest that only small-scale molecular motions accomplish glutamate uptake. To solve this controversy and further investigate the conformational changes accompanying ion and glutamate transport, we improve the FRET technique by introducing transition metal FRET on EAAT3 transporters expressed in living cell membranes. We find that the extracellular gate of the human glutamate transporter EAAT3 is in the open conformation in the presence of the competitive transporter blocker L-TBOA, corresponding well with L-TBOA bound Glpth crystal structure. In contrast, HP2 is in a relatively closed conformation in Apo, Na+, K+, or Na+ plus glutamate conditions, suggesting that to function as the extracellular gate HP2 does not require as large an opening movement as sterically created by L-TBOA. For the outward/inward conformations of EAAT3, we find that the transporter favors the outward-facing state in the presence of the extracellular competitive transporter blocker L-TBOA, favors more the inward-facing state in K+, while the outward-facing and inward-facing states distribution is in an intermediate range in Apo (ion and substrate free), Na+, or Na+ plus glutamate. Our FRET experiments reveal large conformation changes of the transport domain relative to the trimerization domain during substrate translocation in human glutamate transporter. Interestingly, our FRET result suggests that the Na+ bound only transporter in the absence of the substrate glutamate can also isomerize between outward-facing and inward-facing conformations. Results for Aim 2 are presented in Chapter 3.
glutamate transporter; EAAT3; transition metal FRET; transport mechanism; Na binding site
Wang, Xiaoyu, "Transport Mechanism of Human Glutamate Transporter EAAT3" (2014). Open Access Dissertations. 1232.