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A TRIP AROUND THE TRANSPORT CYCLE: THE STRUCTURAL BASIS OF GLUTAMATE TRANSPORTER FUNCTION

Jiang, Jie (2011) A TRIP AROUND THE TRANSPORT CYCLE: THE STRUCTURAL BASIS OF GLUTAMATE TRANSPORTER FUNCTION. Doctoral Dissertation, University of Pittsburgh.

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Abstract

Neuronal and glial glutamate transporters function to clear synaptically released glutamate from the extracellular space. This process not only ensures the spatial and temporal fidelity of excitatory signaling, but also prevents neuronal death triggered by excess glutamate. In addition, glutamate transporters possess a substrate-gated anion channel function, which may play an important role in shaping synaptic transmission. Although much progress has been made on the topology, structure, and function of these carriers, our knowledge of the conformational dynamics that drive glutamate transporter functions is far from complete.
Using biochemical, electrophysiological and functional assays in combination with computational simulations, we identified several large-scale collective motions that are intrinsic to glutamate transporter trimers. These collective motions are functionally important for substrate transport, but not for the substrate-gated anion conductance. Furthermore, we showed that these collective motions are coupled to the inward movement of the transport domain, and thus serve a critical function in the transport cycle. Next, we identified a conserved arginine residue, R388 in human EAAT1, that is involved in both substrate transport and anion conduction. Mutants reversing the positive charge (R388D and R388E) predominantly exist in the anion leak state and abolish the substrate-activated anion current. We also demonstrated that the transport domain in the negatively-charged mutant R388D spends a longer time in the inward-facing orientation in the absence of substrate compared to the wild type transporter. These results not only suggest the characterized arginine residue is an important element in the functional coupling between substrate transport and the anion channel activity, but also indicate the role of the inward transport domain movement in anion permeation. Finally, we constructed a functional glutamate transporter concatemer. Our preliminary results suggested that glutamate transporters are functional as trimers and the individual subunits transport substrate independently.
The work presented in this dissertation provides a greater understanding of the structural determinants of the dual functions of glutamate transporters. Furthermore, it provides a powerful tool to further study contributions of subunit interactions and the inward piston-like movement of the transport domain to channel gating and anion permeation.


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Details

Item Type: University of Pittsburgh ETD
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Jiang, Jiejij12@pitt.eduJIJ12
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairCascio, Michaelcascio@pitt.eduCASCIO
Thesis AdvisorAmara, Susanamaras@pitt.eduAMARAS
Committee MemberJohnson, Jonjjohnson@pitt.eduJJOHNSON
Committee MemberKlein-Seetharaman, Judithjudithks@cs.cmu.edu
Committee MemberTorres, Gonzalogtorres@pitt.eduGTORRES
Committee MemberGrewer, Christofcgrewer@binghamton.edu
Date: 18 November 2011
Date Type: Publication
Defense Date: 20 September 2011
Approval Date: 18 November 2011
Submission Date: 3 November 2011
Release Date: 18 November 2011
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 166
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Neurobiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Glutamate transporter, transport cycle, large collective motions, cooperativity, conformational changes, anion conductance,
Date Deposited: 18 Nov 2011 16:17
Last Modified: 18 Nov 2016 06:15
URI: http://d-scholarship.pitt.edu/id/eprint/6201

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