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Anion Conducting States of Excitatory Amino Acid Transporters

Divito, Christopher B. (2013) Anion Conducting States of Excitatory Amino Acid Transporters. Doctoral Dissertation, University of Pittsburgh.

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Abstract

Excitatory amino acid transporters (EAATs) are secondary active, electrogenic transporters which translocate L-glutamate (glu) against its concentration gradient using the co-transport of 3 Na+, 1 H+, and the counter-transport of 1 K+ ion. In addition, these carriers possess a thermodynamically uncoupled anion channel that fluxes Cl- but is promiscuous with several permeant anionic species. The roles of EAATs are to shape the spatio-temporal profile of released glu in both the synaptic cleft and extra-synaptic regions as well as maintaining a low ambient extracellular concentration of glu. This transport activity regulates activation of glu receptors and thus regulates excitatory neurotransmission.
Using a combination of techniques, we were successful in identifying inward oriented transporter conformations which allow transitions to open channels states. This observation was enabled by our development of a novel method to isolate EAAT1 in the inward facing conformation. While constrained to these conformations, currents with the same macroscopic amplitudes as conducting states mediated by the outward facing, Na+ bound states were observed. The persistence of currents is indicative of a channel gating mechanism that is insensitive to transporter orientation and that the anion channel is open during the majority of the transport cycle. Additional conducting states allows for a larger contribution of the anion channel function of EAATs to shape cellular function then previously assumed.
Next we investigated the gating mechanism of the anion channel. We assayed for the ability of Na+ to gate the anion channel in both glial (EAAT1 and EAAT2) and neuronal (EAAT3 and EAAT4) isoforms. We discovered that the glial isoforms are not gated by Na+ but are leak channels with an open probability and single channel conductance that is insensitive to Na+ concentrations. In contrast, neuronal EAAT isoforms EAAT3 and EAAT4 both display Na+ dependent channel activity. This is the first example of a significant functional difference between glial and neuronal transporter isoforms of the solute carrier 1 (SLC1) family. The research presented here allows for a greater understanding of low open probability channel states and the possible contributions of the EAAT anion channel to the functioning of the nervous system.


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Details

Item Type: University of Pittsburgh ETD
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Divito, Christopher B.cbd8@pitt.eduCBD8
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairJohnson, Jon W.jjohnson@pitt.eduJJOHNSON
Thesis AdvisorAmara, Susan G.susan.amara@nih.gov
Committee MemberSeal, Rebecca P.rpseal@pitt.eduRPSEAL
Committee MemberCascio, Michaelcasciom@duq.edu
Committee MemberMerieny , Stevenmerieny@pitt.eduMERIENY
Committee MemberGalli, AurelioAurelio.galli@vanderbilt.edu
Date: 19 December 2013
Date Type: Publication
Defense Date: 18 November 2013
Approval Date: 19 December 2013
Submission Date: 19 December 2013
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 194
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, EAAT, neurotransmitter transporter, ion channel, chloride, transporter, structure/function
Date Deposited: 19 Dec 2013 19:17
Last Modified: 15 Nov 2016 14:16
URI: http://d-scholarship.pitt.edu/id/eprint/20315

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