Sulfhydryl Modification of V449C in the Glutamate Transporter EAAT1 Abolishes Substrate Transport but not the Substrate-Gated Anion Conductance

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that mo...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2001-12, Vol.98 (26), p.15324-15329
Main Authors: Seal, Rebecca P., Shigeri, Yasushi, Eliasof, Scott, Leighton, Barbara H., Amara, Susan G.
Format: Article
Language:English
Subjects:
Amino acids
anion conductance
Anions
Biological Sciences
Biological Transport
Chlorides
Electric current
Excitatory amino acid transporter
Excitatory Amino Acid Transporter 1 - chemistry
Excitatory Amino Acid Transporter 1 - metabolism
Experiments
Humans
Ion Channel Gating
Ions
Kinetics
Neurotransmitters
Oocytes
Potassium
Protein Conformation
Protons
Sodium
Steady state current
Sulfhydryl Compounds - chemistry
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Summary:Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC50S for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC50for L-glutamate to activate the anion conductance, without affecting the EC50for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.011400198