Fluorometric measurements of conformational changes in glutamate transporters

Hans P Larsson, Anastassios V. Tzingounis, Hans P. Koch, Michael P. Kavanaugh

Research output: Contribution to journalArticle

88 Citations (Scopus)

Abstract

Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent extracellular glutamate concentrations from reaching neurotoxic levels (1). It is thought that glutamate transporters mediate glutamate transport through a reaction cycle with conformational changes between the two major access states that alternatively expose glutamate-binding sites to the extracellular or to the intracellular solution. However, there is no direct real-time evidence for the conformational changes predicted to occur during the transport cycle. In the present study, we used voltage-clamp fluorometry to measure conformational changes in the neuronal excitatory amino acid transporter (EAAT) 3 glutamate transporter covalently labeled with a fluorescent reporter group. Alterations in glutamate and cotransported ion concentrations or in the membrane voltage induced changes in the fluorescence that allowed detection of conformational rearrangements occurring during forward and reverse transport. In addition to the transition between the two major access states, our results show that there are significant Na+-dependent conformational changes preceding glutamate binding. We furthermore show that Na+ and H+ are cotransported with glutamate in the forward part of the transport cycle. The data further suggest that an increase in proton concentrations slows the reverse transport of glutamate, which may play a neuro-protective role during ischemia.

Original languageEnglish
Pages (from-to)3951-3956
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number11
DOIs
StatePublished - Mar 16 2004
Externally publishedYes

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Amino Acid Transport System X-AG
Glutamic Acid
Excitatory Amino Acid Transporter 3
Fluorometry
Protons
Ischemia
Fluorescence
Binding Sites
Ions
Neurons
Membranes

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Fluorometric measurements of conformational changes in glutamate transporters. / Larsson, Hans P; Tzingounis, Anastassios V.; Koch, Hans P.; Kavanaugh, Michael P.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 11, 16.03.2004, p. 3951-3956.

Research output: Contribution to journalArticle

Larsson, Hans P ; Tzingounis, Anastassios V. ; Koch, Hans P. ; Kavanaugh, Michael P. / Fluorometric measurements of conformational changes in glutamate transporters. In: Proceedings of the National Academy of Sciences of the United States of America. 2004 ; Vol. 101, No. 11. pp. 3951-3956.
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