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The Journal of Neuroscience, December 1, 1998, 18(23):9620-9628
Stoichiometry of the Glial Glutamate Transporter GLT-1 Expressed
Inducibly in a Chinese Hamster Ovary Cell Line Selected for Low
Endogenous Na+-Dependent Glutamate Uptake
Line M.
Levy1,
Orpheus
Warr2, and
David
Attwell2
1 Department of Anatomy, University of Oslo, Blindern,
N-0317 Oslo, Norway, and 2 Department of Physiology,
University College London, London, WC1E 6BT, United Kingdom
Glutamate transport across the plasma membrane of neurons and glia
is powered by the transmembrane electrochemical gradients for sodium,
potassium, and pH, but there is controversy over the number of
Na+ cotransported with glutamate. The stoichiometry
of glutamate transporters is important because it determines a lower
limit to the extracellular glutamate concentration,
[glu]o, in both normal and pathological
conditions. We used whole-cell clamping to study the stoichiometry of
the glial transporter GLT-1, the most abundant glutamate transporter in
the brain, expressed under control of the Tet-On system in a Chinese
hamster ovary (CHO) cell line selected for low endogenous glutamate
transport. After the induction of GLT-1 expression with doxycycline,
glutamate evoked a Na+-dependent inward current with
the voltage dependence and pharmacology of GLT-1 and acidified the cell
cytoplasm. Raising [K+]o around cells
clamped with electrodes containing sodium and glutamate evoked an
outward reversed uptake current. These responses were reduced by the
specific GLT-1 blocker dihydrokainate (DHK). DHK evoked an outward
current with NO3 , but not with
Cl, as the main intracellular anion, suggesting
that the anion conductance of the transporter is active even without
external glutamate but generates little current in the absence of
highly permeable anions like NO3.
Measuring the reversal potential of the transporter current in various
ionic conditions suggested that the transport of one glutamate anion is
coupled to the cotransport of three Na+ and one
H+ and to the countertransport of one
K+. This suggests that in ischemia, when
[K+]o rises to 60 mM, the
reversal of glutamate transporters will raise [glu]o to
>50 µM.
Key words:
glutamate; uptake; inducible expression; CHO cell line; GLT-1; transport
Copyright © 1998 Society for Neuroscience 0270-6474/98/18239620-09$05.00/0
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