Abstract:
Glutamate transporters actively take up
glutamate into the cell, driven by the co-transport
of sodium ions down their transmembrane
concentration gradient. It was proposed that
glutamate binds to its binding site and is
subsequently transported across the membrane in
the negatively charged form. With the glutamate
binding site being located partially within the
membrane domain, the possibility has to be
considered that glutamate binding is dependent on
the transmembrane potential, and, thus,
electrogenic. Experiments presented in this report
test this possibility. Rapid application of
glutamate to the wild-type glutamate transporter
subtype EAAC1 (excitatory amino acid carrier 1)
through photo-release from caged glutamate
generated a transient inward current, as expected
for the electrogenic inward movement of cotransported Na+
. In contrast, glutamate application
to a transporter with the mutation A334E induced
transient outward current, consistent with
movement of negatively-charged glutamate into its
binding site within the dielectric of the membrane.
These results are in agreement with electrostatic
calculations, predicting a valence for glutamate binding of -0.27. Control experiments further
validate and rule out other possible explanations
for the transient outward current. Electrogenic
glutamate binding can be isolated in the mutant
glutamate transporter because reactions such as
glutamate translocation and/or Na+
binding to the
glutamate-bound state, are inhibited by the A334E
substitution. Electrogenic glutamate binding has
to be considered together with other voltage
dependent partial reactions to cooperatively
determine the voltage dependence of steady-state
glutamate uptake and glutamate buffering at the
synapse.
