LOOKING FOR THE INWARD FACING STATE OF GLUTAMATE TRANSPORTERS
Xiaoyu Wang, Marta Perez, and H. Peter Larsson
Department of Physiology and Biophysics, University of Miami, USA
ABSTRACT
METHODS
To test the nature of conformational changes
that occur during glutamate uptake, we determined
the relative positions of different domains in the
human neuronal glutamate transporter EAAT3 under
conditions that favor different conformations using
fluorescence resonance energy transfer (FRET)
analysis on EAAT3 transporters expressed in Xenopus
oocytes. FRET is a phenomenon by which a donor
fluorophore, in its excited state, transfers its excitation
energy to a nearby acceptor fluorophore. The
efficiency of FRET tapers off by the sixth power of
the distance separating the donor and acceptor
fluorophore, allowing an estimation of the relative
distance between pairs of acceptor and donor
fluorophores.
Conductance (nS)
Conductance (nS)
1
No large horizontal conformational
changes detected in EAAT3 in previous
0.4
FRET experiments
0.2
0
50
1
3
0.1
0.01
1E-3
50
50
Voltage (mV)
14
10
0
50
Voltage (mV)
0
Question: Can we find a new strategy 30
to
acquire the inward conformation of
glutamate transporters?
2
Optimizing the external solution does not
reveal the inward state of glutamate
transporters
A430C
Alexa-488
Zd=5.9eo
zd
8
Fig. 1. Distance measured between intra-subunit and inter-subunit FRET
6 pairs in
100mM CholineCl and 100mM NaCl + 1mM glutamate. Alexa-488-maleimide was
4
used as FRET donor fluorophore and TetraMethylRhodamine-MTS (TMR-MTS)
was
used as FRET acceptor molecule. (Larsson and Koch, 2006)
2
L387C
Alexa-488
FRET efficiency is measured with donor quenching
method. Distance between FRET pairs for a trimeric
transporter structure with one donor fluorophore and
two acceptor fluorophores was calculated.
0
Different FRET pairs do not detect the
inward conformation of glutamate
transporters
A430C
Fig. 3. Fluorescein or
Zd=4.6eo
Oregon Green used as
Fluorescein
FRET donor fluorophore.
12
G/Gmax
A recent crystal structure of the bacterial
glutamate transporter homologue GltPh (Reyes et
al., 2009) was interpreted as the inward facing
state of glutamate transporters. Comparing this
inward facing structure to the earlier outward
facing structures of GltPh suggested that
glutamate transporters undergo a large
conformational change between these two states.
In contrast, earlier FRET experiments from our
group (Koch and Larsson, 2005) suggested that
the mammalian glutamate transporter EAAT3
does not undergo large conformational changes
during the uptake cycle. We here tested the
proposed inward facing state of glutamate
transporters using different FRET techniques
under different ion and substrate conditions that,
we assume, forces EAAT3 to mainly occupy the
inward facing state or the outward facing state.
Our results suggest that the opening of the
external gate is the rate limiting step of the
whole transporter cycle. We also proposed a
kinetic model that is consistent with our FRET
results on glutamate transporters.
RESULTS
20
4
10
A430C
Oregon Green
0
Internal application of TFB-TBOA blocks
glutamate currents, but does not trap the
inward state of glutamate transporters
a
Fig. 2. FRET efficiency (E)
measured
differences
in
EAAT3 A430C or L387C
between K+ and Na+ (K-Na),
Choline and Na+ (Ch-Na), or
Na+ +Glutamate and Na+
(Glu-Na) external solutions.
Ti-To is the expected change
in the FRET efficiency E for
the homologous residue in the
GltPh
crystal
structures
between the outward- and
inward-facing states. Alexa488-maleimide was used as
FRET
donor
and
TetraMethylRhodamine-MTS
(TMR-MTS) was used as
FRET acceptor.
FRET
efficiency
(E)
measured differences in
EAAT3 A430C between
K+ and Na+ (K-Na),
Choline and Na+ (Ch-Na),
or Na+ +Glutamate and
Na+ (Glu-Na). external
solutions. Ti-To is the
expected change in the
FRET efficiency E for the
homologous residue in the
GltPh crystal structures
between the outward- and
inward-facing states.
TetraMethylRhodamineMTS (TMR-MTS) was
used as FRET acceptor.
b.
A430C
Alexa-488
+TFB-TBOA
Fig. 4. a Intracellular application
of 1mM TFB-TBOA blocks the
glutamate-activated
EAAT3
currents.
TFB-TBOA
was
injected into the cytosol of the
oocyte. b FRET efficiency (E)
measured differences in EAAT3
A430C between oocytes injected
with TFB-TBOA, in the
presence of Na (Na) or
glutamate (Glu), and oocytes not
injected
with
TFB-TBOA
(Na(o)). Ti-To is the expected
change in the FRET efficiency E
for the homologous residue in
GltPh crystal structures between
the outward- and inward-facing
states. Alexa-488-maleimide was
used as FRET donor and
TetraMethylRhodamine-MTS
(TMR-MTS) was used as FRET
acceptor.
5
Model with short-lived inward state can
explain the FRET data
a.
b.
c.
A430C
L387C
Fig. 5. a Eight-state model of glutamate transporter
cycle with the opening of the external gate as the
slowest step in the cycle. b Simulated FRET
efficiency (E) differences in EAAT3 A430C or
L387C between K+ and Na+ (K-Na), or Na+
+Glutamate and Na+ (Glu-Na) external solutions.
Ti-To is the expected change in the FRET efficiency
E for the homologous residue in GltPh crystal
structures between the outward- and inward-facing
states. c Simulated glutamate affinity at
-30mV, pH= 7.5.
CONCLUSIONS
•No large FRET efficiency change detected
in EAAT3 under multiple conditions.
•Internal TFB-TBOA does not trap the
inward state of glutamate transporters.
• Both experimental result and model
suggest inward state of
glutamate
transporters is short lived.