 |
 Previous Article | Next Article 
Volume 17, Number 10,
Issue of May 15, 1997
pp. 3401-3411
Copyright ©1997 Society for Neuroscience
Drosophila Serotonin Transporters Have
Voltage-Dependent Uptake Coupled to a Serotonin-Gated Ion Channel
A. Galli,
C.I. Petersen,
M. deBlaquiere,
R.D. Blakely, and
L.J. DeFelice
Center for Molecular Neuroscience, Department of Pharmacology,
Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Serotonin (5HT) transporters (SERTs) couple to existing ion
gradients to transport 5HT into presynaptic terminals. In mammalian SERTs, the transport cycle is reported as electroneutral, with a
translocation of zero net charge, and 5HT uptake is independent of
membrane voltage. Yet mammalian SERTs exhibit 5HT-induced currents, and
Drosophila SERTs (dSERTs) show voltage-dependent uptake.
Thus, the relationship between uptake and current remains
controversial; furthermore, the number of 5HT molecules translocated
per ion channel event is unknown. To investigate this, we have used
heterologous expression of cloned dSERTs to measure 5HT flux and dSERT
currents concurrently under voltage clamp, and we have used fluctuation analysis to measure the size of the elementary ionic events in the same
cells. RNA-injected Xenopus oocytes accumulate 5HT, and paroxetine or desipramine inhibit this uptake. RNA-injected oocytes also display paroxetine-sensitive 5HT-induced currents and
5HT-independent leak currents. Na replacement decreases the uptake and
the induced currents. 5HT-induced current and 5HT uptake both increase
at negative potentials, where 5HT carries ~5% of the induced
current. Recently, several groups have reported similar phenomena for
other transporters, in which transmitter-induced currents exceed the predictions of coupled transport. We now provide evidence that in
dSERT, ~500 5HT molecules are translocated per channel opening, which, at  20 mV, carries ~10,000 electronic charges. These data support a model in which 500 SERT cycles occur for each 5HT-induced channel opening or a model in which 500 5HT molecules and 10,000 electronic charges pass through a common pore.
Key words:
serotonin;
transporter;
uptake;
antidepressants;
channels;
Xenopus oocytes
INTRODUCTION
Neurotransmitter transporters couple the
gradients of ions to the flux of the transmitter (Amara and Kuhar,
1993 ; Lester et al., 1994). Na- and Cl-coupled transporters constitute
a large family of related proteins that take up GABA, catecholamines, and serotonin (5HT). Na-coupled transporters make up a separate gene
family for the uptake of glutamate and aspartate. Recently, several
groups have described transporter-associated currents that exceed the
predictions of stoichiometric transport (DeFelice and Blakely, 1996;
Sonders and Amara, 1996). Excess currents have been observed in
heterologous expression systems for 5HT (Corey et al., 1994; Mager et
al., 1994), glutamate (Vandenberg et al., 1995; Wadiche et al.,
1995a,b), norepinephrine (Galli et al., 1995, 1996), GABA (Cammack et
al., 1994, 1996), and dopamine (Sonders et al., 1997) transporters.
Similar phenomena appear in native preparations for 5HT (Bruns et al.,
1993), GABA (Cammack and Schwartz, 1993), and glutamate (Larson et al.,
1996) transporters. Here we report the first data to correlate in the
same cells the movement of transmitter with the channel activity
underlying the transmitter-induced current. We propose two models that
relate this information to voltage-dependent uptake.
Serotonin uptake has been characterized by radiolabeled 5HT flux
(Rudnick, 1996). Based on this method, Rudnick and Clark (1993) modeled
mammalian SERTs as electroneutral with stoichiometry: Na+/Cl /5HT+ and K+
countertransported. This process is apparently voltage-independent (Rudnick and Nelson, 1978) and does not rely on a K gradient (Nelson and Rudnick, 1979). Although Reith et al. (1989) and Cool et al. (1990)
corroborate this result, Kanner and Bendahan (1985) report K-diffusion-potential-stimulated 5HT uptake. Therefore, discrepancies exist concerning the role of voltage in 5HT uptake. In a heterologous expression system, in which Xenopus oocytes were used to
express rat SERTs, Mager et al. (1994) observed that 5HT uptake was
voltage-independent. In this system, it was also shown that 5 to 12 Na+ ions could "escape" into the cell during each
transport cycle, thus generating an ionic current in excess of the
predicted electroneutrality of coupled transport in mammalian
SERTs.
Corey et al. (1994) and Demchyshyn et al. (1994) cloned the
Drosophila serotonin transporter (dSERT) and studied its
ionic dependence and pharmacological characteristics. In
Xenopus oocytes expressing dSERT, Corey et al. report that
5HT uptake depends on Na and Cl. Changing the membrane from 40 to
80 mV increased 5HT uptake by a factor of three. Moreover, the
induced currents in dSERT are larger than those in mammalian SERTs
under comparable conditions. It thus appeared possible to investigate
the channels underlying the dSERT currents and to study their
relationship to voltage-dependent uptake in the same cells. We have
combined radioactive flux assays and voltage-clamp and fluctuation
analysis to quantify the relationship between uptake and membrane
potential. These data also provide a measure of 5HT translocation per
5HT-induced channel opening, and they are consistent with two models of
the transporter. In one model, hundreds of coupled transport cycles must occur for each channel opening. In this case, voltage-dependent 5HT uptake resides in the transport cycle, and 5HT accumulates by
coupling to the gradients of the co-transported ions. A second model
envisions that 5HT gates a channel permeable to small inorganic ions
and to 5HT. In this case, electrochemical gradients acting on 5HT
cations explain voltage-dependent uptake. If the transporter is a 5HT
channel and no coupling occurs in the pore, 5HT can accumulate against
its chemical gradient only as predicted by the voltage gradient. We
evaluate these predictions and further compare the two models in the
Discussion.
MATERIALS AND METHODS
Electrophysiology. We performed voltage-clamp
experiments using the two-electrode voltage-clamp technique. A
GeneClamp 500 (Axon Instruments, Foster City, CA), band-limited at 2000 Hz, was used to measure current. The recorded current was stored for analysis on videotape (Panasonic, Secaucus, NJ). Voltage-clamp electrodes filled with 3 M KCl solution, were pulled using
a programmable puller (Sachs-Flaming, P87, Sutter Instruments, Novato,
CA). The voltage steps ranged from 140 to 40 mV and lasted 500 msec.
All the data were obtained at room temperature. The recording solution was as follows (in mM): Na+ Ringer's solution
(96 NaCl, 2 KCl, 5 MgCl2, 5 HEPES, and 0.6 CaCl2); the ionic substitutions were NMDG+ for
Na+, 96 NMDGCl; Li+ for Na+, 96 LiCl; acetate for Cl, acetate salts of Na+.
Bath solutions were changed by a gravity pump at a rate of 1 ml/min.
[3H]5HT flux. Current measurements and flux
assays were measured simultaneously while perfusing the voltage-clamped
oocytes with 1.8 µM 5HT (200 nM
[3H]5HT, 100 Ci/mmol, Amersham, Arlington Heights, IL)
final concentration. The experiments were terminated by washing the
oocytes in Ringer's solution three times over a period of 20 sec, and
then solubilized in 1% SDS. The 5HT uptake was determined by liquid
scintillation counting. We performed control experiments by injecting
the oocytes with 100 pmol of [3H]5HT, and measuring the
release of radiolabeled 5HT during a 1 min experimental time. During
this time, no significant efflux of [3H]5HT was detected.
Furthermore, in uninjected oocytes, nonspecific uptake, during 1 min
experimental time, was not voltage-dependent in the range of potentials
studied and was <4% of the specific uptake at 20 mV.
Oocytes and cRNA. The 2.3 kb dSERT BglII fragment
was blunt-ended and cloned into the blunt-ended BamHI site
of pBluescript KS+ plasmid (Stratagene, La Jolla, CA)
downstream of the T7 promotor (Demchyshyn et al., 1994). cRNA was
transcribed in vitro using the Ambion mMessage mMachine T7
In Vitro Transcription Kit. Stage V and VI defolliculated
oocytes were injected with 42 nl (1 µg/µl) of dSERT cRNA and then
incubated at 18°C in Ringer's solution, pH 7.6, supplemented with
1% penicillin-streptomycin stock (10,000 U/ml, Gaithersburg, MD), and
5% horse serum (Life Technologies) (Quick et al., 1992). Peak
expression of dSERT varied from batch to batch ranging from 4 to 9 d.
RESULTS
5HT activates paroxetine-sensitive currents in
dSERT-cRNA-injected oocytes
RNA-injected oocytes display saturable 5HT accumulation,
5HT-induced currents, and transporter-associated but 5HT-independent currents that are not present in uninjected controls. We begin with a
description of the 5HT-induced and 5HT-independent (leak) currents. In
96 mM NaCl, oocytes injected with cRNA for dSERT exhibited
an inward current when held at negative potentials and 5HT was added to
the bath. In the experiment shown in Figure 1, we held
an oocyte at 80 mV and added 5 µM 5HT to the perfusing Ringer's solution. We refer to the difference between the holding current and the current in the presence of 5HT as the 5HT-induced current. Uninjected or mock-injected oocytes have no 5HT-induced current (data not shown). The 5HT-induced current typically reaches a
steady-state in <2 sec after 5HT application (reflecting primarily the
speed of the perfusion system). In RNA-injected oocytes, replacing NaCl
with LiCl activates an inward current at 80 mV with no 5HT present;
in this experiment, the inward current in 96 mM Li was 142% of the 5HT-induced current in 96 mM Na. We refer to
this as the Li leak current, meaning the transporter-associated current generated in the presence of Li and in the absence of 5HT. The Li leak
is not seen in uninjected oocytes. In agreement with other studies,
adding 5HT reduced the Li leak by 30 ± 7% (6 oocytes). Simultaneous application of 5HT (5 µM) and LiCl (96 mM) induced an inward current that was 70 ± 17% (6 oocytes) of the Li leak current. This suggests that the Li leak current
does not correlate with 5HT transport, and Figure 3A shows
that in the presence of Li and 5HT, there is no 5HT transport. Finally,
we note that 1 µM paroxetine reduces the 5HT-induced
current 90-100% and the Li leak current by 70-80%; paroxetine has
no effect on uninjected oocytes, thus both categories of current are
associated with the presence of dSERT in the membrane.
Fig. 1.
Serotonin induces paroxetinesensitive
currents in dSERT-cRNA-injected oocytes. Oocytes injected with cRNA for
the Drosophila serotonin transporter (dSERT) have a
5HT-induced inward current when oocytes are clamped to negative
potentials. The holding potential in these experiments was 80 mV. In
96 mM NaCl, 5 µM 5HT induces current in
oocytes injected with Drosophila serotonin transporter cRNA. Serotonin induces no current in uninjected or mock-injected oocytes (data not shown). Substituting Na with Li in the absence of 5HT
stimulates a large paroxetine-sensitive inward current (the Li leak).
In 96 mM LiCl solution, 5 µM 5HT partially
blocks the leak current. The addition of 1 µM paroxetine,
a 5HT-transporter inhibitor, to the Ringer's solution perfusion medium
decreases the holding current. We obtained a similar decrease using
desipramine (10 µM) or substituting NaCl with CholineCl
(data not shown).
[View Larger Version of this Image (12K GIF file)]
Fig. 3.
Na dependence of [3H]5HT uptake and
5HT-induced current. A, 5HT uptake was measured after 5 min incubation with 200 nM [3H]5HT. To assess
Na-dependent uptake, the standard Ringer's solution was modified by
replacing NaCl with LiCl or NMDG-Cl to maintain osmolarity. In the
absence of Na, 5HT uptake is strongly reduced in three batches of
oocytes (4 oocytes for each ion replacement). Nonspecific uptake in
uninjected oocytes was <8% of the blockable uptake. B,
The 5HT-induced current depends on the external Na concentration. 5HT
was held constant at a saturating concentration (5 µM)
(see Fig. 6), and the membrane potential was held at 30 mV. The
substitution of NaCl with NMDG-Cl reduces the steady-state current at
this potential. The inset shows the ratio of the
transporter current obtained in 0 mM NaCl + 5 µM 5HT to the current obtained in Ringer's solution + 5 µM 5HT at different voltages.
[View Larger Version of this Image (15K GIF file)]
Although we have not further characterized the leak, the presence of a
transporter-associated leak pathway most evident in Li solutions raises
the possibility of a similar pathway in Na solutions, in which we
perform all experiments below. Indeed, Figure 1 shows that in 96 mM NaCl, and in the absence of 5HT, 1 µM
paroxetine reduces the holding current in RNA-injected oocytes. There
is no similar effect in uninjected oocytes. We obtain approximately the
same change in holding current with 1 µM paroxetine, with 10 µM desipramine, or by substituting NaCl with
CholineCl. We refer to the paroxetine- or desipramine-revealed current
as the Na leak current, implying the transporter-associated current in the presence of Na, but in the absence of 5HT. This does not imply, as
the name might suggest, that the Na leak current is carried by Na.
Indeed, we have not characterized the Na leak with regard to the
permeating ions. Rather, we seek to minimize the leak current by
working near its reversal potential, which we measure in Figure 2.
Fig. 2.
Current-voltage relationship of Na leak current.
Steady-state I-V curve for Na leak
current in Ringer's solution (96 mM Na). The membrane
potential was stepped for 500 msec between 140 and +40 mV from a
holding potential of 40 mV. After adding 10 µM desipramine to the perfusion medium, the current decreased to a new
steady-state value at each voltage. We define leak current as the
difference between the background current before adding desipramine and
the current in the presence of desipramine. Data were normalized at
140 mV (4 oocytes). The Na leak current at 140 mV has an average
value of 9.05 ± 5.2 pA, ~10% of the 5HT-induced current at this potential.
[View Larger Version of this Image (10K GIF file)]
Current-voltage relationship of Na leak current
Figure 2 shows the average current-voltage relationship of the
leak current (96 mM Na, no 5HT) obtained from RNA-injected oocytes. We held the oocytes at 40 mV and stepped the voltage between
140 and 40 mV in 20 mV increments, measuring the currents in the
steady state. The Na leak current is defined as the difference between
the control current (96 mM Na, no desipramine) and the current after adding desipramine (96 mM Na, 10 µM desipramine). Desipramine (10 µM) has no
effect on uninjected oocytes. Within the same batch of oocytes, Na leak
currents are ~10% (9.02 ± 5.2 nA, 4 oocytes) of 5HT-induced
current (89.7 ± 29 nA, 4 oocytes) at 140 mV. We fit the
experimental points in Figure 2 (normalized to 140 mV) with a cubic
polynomial using a nonlinear least-squares procedure, obtaining
Vrev = 23 mV. Fitting a straight line to the
four data points surrounding zero current in Figure 2 gives a reversal
of 26 mV. The Nernst potentials for K, Cl, and Na in native oocytes
are, respectively, 95, 28, and 61 mV (Dascal, 1987; Costa et al.,
1989). Although the leak Vrev is near the literature value of the Cl reversal potential, we have not investigated the ion selectivity of the leak in RNA-injected oocytes. We reveal approximately the same magnitude of leak current at 80 mV by substituting Na with choline (see above), indicating that at negative potentials, Na may be a major carrier. (For additional discussion of
the leak current in hSERT, see Mager et al., 1994.) In this article, we
merely use the empirical value of the reversal potential to reduce the
leak when determining the elementary event underlying 5HT-induced
currents.
Ionic dependence of 5HT uptake and 5HT-induced current
To verify the Xenopus oocytes expression system
for studying the electrophysiological characteristics of dSERT, we
studied the ionic selectivity of [3H]5HT uptake to
compare with previous studies (Corey et al., 1994). Levels of
expression were similar in oocytes from the same injection batch, even
though they differed significantly from batch to batch. A marked batch
deviation also exists in the number of days that the oocytes require to
reach the peak expression level (between 4 and 9 d). For the
uptake measurements presented in Figures 3 and
4, the oocytes were not impaled for voltage clamp.
Figure 3 shows the Na dependence of 5HT uptake and 5HT-induced current. The oocytes were incubated in a 180 µl bath, and 5HT was added to the
medium solution and briefly stirred to reach 1.8 µM final concentration. The solution contained a final concentration of 200 nM [3H]5HT. An incubation time of 5 min was
allowed before beginning the uptake measurements. The uptake velocities
that we obtained in our expression system are in approximate agreement
with Corey et al. (1994), although as mentioned, considerable
variability exists from batch to batch. Complete replacement of Na with
either Li or NMDG reduced the uptake by an average of 81.5 ± 10%
and 78 ± 17%, respectively (three different batches, 3 to 5 oocytes for each Na replacement). Figure 3A shows the effect
of Na substitution on 5HT uptake for a representative batch. Replacing
Na reduces 5HT uptake to nonspecific levels seen in uninjected oocytes.
Figure 3B shows 5HT-induced current obtained after applying
5 µM 5HT to an oocyte held at 30 mV. A comparison of
B and A is not strictly valid, because the
oocytes in A were not voltage-clamped; 30 mV was selected,
because it is near the resting potentials of RNA-injected oocytes,
which tend to have fewer negative resting potentials than uninjected
oocytes. Nevertheless, we compare A and B to show
that when the oocytes used for flux analysis have not been impaled,
there is still a qualitative correlation between the ionic dependence
of uptake and the ionic dependence of the transmitter-induced current.
The substitution of Na with NMDG reduced the 5HT-induced current by an
average of 80 ± 7% (4 oocytes), in approximate agreement with
the reduction in uptake. The percent reduction that was induced by Na
exchange is not strongly voltage-dependent in the range of 90 to 30
mV (inset, 4 to 6 oocytes for each point). Figure
4A illustrates that replacing Cl with acetate lowers 5HT uptake by 76 ± 7% (four batches, 4 to 5 oocytes per batch). Figure 4B shows 5HT-induced current obtained after
applying 5 µM 5HT to an oocyte held at 30 mV. The
substitution of Cl with acetate reduced the 5HT-induced currents to an
average of 32 ± 20% (3 oocytes). The reduction in induced
current by Cl exchange is voltage-independent in the range 90 to 30
mV (inset, 4 to 6 oocytes for each point). We interpret the
antagonist-block and ion-replacement data for Na and Cl as indicative
of the coupling between 5HT uptake and 5HT-induced current. However,
another explanation for the observed differences between A
and B in Figures 3 and 4 is that nominally Na- or Cl-free
solutions may be contaminated by these ions leaking from the oocyte
through channels or pumps not associated with the transporter. We have
not attempted protocols that would eliminate such buildup.
Fig. 4.
Cl dependence of [3H]5HT uptake and
5HT-induced current. A, 5HT uptake was measured after 5 min incubation with 200 nM [3H]5HT. To assess
Cl-dependent uptake, the standard Ringer's solution was modified by
replacing NaCl with Na-acetate to maintain osmolarity. In the absence
of Cl, 5HT uptake is strongly reduced in three batches of oocytes (4 oocytes for each ion replacement). In experiments performed without
external Cl, 5HT uptake was strongly reduced. B, The
oocytes were clamped at 30 mV and perfused with Ringer's solution
and 5 µM 5HT. When the external Cl was exchanged with acetate, the current decreased. The inset shows the
ratio of the transporter current obtained in solution containing no Cl + 5 µM 5HT to the current obtained in Ringer's solution + 5 µM 5HT at different voltages.
[View Larger Version of this Image (21K GIF file)]
Voltage and concentration dependence of the
5HT-induced current
To investigate the 5HT dependence and voltage dependence of the
induced current, the level of substrate in the bath was increased incrementally from 0 to 10 µM at three voltages between
100 and 60 mV. At a fixed voltage, the amplitude of the current
saturates with increasing 5HT concentration. Normalizing to the current obtained at 10 µM 5HT, the data were fit by nonlinear
regression to the equation:
which gave (3 oocytes for each voltage) for 60 mV,
Km = 1.69 ± 0.24 µM and
n = 1.38 ± 0.19; for 80 mV,
Km = 0.8 ± 0.12 µM and
n = 1.32 ± 0.25; for 100 mV,
Km = 1.16 ± 0.23 µM and
n = 0.95 ± 0.17. The average values are
Km = 1.21 µM and n = 1.21. Km and n were also obtained
by pooling the experiments. In this case, Km = 1.26 ± 0.20 µM and n = 1.19 ± 0.19. We conclude that there is no significant difference between
affinity constants or Hill coefficients in the range of 100 to 60
mV. Thus, the shape of the I(V) curve in
this range is unlikely to be influenced by Km
measured for 5HT titration. However, because we have not measured
Km for Na or Cl, we cannot eliminate
voltage-dependent binding or variable stoichiometry as possible
explanations of voltage-dependent uptake.
5HT uptake depends on voltage
The comparison between uptake and induced current
demonstrated in Figures 3 and 4 suggests that they are associated
events. To investigate the voltage dependence of uptake, we initially performed radiolabeled 5HT flux experiments on batches of oocytes expressing different levels of transporter. We held oocytes at 30 mV
and perfused them with 1.8 µM 5HT (100 Ci/mmol). After 5 min, the oocytes were washed, lysed, and assayed for accumulated 5HT.
This procedure was repeated for another set of oocytes from the same
batch, but held at 100 mV. We duplicated these experiments for three
batches, including one batch with low expression. These data are shown
in Figure 5; the shading of the bar identifies a
particular batch. Even including a batch with low levels of uptake, the
average accumulation of 5HT across batches was 2.24 ± 0.8 times
higher at 100 than at 30 mV (3 or 4 oocytes in each batch). The
same pattern exists at lower 5HT concentrations. This is shown in the
inset, in which we used 0.1 µM 5HT and obtained 3.36 ± 0.9 times higher 5HT uptake by dSERT at 100 than at 30 mV (one
batch, 3 oocytes per voltage). These data are in approximate agreement
with Corey et al., who performed similar experiments at 40 and 80
mV. From our data, we conclude that negative voltages stimulate 5HT
uptake, and this stimulation is approximately independent of the level
of expression or the concentration of 5HT.
Fig. 5.
Membrane potential regulates 5HT uptake. In these
experiments, the membrane potential was held at 30 or 100 mV while
the oocytes were exposed for 5 min to 1.8 µM 5HT or 0.1 µM 5HT (inset). Uptake in oocytes not
under voltage clamp was measured as in Figures 3 and 4. In three
batches of oocytes (at least 3 oocytes per batch per voltage), we found
that the uptake of 5HT is significantly increased at the negative
potential. Each bar represents a different batch, and
the variation in absolute magnitude reflects different levels of
expression.
[View Larger Version of this Image (19K GIF file)]
Number of ions translocated with 5HT varies with
membrane potential
Figure 5 demonstrates that negative voltages stimulate 5HT uptake
in dSERT; however, the protocol prohibits alteration of voltage in the
same cell, because the cells are lysed after each measurement.
Furthermore, differences in expression levels force reliance on batch
averages to measure uptake as a function of voltage, as in a previous
study of dSERT (Corey et al., 1994). We require a method that compares
oocytes from different batches, without regard to the level of
expression. To achieve this, we measured 5HT uptake and 5HT-induced
current simultaneously for single oocytes held at a specific potential.
Each oocyte was voltage-clamped and bathed for 50 sec in
[3H]-labeled 1.8 µM 5HT Ringer's solution.
The integration of the 5HT-induced current measures the total charge
movement Q at a particular voltage. We also measured
Q5HT from the same voltage by scintillation
counting of lysed oocytes. The derived quantity  (V) is the ratio
Q/Q5HT, which we obtain from
different oocytes at different voltages. Thus, (V)
is independent of expression level. We distinguish the charge
Q5HT carried by 5HT+ (assuming 5HT
is translocated as a monovalent cation at pH = 7.6) from the
charge Qe carried by other ions. Thus,
Q = Qe + Q5HT. If each transporter carries the same
proportion of 5HT and non-5HT charge, then:
where qe and q5HT
represent charge movements through individual transporters. Thus,
(V) is a microscopic property of individual transporters, and 1/ represents the fraction of charge carried by
5HT. Figure 6A plots (V)
as a function of voltage for data from 54 oocytes (4 to 11 oocytes for
each point). We find translocated charge in excess of 5HT accumulation,
and this ratio is voltage-dependent. The minimum ratio is = 17 ± 3 at 20 mV, and the maximum ratio is = 67 ± 15 at 80 mV
(mean ± SD). For example, at 80 mV, 5HT induces the
translocation of 67e for every 5HT molecule that passes
through dSERT (e = 1 electronic charge unit). We fit
the average points (solid squares) and the upper and lower
SDs (bars) with a cubic polynomial. These fits are the solid
curves in Figure 6A. To estimate the absolute current
carried by the movement of 5HT+ alone, we now compare
(V) with I(V) curves
measured separately for particular oocytes. Figure 6B shows
the I(V) curve for 5HT-induced currents
from the same batch. The voltage was stepped for 500 msec to 140 to
40 mV in 20 mV increments, holding the oocyte at 40 mV, and currents
were measured between 400 and 500 msec after the initiation of the test
pulse. We defined the I(V) curve by
subtracting the control current (no 5HT) from the 5HT-induced current.
The I(V) curve displays inward
rectification and does not reverse for voltages up to 40 mV, in
agreement with Corey et al. (1994). These representative oocytes had
nearly equivalent expression levels, judging by the small SD in
5HT-induced currents. We fit these experimental points (solid
triangles) with the quadratic polynomial. We may also write as
the ratio = I/I5HT, because Q and Q5HT are measured in the same
time interval. The current carried by 5HT in these oocytes is then
given by:
Note that in this formula, I(V) and
(V) come from two different data sets;
I(V) varies with expression level and is
measured for an individual oocyte, and (V) is
independent of expression level in the range we have studied and is
measured one voltage at a time for separate oocytes. The result of this
division appears in Figure 6C. The solid line is the ratio
of averages from A and B and is the current
carried by 5HT for the oocytes in B. The dotted lines in
C reflect the same calculation using fits to the SDs in
A and B. These data are consistent with Figure 5,
which shows that 5HT uptake increases at negative voltages.
Fig. 6.
The number of charges translocated per 5HT varies
with membrane potential. A, Ion translocation and
substrate uptake were simultaneously measured during a 50 sec
application of 1.8 µM 5HT at different voltages. The
ratio () of the total charge translocated (obtained by integrating
the 5HT-induced current) and the charge measured by the substrate
accumulation is voltage-dependent. We fit the average ratio, and the
upper and lower SDs, with a third-order polynomial
pV = p1V3 + p2V2 + p3V + p4 (solid lines).
B, Currents obtained by applying 1.8 µM
5HT to the bath perfusion and clamping at different voltages (3 oocytes
per point from the same batch). The average of the difference currents
is plotted against voltage. We fit the experimental points with the
quadratic polynomial I(V) = p1V2 + p2V + p3. C, Dividing the absolute
currents (the solid line in B), by (the three solid lines in A), determines
the current carried by 5HT in these particular oocytes. The
solid line represents the average current carried by 5HT
ions as a function of voltage, and the dashed lines
represent the SDs.
[View Larger Version of this Image (12K GIF file)]
Number of 5HT molecules translocated per 5HT-induced
channel opening
I5HT does not give the relationship
between the movement of 5HT and the elementary event underlying the
induced current. For this, we must know the charge carried in a typical
channel opening, which will enable us to estimate how many 5HT
molecules are translocated per channel event. First note that we may
infer from Figure 6A the relative charge translocated
per 5HT molecule. In particular:
This ratio does not indicate the absolute number of charges
translocated per transporter event, only the relative number. For
example, at 20 mV, = 17 implies 16e for each 5HT
molecule. If we assume, for example, that one 5HT corresponds to one
transporter cycle, we might conclude that 16e
translocate/cycle. However, the data are also consistent with
160e moving every 10 cycles, or any other ratio of 16:1. To
know the absolute ratio, we need the charge carried by the channels
that underlie the current. To estimate this, we measured the mean and
the variance of the 5HT-induced currents, as indicated in Figure
7. In these experiments, oocytes were held at 20 mV to
reduce contamination from the Na leak pathway (see Fig. 2).
Fig. 7.
Fluctuation analysis of the 5HT-induced current.
The record shows a typical experiment in which the oocyte was held at
20 mV, and 1.8 µM serotonin was added to the bath at
the indicated time. The insets show the current
fluctuations on an expanded scale. The 5HT-induced current is taken as
the average current difference at the quasi steady-state level. The
5HT-induced fluctuations are measured as the difference in variance
between the two traces (inset), calculated over 16 sec
in the bandwidth of 10-1000 Hz.
[View Larger Version of this Image (28K GIF file)]
From shot noise theory (DeFelice, 1981), the ratio of the variance to
the mean is independent of the frequency of events. If a unitary event
is approximated by a  function, and if the fluctuations caused by
the random arrival of such events are measured in a bandwidth B, the
ratio of the variance to the mean is given by:
In this formula, I indicates the 5HT-induced current,
 2 is the difference variance in the presence and
absence of 5HT, and q is the net charge per shot. From seven
experiments similar to that in Figure 7, we measured
2/I = 2.75 ± 0.92 pA at
20 mV. The value of I in these experiments ranged between
8.4 and 25 nA, indicating that the level of expression in these
oocytes varied threefold. Thus, 5HT-induced noise is proportional to
the 5HT-induced current in this expression range. No comparable
increase in the variance occurs on injecting currents up to 25 nA in
control oocytes. 5HT increases neither the current nor the variance in
uninjected control oocytes, and 1 µM paroxetine blocks
both the 5HT-induced current and the 5HT-induced fluctuations in
RNA-injected oocytes, indicating further that the fluctuations are
associated with the transporter. In these same oocytes, we also
measured at 20 mV, as in Figure 6A. For the
bandwidth between 10 and 1000 Hz, the charge q obtained from
2/2BI has the average value: q = 8250 ± 2870e (7 oocytes). We also determined
q by fitting a linear regression to
2/I = io + 2qB, using four-pole Butterworth filters to vary the upper
cutoff between 100 and 1000 Hz. For three oocytes and four values of
B, this plot yielded io = 0.102 ± .12 pA and 2q = 13,510 ± 1005e
(q = 6755e), which is in reasonable
agreement with q = 8250e. This result
implies that the frequency spectrum of the induced noise is
approximately flat. In the following discussion, we use the larger
value of q obtained from the full bandwidth. The value of
q is unexpectedly large and leads to the conclusion that
~500 transport cycles occur for each channel opening. In the
Discussion, we show that relaxing the assumption of the function
leads to a similar conclusion. For a model in which 5HT translocation
occurs exclusively through coupled transport, q/ estimates of the number of cycles ( ) per 5HT-induced channel. Dividing q = 8250 by = 18 ± 2.7 (the value
obtained at 20 mV in the oocytes used for fluctuation analysis) gives
458 ± 159e. Assuming that 5HT is a monovalent cation
and assuming one 5HT per cycle imply that = 458 cycles. Thus, the
combination of flux data and noise analysis provides a novel measure of
transporter cycles per channel opening, assuming that coupled transport
and so-called uncoupled currents are separate transporter states. We
stress that the coupled and uncoupled states are likely to be linked
because of similar 5HT and ionic dependence and antagonist sensitivities. These data are also consistent with a model in which
transport and channel openings are not separate events. In this case,
q/ has a different interpretation, that is, the number of
5HT molecules that translocate per channel opening is q5HT = q/. Below, we use the
nominal values: (or q5HT) = 500, and
q = 10,000 when considering the implications of these
data for 5HT transport and ion permeation through dSERT.
DISCUSSION
RNA-injected oocytes display 5HT uptake that is absent in
uninjected or mock-injected oocytes, and they have a pronounced 5HT-induced current. The 5HT flux and 5HT-induced current are ion-dependent. Replacing Na with Li or NMDG or replacing Cl with acetate reduces uptake and current, and flux and current are sensitive to paroxetine and desipramine. Moreover, Km
obtained from induced currents are in approximate agreement with
Km for dSERT determined by uptake assays in
Xenopus oocytes, for which Km = 0.637 µM (Corey et al., 1994). The value we obtain,
Km = 1.26 µM, is independent of
voltage between 100 and 60 mV. Corey et al. (1994) show that both
uptake and current increase with hyperpolarization, in agreement with
our results. Comparable ionic dependence, voltage dependence, 5HT and
antagonist sensitivities, and Km values indicate
that 5HT flux and 5HT-induced current are coupled mechanisms.
Recent findings from a number of laboratories (see introductory
remarks) make it necessary to consider at least three types of current
associated with neurotransmitter transporters: (1) channels that open
in the absence of transmitter (leak), (2) transmitter-gated channels
without transport, and (3) current produced during transmitter translocation. It is simple to isolate the leak current (1) but difficult to separate transmitter-gated current (2) from
transmitter-translocation current (3). We have isolated the current
carried by the charged neurotransmitter 5HT+ and estimated
the size of the transmitter-gated channels, eliminating leak pathways
by working at the leak-reversal potential. The data have two
interpretations. First, the transmitter-gated channel may represent an
ion pathway through which there is no translocation of transmitter. In
this case, we have measured the number of transport cycles (which may
generate a fraction of the current, depending on stoichiometry) that
occur per channel opening (which generate the major current). Note that
the ratio = transport cycles/channel is distinct from ratios
published previously ( = charges/translocated transmitter). The
results presented here also agree with the combined movement of 5HT and
other ions through a 5HT-gated channel that combines 2 and 3 in the
same state.
Current carried by leak pathways
Leak current is defined as transporter-associated current in the
absence of transmitter. In mammalian SERTs, 5HT reduces the Li leak
(LiCl replacing NaCl) (Mager et al., 1994), and we observe a similar
phenomenon in dSERT. A leak is also present in NaCl, which we reveal by
application of paroxetine or desipramine or by the replacement of Na
with choline. The Na leak exists in the presence of 5HT, but 5HT
partially blocks it. The Na leak is 10% of the induced current at
140 mV and reverses near 20 mV. The induced current does not
reverse, and we interpret this as large outward leak current
compared with outward induced current; thus, plotting
(induced current) (leak) results in net inward current. Using this
convention, the inward current positive to the reversal potential of
the leak represents 5HT-induced current and, primarily, the outward
leak current that is blocked by 5HT. A similar interpretation was made
for the dopamine transporter (Sonders et al., 1997).
The leak current
We now consider how the leak current affects the measure of
I5HT and, therefore, our estimate of or
q5HT. Define Na leak in the absence of 5HT as
Ileak, and the comparable pathway in 5HT as
I leak. If 5HT partially blocks
Ileak, then
Ileak < Ileak, and (V) is larger than we would estimate if we
could exclude the leak. This is because the (V)
calculation involves subtracting currents in the absence of 5HT from
currents in the presence of 5HT, and we may be subtracting too much.
However, the leak does not affect the calculation
I5HT = I/, because total current
divides out in this ratio. This does not hold for = q/; thus, for this estimate, we must eliminate the leak.
Although Ileak is only 10% of the 5HT-induced
current at 140 mV, it may be substantially greater at more positive
potentials and could affect our estimate of . For this reason, we
performed fluctuation experiments near the leak reversal potential,
where the current from this pathway should be minimal. The value of at 20 mV therefore is an approximation of the 5HT-induced ratio of
current to flux in the absence of leak. At 20 mV, = 17, which
greatly exceeds one net charge per transport cycle, assuming a
stoichiometry of Na+/Cl/5HT+.
Several groups have reported similar data for other transporters, in
which currents exceed the predictions of coupled transport. For
example, Sonders et al. (1996) found that in dopamine transporters, = 3 at 20 mV and = 7 at 120 mV.
The shot noise model
At 20 mV, we show that 500 5HT molecules enter the cell per
5HT-induced channel opening and that each channel opening carries 10,000e. This estimate does not rely on the turnover rate or
the number of transporters and is in approximate agreement with
channels recorded from oocyte patches expressing a mammalian SERT (Lin et al., 1996). The channels they record are 0.4 pA at 20 mV, with an
average open time of 2.5 msec, implying a charge transfer of
6200e. Using the shot noise model and whole-cell
fluctuations to estimate channel size enabled us to perform flux assays
on the same cell, which presently would be impossible in a cell-free patch. The theory assumes that every shot is i(t) = q(t), in which case
2/I = 2qB. If we
assume that events have an average width of  t, then
2/I ~ q/t, and q represents an average
charge per event. The smallest t that could contribute to
fluctuations that are cut off at 1000 Hz is t ~ 0.1 msec.
t > 0.1 msec implies q > 3000e. This gives a lower limit to the number of 5HT
molecules transferred per channel opening; for = 17, >176.
Transporter models
Figure 8 describes two possible interpretations of
these data. For simplicity, we show only the binding of 5HT; the
binding of obligatory Na and Cl ions is implied. In A, we
presume that 500 coupled transport cycles, , occur for every
elementary current. In this model, we envision two classes of events,
the coupled transport of 5HT and co-transported ions, which is a small
and perhaps even silent electrical event, and a 5HT-gated ion channel. The transporter flips back and forth between transporter mode and
channel mode, and the channel mode occurs with low probability compared
with coupled transport (Galli et al., 1995). In such a model, we would
expect the ratio of current to transport to be given by
Nip/N  , where N is the number of
transporters, is the cycle rate, is the net charge transferred
per cycle, i is the elementary current through the open
5HT-gated channel, and p is the probability of this channel
being open. Thus, for the model in A, = ip/, emphasizing that is a relative value requiring an independent knowledge of i and p to
relate to . Nevertheless, one can use the measured value of to calculate another transporter characteristic, = q/, the number of transporter cycles that occur per
channel opening. The model illustrated in Figure 8B
raises the possibility that there is only one class of elementary
events and that 5HT moves through the channel with other ions in a
ratio determined by . We reconcile the conduction of hundreds of
5HTs with an apparent stoichiometry of one 5HT binding to dSERT by
assuming that whereas one 5HT molecule opens the transporter,
10,000e pass each opening, ~5% of which are 5HT.
Fig. 8.
Alternative models of dSERT. In A,
two classes of events occur. The transporter is either in the coupled
mode, in which 5HT translocates in fixed stoichiometric ratio with
co-transported ions. On average, once every 500 cycles, a 5HT-gated
channel opens, allowing 10,000e to pass through but not
5HT+. In the second model, illustrated in B,
5HT gates a channel through which 10,000e pass, ~5%
of which are 5HT+. For simplicity, we have shown only 5HT
binding in A and B; obligatory binding of
Na and Cl ions is implied in both models.
[View Larger Version of this Image (26K GIF file)]
The relationship we have measured between uptake and currents not only
provides novel data for a particular transporter, but it suggests a new
strategy the combination of flux and noise analysisto study other
transporters. If 5HT translocates exclusively via coupled transport and
the major current flows through a separate state, voltage-dependent
uptake must reside in the rate constants of the transport cycle, the
binding affinities of 5HT or co-transported ions, or voltage-dependent
stoichiometry. On the other hand, if transport and current occur in the
same state, electrochemical potentials across a pore permeable to 5HT
cations determine voltage-dependent uptake. In this case, a charged
transmitter could move against its concentration gradient only at
sufficiently negative voltages. This is a departure from models that
assume substrate-induced currents through a pore-like structure, but
retain fixed or variable stoichiometry for coupled transport (Cammack
et al., 1994; Wadiche et al., 1995; Lin et al., 1996; Sonders et al.,
1997). However, it relates to a recent model proposed by Su et al.
(1996). A 5HT channel would imply that in some cases, clearance may
result from the conduction of transmitters through an open pore.
FOOTNOTES
Received Dec. 3, 1996; revised Jan. 31, 1997; accepted Feb. 22, 1997.
This work was supported by National Institutes of Health Grants
NS-34075, NS-33373, and DA-07390, and National Alliance for Research on
Schizophrenia and Depression Established Investigator Awards to L.J.D.
and R.D.B. We thank Scott Ramsey for his assistance in setting up the
initial experiments for dSERT cRNA-injected oocytes and Dawn Borromeo
for technical assistance. The dSERT clone was a generous gift of Dr. H. Niznik of the Clarke Institute.
Correspondence should be addressed to Professor Louis J. DeFelice,
Department of Pharmacology, Vanderbilt University Medical Center,
Nashville, TN 37232-6600.
REFERENCES
-
Amara SG,
Kuhar MJ
(1993)
Neurotransmitter transporters: recent progress.
Annu Rev Neurosci
16:73-93[ISI][Medline].
-
Blakely RD,
Berson HE,
Fremeau Jr RT,
Caron MG,
Peek MM,
Prince KH,
Bradley CC
(1991)
Cloning and expression of a functional serotonin transporter from rat brain.
Nature
354:66-70[Medline].
-
Bruns KJ,
Engert F,
Lux HD
(1993)
A fast activating presynapse reuptake current during serotonergic transmission in identified neurons of Hirudo.
Neuron
10:559-572[ISI][Medline].
-
Cammack JN,
Schwartz EA
(1993)
Ions required for the electrogenic transport of GABA by horizontal cells of the catfish retina.
J Physiol (Lond)
472:81-102[Abstract/Free Full Text].
-
Cammack JN,
Schwartz EA
(1996)
Channel behavior in a
 -aminobutyrate transporter.
Proc Natl Acad Sci USA
93:723-727[Abstract/Free Full Text]. -
Cammack JN,
Rakhilin SV,
Schwartz EA
(1994)
A GABA transporter operates asymmetrically and with variable stoichiometry.
Neuron
13:949-960[ISI][Medline].
-
Cool DR,
Leibach FH,
Ganapathy V
(1990)
Modulation of 5HT uptake kinetics by ions and ion gradients in human placental brush-border membrane vesicles.
Biochemistry
29:1818-1822[Medline].
-
Corey YL,
Quick MW,
Davidson QN,
Lester HA,
Guastella J
(1994)
A cocaine-sensitive Drosophila serotonin transporter: cloning, expression, and electrophysiological characterization.
Proc Natl Acad Sci USA
91:1188-1192[Abstract/Free Full Text].
-
Costa PF,
Emilio MG,
Fernandes PL,
Gil Ferreira H,
Gil Ferreira K
(1989)
Determination of ionic permeability coefficients of the plasma membrane of Xenopus laevis oocytes under voltage clamp.
J Physiol (Lond)
413:199-211[Abstract/Free Full Text].
-
Dascal N
(1987)
The use of Xenopus oocytes for the study of ion channels CRC.
Crit Rev Biochem
22:317-387.[ISI][Medline]
-
DeFelice LJ
(1981)
In: Introduction to membrane noise. New York: Plenum.
-
DeFelice LJ,
Blakely RD
(1996)
Pore models for transporters?
Biophys J
70:579-580[Free Full Text].
-
Demchyshyn LL,
Pristupa ZB,
Sugamori KS,
Barker EL,
Blakely RB,
Wolfgang WJ,
Forte MA,
Niznik HB
(1994)
Cloning, expression, and localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from Drosophila melanogaster.
Proc Natl Acad Sci USA
91:5158-5162[Abstract/Free Full Text].
-
Galli A,
DeFelice LJ,
Duke BJ,
Moore KR,
Blakely R
(1995)
Sodium dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants.
J Exp Biol
198:2197-2212[Abstract].
-
Galli A,
Blakely R,
DeFelice LJ
(1996)
Norepinephrine transporters have channel modes of conduction.
Proc Natl Acad Sci USA
93:8671-8676[Abstract/Free Full Text].
-
Kanner BI,
Bendahan A
(1985)
Transport of 5HT in membrane vesicles from rat basophilic leukemia cells.
Biochim Biophys Acta
816:403-410[Medline].
-
Larson HP,
Picaud SA,
Werblin FS,
Lecar H
(1996)
Noise analysis of the glutamate-activated current in photoreceptors.
Biophys J
70:733-742[Abstract/Free Full Text].
-
Lester HA,
Mager S,
Quick MW,
Corey JL
(1994)
Permeation properties of neurotransmitter transporters.
Annu Rev Pharmacol Toxicol
342:219-249.
-
Lin F,
Lester HA,
Mager S
(1996)
Single-channel currents produced by the serotonin transporter, and analysis of a mutation affecting ion permeation.
Biophys J
71:3126-3135[Abstract/Free Full Text].
-
Mager S,
Churl M,
Henry DJ,
Chavkin C,
Hoffman BJ,
Davidson N,
Lester HA
(1994)
Conducting states of a mammalian serotonin transporter.
Neuron
12:845-859[ISI][Medline].
-
Nelson PJ,
Rudnick G
(1979)
Coupling between platelet 5-HT and K transport.
J Biol Chem
254:10084-10089[Free Full Text].
-
Quick MW,
Naeve J,
Davidson N,
Lester HA
(1992)
Incubation with horse serum increases viability and decreases background neurotransmitter uptake in Xenopus oocytes.
Biotechniques
13:357-361[ISI][Medline].
-
Ramamoorthy S,
Bauman AL,
Moore KR,
Han H,
Yang-Feng T,
Chang AS,
Ganapathy V,
Blakely R
(1993)
Antidepressant and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization.
Proc Natl Acad Sci USA
90:2542-2546[Abstract/Free Full Text].
-
Reith ME,
Zimanyi I,
O'Reilly
(1989)
Role of ions and membrane potential in uptake of serotonin into plasma membrane vesicles from mouse brain.
Biochem Pharmacol
38:2091-2097[ISI][Medline].
-
Rudnick G
(1996)
Mechanisms of biogenic amine neurotransmitter transporters.
In: Neurotransmitter transporters: structure, function, and regulation (Maarten EA,
Reith,
eds), pp 73-100. Totowa, NJ: Humana.
-
Rudnick G,
Clark J
(1993)
From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters.
Biochim Biophys Acta
1144:249-263[Medline].
-
Rudnick G,
Nelson PJ
(1978)
Platelet 5-hydroxytryptamine transport.
J Biol Chem
254:4631-4635[Abstract/Free Full Text].
-
Sonders M,
Amara SG
(1996)
Channels in transporters.
Curr Opin Neurobiol
6:294-302[ISI][Medline].
-
Sonders M,
Zhu S-J,
Zahniser N,
Kavanaugh MP,
Amara S
(1997)
Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants.
J Neurosci
17:960-974[Abstract/Free Full Text].
-
Su A,
Mager S,
Mayo SL,
Lester H
(1996)
A multi-substrate single file model for ion-coupled transporters.
Biophys J
70:762-777[Abstract/Free Full Text].
-
Vandenberg RJ,
Arriza JL,
Amara SG,
Kavanaugh MP
(1995)
Constitutive ion fluxes and substrate binding domains of human glutamate transporters.
J Biol Chem
270:17668-17671[Abstract/Free Full Text].
-
Wadiche JI,
Amara SG,
Kavanaugh MP
(1995a)
Ion fluxes associated with excitatory amino acid transport.
Neuron
15:721-728[ISI][Medline].
-
Wadiche JI,
Arriza JL,
Amara SG,
Kavanaugh MP
(1995b)
Kinetics of a human glutamate transporter.
Neuron
14:1019-1027[ISI][Medline].
This article has been cited by other articles:
|
|
|
|
 
H. Iwamoto, R. D. Blakely, and L. J. De Felice
Na+, Cl-, and pH Dependence of the Human Choline Transporter (hCHT) in Xenopus Oocytes: The Proton Inactivation Hypothesis of hCHT in Synaptic Vesicles
J. Neurosci.,
September 27, 2006;
26(39):
9851 - 9859.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. M. Korkhov, M. Holy, M. Freissmuth, and H. H. Sitte
The Conserved Glutamate (Glu136) in Transmembrane Domain 2 of the Serotonin Transporter Is Required for the Conformational Switch in the Transport Cycle
J. Biol. Chem.,
May 12, 2006;
281(19):
13439 - 13448.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Krause and W. Schwarz
Identification and Selective Inhibition of the Channel Mode of the Neuronal GABA Transporter 1
Mol. Pharmacol.,
December 1, 2005;
68(6):
1728 - 1735.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. D. Blakely, L. J. DeFelice, and A. Galli
Biogenic Amine Neurotransmitter Transporters: Just When You Thought You Knew Them
Physiology,
August 1, 2005;
20(4):
225 - 231.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Kahlig, F. Binda, H. Khoshbouei, R. D. Blakely, D. G. McMahon, J. A. Javitch, and A. Galli
Amphetamine induces dopamine efflux through a dopamine transporter channel
PNAS,
March 1, 2005;
102(9):
3495 - 3500.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. B. Larsen, B. Elfving, and O. Wiborg
The Chicken Serotonin Transporter Discriminates between Serotonin-selective Reuptake Inhibitors: A SPECIES-SCANNING MUTAGENESIS STUDY
J. Biol. Chem.,
October 1, 2004;
279(40):
42147 - 42156.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Gendreau, S. Voswinkel, D. Torres-Salazar, N. Lang, H. Heidtmann, S. Detro-Dassen, G. Schmalzing, P. Hidalgo, and C. Fahlke
A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters
J. Biol. Chem.,
September 17, 2004;
279(38):
39505 - 39512.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
![I=I<SUB><UP>max</UP></SUB>[<UP>5HT</UP>]<SUP><UP>n</UP></SUP><UP>/</UP>(K<SUP><UP>n</UP></SUP><SUB><UP>m</UP></SUB>+[5<UP>HT</UP>]<SUP><UP>n</UP></SUP>)<UP>,</UP>](/content/vol17/issue10/fulltext/3401/img001.gif)
