(Received for publication, October 16, 1995; and in revised form, January 16, 1996)
From the
We prepared membrane vesicles from stable LLC-PK cells expressing serotonin (5-HT)
-aminobutyric acid (GABA)
and norepinephrine (NE) transporters (SERT, GAT-1, and NET). These
vesicles accumulate transport substrates when the appropriate
transmembrane ion gradients are imposed. For NET, accumulation of
[
H]dopamine (DA) was stimulated by imposition of
Na
and Cl
gradients (out > in)
and of a K
gradient (in > out). The presence of
Na
or Cl
, even in the absence of a
gradient, stimulated DA accumulation by NET, but K
had
little or no effect in the absence of a K
gradient.
Stimulation by a K
gradient was markedly enhanced by
increasing the K
permeability with valinomycin,
suggesting that net positive charge is transported together with DA.
Cationic DA is likely to be the major substrate for NET, since varying
pH did not affect K
. We estimated the
Na
:DA stoichiometry by measuring the effect of the
transmembrane Na
gradient on peak DA accumulation. The
results suggest a 1:1 cotransport of Na
with DA. Taken
together, the results suggest that NET catalyzes cotransport of one
cationic substrate molecule with one Na
ion, and one
Cl
ion, and that K
does not
participate directly in the transport process.
The synaptic action of neurotransmitters released by nerve cells
is terminated by a reuptake process in which the transmitters are
transported back inside the nerve endings from which they were
released. Recently, cDNAs encoding transporters for neurotransmitters,
amino acids, and other substrates have been cloned. These include
-aminobutyric acid (GABA) (
)transporters and
transporters for serotonin (5-HT) and the catecholamines norepinephrine
(NE) and dopamine
(DA)(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
Many of these transporters share extensive sequence homology and
constitute a multigene
family(17, 18, 19, 20, 21, 22) .
In addition to the similarity in primary sequence, these transporters
share a functional dependence on Na
and
Cl
(23, 24, 25, 26, 27) .
In some cases, this dependence has been shown to reflect the
cotransport of Na
and Cl
with the
neurotransmitter substrate(28, 29, 30) . This
cotransport leads to a coupling of the downhill influx of Na
and Cl
with the uphill influx of substrate and
leads to the internal accumulation of substrate to concentrations
hundreds of times higher than in the medium.
In addition to the
difference in substrate specificities between members of this family,
there are also differences in ion coupling mechanisms. The 5-HT and
GABA transporters (SERT and GAT-1) have been studied extensively in
membrane vesicle preparations from platelets and synaptosomes,
respectively(31, 32) . These studies reveal that SERT
couples transport of its cationic substrate with one Na ion while GAT-1 mediates zwitterionic GABA transport together
with two Na
ions. Both transporters apparently use a
single Cl
ion, but SERT couples 5-HT influx to efflux
of one K
, while GAT-1 mediated transport does not
directly involve K
. The net result is that 5-HT
transport by SERT is an electrically neutral process, and that GABA
influx generates a net inward current.
It has become apparent
recently that in addition to the coupled flux of Na,
K
, and Cl
the 5-HT, GABA, and NE
transporters also catalyze uncoupled ion
flux(33, 34, 35) . This uncoupled flux is
likely to represent rare events in which the transporter transiently
behaves like an ion channel(36) . In studies that attempt to
measure solute transport by recording the electrical current associated
with transport, the uncoupled ion flux can confound determination of
transport stoichiometry and electrogenicity. For example, even though
5-HT transport is electroneutral, addition of 5-HT to oocytes
expressing SERT leads to an inward current(33) . Substrate
accumulation, in contrast, depends on imposed ion gradients and
electrical potentials but is not likely to be influenced by uncoupled
currents carried by the transporter. Thus, flux studies performed with
membrane vesicle preparations remain the experimental system of choice
for determination of ion coupling stoichiometry. In this paper we use
the term ``stoichiometry'' to refer only to the substrates
and ions transported in the carrier cycle and not the variable number
of ions translocated during uncoupled ion flux.
Other members of the
NaCl-coupled transporter family have not been studied as thoroughly as
SERT and GAT-1, partly due to the lack of suitable membrane vesicle
preparations. One exception is the NE transporter (NET), which has been
studied in membrane vesicles prepared from PC12 cells and placental
syncytiotrophoblast(37, 38) . Harder and Bonisch (37) concluded that NE transport into PC12 vesicles was coupled
to Na and Cl
, and was electrogenic,
but they failed to arrive at a definitive coupling stoichiometry
because of uncertainties about the role of K
.
According to their analysis, stimulation of NE influx by internal
K
resulted either from direct K
countertransport as occurs with SERT (39) , or from a
K
diffusion potential which drives electrogenic NE
influx, as with GAT-1(26) . Ganapathy and co-workers (38) studied NET mediated transport of both NE and DA into
placental membrane vesicles (both catecholamines are substrates for the
cloned transporter(27) ). They reached a similar conclusion
regarding ion coupling, but also were left with some ambiguity
regarding K
. In fact, the effects of ions on
NET-mediated DA accumulation were similar to those observed with
SERT-mediated 5-HT transport in the same membranes and the two
activities were distinguished only by their inhibitor
sensitivities(40) . Part of the difficulty in interpreting and
comparing these data stems from the fact that they were obtained in
different cells, with unknown, and potentially very different
conductances to K
.
Two further problems make it
difficult to interpret existing data on NET ion coupling. Both previous
studies assumed that the cationic form of the catecholamine substrate
was transported(37, 38) . However, both cationic and
neutral forms of catecholamine substrates are present at physiological
pH, and there is no previous evidence indicating that one or the other
is the true substrate. In the case of the vesicular monoamine
transporter, the ionic form of the substrate is a matter of some
controversy(41, 42, 43) . Furthermore, the
number of Na ions cotransported with substrate was
estimated from the dependence of transport rate on Na
concentration(37, 38) . This method is capable
of detecting the involvement of multiple Na
ions only
if they have similar binding affinity and kinetics. If two
Na
ions (for example) with widely different affinities
or binding kinetics are cotransported, the dependence of transport rate
on Na
may reflect only binding of the lowest affinity
or most slowly associating Na
ion.
We have recently
established LLC-PK cell lines stably expressing the
biogenic amine transporters SERT, NET, and DAT as well as the GABA
transporter GAT-1. Using these cell lines, we have characterized and
compared the transporters under the same conditions and in the same
cellular environment(27) . One attractive advantage of
LLC-PK
cells is that it has been possible to prepare plasma
membrane vesicles that are suitable for transport studies(44) .
We took advantage of this property to prepare membrane vesicles
containing transporters for GABA, 5-HT, and NE, all in the same
LLC-PK
background. These vesicles should have identical
composition except for the heterologously expressed transporter.
Moreover, these vesicles are suitable for estimating equilibrium
substrate accumulation in response to imposed ion gradients. In this
paper, we describe experiments that define the ion coupling
stoichiometry for NET using the known stoichiometries for GAT-1 and
SERT mediated transport as internal controls.
Figure 1:
Time course of
substrate accumulation into membrane vesicles. Membrane vesicles
prepared from LLC-GAT (top panel), LLC-SERT (middle
panel), and LLC-NET (lower panel) were equilibrated with
buffer B (see Table 1). The reactions then were started by
diluting vesicle suspensions into buffer A containing H-labeled 5-HT, GABA, or DA. At the times indicated,
transport was terminated by dilution and filtration as described under
``Experimental Procedures.'' The open symbols represent control incubations with buffer G replacing buffer A for
LLC-GAT vesicles, and in the presence of 100 µM cocaine
for LLC-SERT and LLC-NET vesicles.
Figure 2:
Ion requirements for transport. To test
whether K is required by the transporters, substrate
accumulation in 5 min was measured when inward Na
and
Cl
gradients were imposed across the vesicle
membranes with K
absent (column 3) or present
on both sides of the membrane at equal concentration (column
2). Similarly the requirements of Cl
and
Na
were tested with an inward Na
gradient and an outward K
gradient imposed with
Cl
absent (column 5) or present on both
sides of the membrane at equal concentration (column 4), and
with inward Cl
gradient and outward K
gradient imposed with Na
absent (column
7) or present on both sides of the membrane at equal concentration (column 6). Controls with all and none of the three gradients
imposed are shown in columns 1 and 8, respectively. GAT, SERT, and NET represent vesicles prepared from
LLC-GAT, LLC-SERT, and LLC-NET cells. The buffer conditions from left
to right are (internal medium/external medium): column 1,
buffer B/buffer A; column 2, buffer B/buffer D; column
3, buffer C/buffer A; column 4, buffer E/buffer A; column 5, buffer B/buffer F; column 6, buffer
H/buffer A; column 7, buffer B/buffer G; column 8,
buffer C/buffer C.
For Na,
Cl
, and K
, we tested whether each
ion was required for transport and if a gradient of that ion stimulated
accumulation. The results for NET, GAT-1, and SERT are presented in columns 2-7 of Fig. 2. Adding K
to the external buffer to eliminate the K
gradient decreased accumulation by NET vesicles but had a smaller
effect on SERT vesicles and essentially no effect on GAT-1 vesicles
(compare column 2 to column 1). SERT is known to
countertransport K
with 5-HT(39) , but such a
direct interaction between K
and NET had not been
demonstrated in previous studies(37, 40) . Removing
K
entirely from both internal and external media had
no further effect on NET vesicles, and little effect on GAT-1 vesicles,
but significantly decreased uptake by SERT vesicles. The decreased
uptake by SERT vesicles is consistent with the known participation of
K
in 5-HT transport, and the lack of GAT-1 inhibition
by K
removal also was consistent with evidence that
K
does not participate directly in that reaction (46) (see (47) ). The observation that K
removal does not affect DA accumulation strongly suggests that
K
is not coupled directly to NET-mediated
catecholamine transport.
Column 4 of Fig. 2shows
the results obtained when the Cl gradient was
eliminated by preloading LLC-GAT, LLC-SERT, and LLC-NET vesicles with
buffer E, containing Cl
. For each transporter, there
is a dramatic decrease in the amount of substrate accumulated,
consistent with the proposal that Cl
serves as a
driving force for accumulation. Removal of Cl
entirely from both internal and external media (column
5) caused a further reduction in transport by each transporter, as
expected if Cl
is directly involved in the transport
reaction as a cotransported ion(28) . Na
cotransport is also a common theme for neurotransmitter
transporters, including these three
transporters(23, 26, 48) . As shown in column 6 of Fig. 2, ablation of the Na
gradient by the use of internal buffer H, which contained
Na
, dramatically decreased accumulation in all three
cases. Removal of Na
from both internal and external
buffer reduced transport even further (column 7) indicating
that both the presence of Na
and a Na
gradient stimulate accumulation, consistent with the cotransport
of Na
by GAT-1, SERT, and NET.
Figure 3:
Response of transport to imposed
K diffusion potentials. Substrate accumulation in 5
min was measured as described under ``Experimental
Procedures.'' Inward Na
and Cl
gradients and outward K
gradient were imposed
(B, in; A, out) with (shaded bars) or without (open
bars) addition of 1 µM valinomycin to the external
medium.
Figure 4:
Transport dependence on
Na gradient. Substrate accumulation in a 5-min
incubation was measured as described under ``Experimental
Procedures.'' Inward Na
and Cl
gradients and outward K
gradient were imposed,
and the Na
gradient was varied by replacing varying
amounts of Li
in the internal medium with
Na
. The external buffer contained 210 mM NaCl, 5 mM K
SO
, and 45 mM Li
SO
. The internal buffers contained 100,
75, 50, 25, and 0%, respectively, of 100 mM Na
SO
, 50 mM K
SO
, and 10 mM NaCl with the
remainder consisting of 100 mM Li
SO
,
50 mM K
SO
, and 10 mM NaCl.
All above buffers also contained 10 mM lithium phosphate, pH
6.7, and 1 mM MgCl
. GAT, SERT, and NET represent vesicles prepared from LLC-GAT, LLC-SERT, and
LLC-NET cells.
Figure 5:
pH dependence of NET transport activity.
LLC-NET cells were plated in 48-well plates.
[H]DA transport assays were performed as
described under ``Experimental Procedures'' using an
incubation time of 2 min. The external buffers consisted of 150 mM NaCl, 1 mM MgSO
, and 10 mM Tris-Cl
adjusted to pH 6.5 (open circles) or 7.5 (filled
circles). The buffer contained 40 nM [
H]DA and sufficient unlabeled DA to give
the indicated total DA concentration.
We have taken a novel approach to understand the ion coupling
stoichiometry of the NET. We are able to express and compare, in the
same parental cell line, transporters whose stoichiometry is known and
also the transporter of interest. This system allows us to manipulate
transmembrane ion gradients and electrical potentials directly, and to
test the effects on transport. For comparison with NET we have chosen
the 5-HT and GABA transporters (SERT and GAT-1) since the ion coupling
stoichiometries for these two transporters are well understood and is
different from one another(31, 32) . SERT cotransports
cationic 5-HT with Na and Cl
and
countertransports K
. The 1:1:1:1 stoichiometry of this
process results in electrically neutral 5-HT transport. In contrast,
GAT-1 cotransports GABA with two Na
ions and one
Cl
and does not transport K
. The
resulting 1:2:1 stoichiometry leads to positive charge movement in the
direction of GABA transport. Our results suggest that the stoichiometry
of NET-mediated DA transport is different from those for both SERT and
GAT-1.
The recent demonstration that neurotransmitter transporters also mediate uncoupled ion flux (33, 34, 35, 52) has cast a measure of confusion on the terms used to describe these proteins and their properties. If, as it seems likely, these transporters transiently form conductive channels through the membrane, a distinction must be made between the types of electrical currents due to channel activity and substrate transport. The term ``electrogenic'' has traditionally been used to describe a coupled transport process in which net charge crosses the membrane. In the absence of ion gradients, an electrogenic transporter should generate an electrical potential in response to an imposed transmembrane substrate gradient. In contrast, the channel activity of such a transporter can only mediate energetically downhill ion flux. Thus, while SERT and GAT-1 both conduct ions by virtue of their intermittant channel activity, GAT-1 is an electrogenic transporter because it transports net charge with GABA and SERT is electroneutral because the 5-HT transport cycle itself does not move net charge across the membrane.
We previously described the
generation of stable cell lines based on LLC-PK cells that
express biogenic amine transporters (27) . We characterized the
transporters with respect to their requirements for external
Na
and Cl
by varying the
cells' external ion compositions. We observed that NET, as well
as SERT and the DA transporter (DAT) require Na
and
Cl
from the outside of the cells for full activity.
These experiments could not determine whether Na
and
Cl
were cotransported or merely required to activate
the transporter. However, the response of transport rate to
Na
was sigmoidal for DAT and hyperbolic for NET,
suggesting that a different number of Na
ions
participated in the two reactions. These differences had been observed
previously(38, 53) , however, they had not been
observed using transporters expressed in the same cell.
While
kinetic measurements of Na dependence can suggest a
cotransport stoichoimetry, their interpretation is not straightforward.
For example, if two Na
ions are cotransported with DA
by NET, the Na
dependence is expected to be sigmoidal.
However, if one of those two Na
ions binds much slower
or more weakly than the other, the Na
dependence might
appear hyperbolic, giving the false impression that only one
Na
ion is involved in the process. Furthermore, a
sigmoidal dependence on Na
is expected if two
Na
ions are required for transport, even if only one
of them actually is cotransported with substrate. To avoid these
potential problems, we estimated the Na
cotransport
stoichiometry by varying the Na
gradient and measuring
the accumulation of substrate at a time when it is close to being in
equilibrium with the imposed gradients.
Since the peak of substrate
accumulation occurs at a time when transmembrane ion gradients are
decaying, the absolute value for internal Na is higher
than that initially present in the internal buffer. Rather than attempt
to estimate internal Na
, we have taken advantage of
identical membrane vesicles from cells expressing SERT and GAT-1. At
the time of peak uptake, we expect the Na
gradient to
have decayed to the same extent in each vesicle preparation. Transport
into vesicles from cells expressing GAT-1 is much more sensitive to
dissipation of the Na
gradient than is transport into
SERT vesicles (Fig. 4). This is expected since the Na
gradient contributes more to the overall driving force for GABA
accumulation than it does for 5-HT accumulation(32) .
Dissipating the Na
gradient has a similar effect on
NET and SERT vesicles. This similarity strongly argues that NET, like
SERT transports with a Na
:substrate coupling
stoichiometry of 1:1.
Previous studies examining the stoichiometry
of NE transport in membrane vesicles from PC12 cells (37) and
placental syncytiotrophoblast (38) used kinetic measurements of
NE or DA influx. Although those studies came to similar conclusions
concerning the overall stoichiometry, there were significant
uncertainties concerning the role of K. Internal
K
stimulated the rate of transport and the extent of
accumulation. We also observed a stimulatory effect of the K
gradient (in > out) on DA accumulation (Fig. 2, lower panel, columns 1 and 2). However, the
results in Fig. 2, lower panel columns 2 and 3, indicate that this effect is not likely to result from an
obligatory interaction of K
with the transporter. In
the absence of a K
gradient, the presence of
K
had no effect on peak [
H]DA
accumulation by NET. In contrast, removal of K
significantly impaired [
H]5-HT accumulation
by SERT (Fig. 2, middle panel, columns 1 and 2), which is known to directly couple 5-HT and K
countertransport(39) . These results argue that the
effects of K
were indirect, and that K
was not coupled to DA transport by NET.
Although K was not directly coupled to DA transport, an outwardly directed
K
gradient stimulated accumulation (Fig. 2).
The most likely reason for this effect is that an endogenous
K
conductance in the membrane allowed K
efflux to generate a membrane potential (negative inside) or to
offset any potential (positive inside) generated by Na
influx. Increasing the K
permeability with
valinomycin stimulated DA accumulation, as would be expected if DA
influx was coupled to influx of net positive charge. This stimulation
of NET-mediated transport by K
or H
diffusion potentials had been observed also in previous studies
and had been attributed to coupled movement of substrate and positive
charge(37, 38) .
Although it might be expected that
GABA accumulation also would be stimulated by a K gradient in the absence of valinomycin, this was not observed.
However, GABA accumulation was stimulated less than DA also when
valinomycin was used to increase K
permeability (Fig. 3). Since GAT-1 couples GABA transport with 2
Na
ions, the Na
gradient represents a
much larger portion of the GAT-1 driving force (relative to the
membrane potential) than it does for NET, which uses only one
Na
ion for cotransport with DA. While this would
reduce the influence of a K
diffusion potential on
GAT-1, we cannot explain why the effect on GAT-1 of dissipating the
K
gradient was completely absent. Another potential
explanation is that the K
gradient does not, in the
absence of valinomycin, induce a diffusion potential, and that the
inhibition of NET results from an inhibitory effect of increased
external K
. At present we have no way to distinguish
between these or other possibilities. Nonetheless, the lack of effect
of K
on NET in the absence of a K
gradient argues against a direct role for K
in
NET-mediated DA transport.
The range of possible ion coupling
stoichiometries for NET is constrained by the observations that the
cationic form of DA is cotransported with one Na ion
and that net positive charge accompanies these ions. Additional ion
gradients that could be coupled to transport are the K
gradient (in > out) and the Cl
gradient (out
> in). A maximum of one cotransported Cl
ion or
one countertransported K
ion could be coupled to
transport. One each of Cl
or K
or
two of either ion would force the overall process to be electroneutral.
The results presented here and elsewhere(37, 38) strongly suggest that transport is electrogenic, and that
the presence of Cl
, as well as the Cl
gradient, are required for efficient transport by NET. In
contrast, the presence of K
does not stimulate
transport in the absence of a K
gradient. The most
straightforward conclusion is that NET couples the cotransport of NE
(or DA), Na
, and Cl
with a 1:1:1
stoichiometry.
The three neurotransmitter transporters (GAT-1, SERT,
and NET) in this gene family whose stoichiometry is now known have
three different ion coupling stoichiometries. Moreover, kinetic
measurements suggest that the DAT cotransports two Na ions with DA(53) . Thus, even among the closely related
biogenic amine transporters (SERT, NET, and DAT), ion coupling
stoichiometries probably differ. Current efforts in this laboratory are
directed toward understanding the stoichiometry of DAT using a similar
approach to that described here. The ability to compare different
transporters in identical membrane vesicle preparations has provided a
new tool for understanding the types and mechanisms of ion coupling in
transporters. By comparing the similarities in coupling between members
of this gene family with regions of sequence similarity, it may be
possible to identify structural regions of the transporters responsible
for ion coupling.