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The Journal of Neuroscience, June 15, 1999, 19(12):4705-4717
Transmembrane Domain I Contributes to the Permeation Pathway for
Serotonin and Ions in the Serotonin Transporter
Eric L.
Barker,
Kimberly R.
Moore,
Fariborz
Rakhshan, and
Randy D.
Blakely
Department of Pharmacology and Center for Molecular Neuroscience,
Vanderbilt University School of Medicine, Nashville, Tennessee
37232-6600
 |
ABSTRACT |
Mutation of a conserved Asp (D98) in the rat serotonin (5HT)
transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+
and Cl
, and a selective loss of antagonist
potencies (cocaine, imipramine, and citalopram but not paroxetine or
mazindol) with no change in 5HT Km value.
D98E, which extends the acidic side chain by one carbon, affected the
rank-order potency of substrate analogs for inhibition of 5HT
transport, selectively increasing the potency of two analogs with
shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E
also increased the efficacy of gramine relative to 5HT for inducing
substrate-activated currents in Xenopus laevis oocytes,
but these currents were noticeably dependent on extracellular medium
acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts
ion permeation coupled to 5HT occupancy. The ability of the D98E mutant
to modulate selective aspects of substrate recognition, to perturb ion
dependence as well as modify substrate-induced currents, suggests that
transmembrane domain I plays a critical role in defining the permeation
pathway of biogenic amine transporters.
Key words:
serotonin; monoamine; transporter; biological transport; carrier proteins; molecular structure; permeation channel; selectivity
filter
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INTRODUCTION |
The Na+- and
Cl-dependent neurotransmitter transporters play a
major role in the dynamic regulation of extracellular neurotransmitter concentrations. Not including species variants, this family of transporters is composed of more than 15 distinct members, including carriers for amino acid neurotransmitters as well as the monoamines serotonin (5HT), norepinephrine (NE), epinephrine, and dopamine (DA)
(Nelson, 1998
). The 5HT transporter (SERT, 5HTT) is of particular clinical interest because it is a target for many antidepressants such
as the tricyclic imipramine and the 5HT-selective reuptake inhibitors
(SSRIs), including fluoxetine and paroxetine (Barker and Blakely, 1995;
Tatsumi et al., 1997) as well as abused psychostimulants, including
cocaine and the amphetamines (Rudnick and Wall, 1992; White, 1998).
Initial structural inferences suggest that the biogenic amine
transporters exhibit cytoplasmic NH2 and COOH tails, 12 putative
transmembrane domains (TMDs), and a large extracellular loop between
TMDs III and IV containing multiple sites for N-glycosylation (Nelson,
1998). Although there has been some controversy regarding this model
(Bennett and Kanner, 1997; Clark, 1997), recent studies using the
substituted cysteine accessibility method (SCAM) (Akabas et al., 1992;
Stauffer and Karlin, 1994) have examined and supported the 12 TMD
topology (Chen et al., 1998). Despite a knowledge of the primary
structure of SERTs and related transporters, little data are available
that define how these domains interact with substrates and antagonists.
5HT transport studies of mammalian SERTs have provided mechanistic data
for the kinetics governing the translocation of organic and ionic
substrates through the membrane. 5HT influx is a co-transport process
coupled to the inward movement of Na+ and
Cl with a predicted stoichiometry of 1 5HT/1
Na+/1 Cl for influx along with
the countertransport of 1 K+, thus predicting a
transport cycle that is electrically neutral (Rudnick et al., 1983; Gu
et al., 1994; Rudnick, 1998a). However, additional ion conducting
states have been associated with SERTs (Mager et al., 1994; Lester et
al., 1996) as well as homologous NE (Galli et al., 1995, 1996) and DA
transporters (Sonders et al., 1997). For SERTs, these conducting states
include (1) a constitutive leak pathway and (2) a substrate-activated
current. In Xenopus laevis oocytes expressing rat SERT
(rSERT), both the SERT leak and substrate-gated currents are
potentiated by medium acidification, suggesting that the transporter is
also highly permeable to protons (Cao et al., 1997, 1998). Whether
these ion-conducting states use a common permeation pathway and whether
this pathway is shared with the pathway along which 5HT translocates is
currently unknown.
The availability of cDNAs encoding SERTs provides the molecular tools
necessary for a structural dissection of the transporter to identify
residues or domains critical for substrate recognition and
translocation, ion coupling, and antagonist blockade. In the present
studies, we identified a residue in TMD I with a binary option (Asp or
Gly) among the Na+- and
Cl-dependent transporters and established that
reversal of this residue in either human norepinephrine transporter
(NET) (D75G) or rat SERT (D98G), or several other substitutions,
caused a disruption of amine transport activity. However, the rSERT
mutant D98E retained significant 5HT transport activity, allowing us to
explore the consequences of this more conservative substitution on
substrate and antagonist recognition, ion dependence, and SERT-mediated ion flow. Our findings link determinants of 5HT, ion recognition, and
nonstoichiometric ion flow, suggesting that TMD I is one of several
domains that may line a common permeation pathway for permeant species
transiting the membrane through biogenic amine transporters.
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MATERIALS AND METHODS |
Site-directed mutagenesis. Point mutations were
introduced into the wild-type rSERT and hNET cDNAs by
oligonucleotide-directed mutagenesis as described previously (Kunkel et
al., 1987). A single-stranded uracil-containing DNA template was
generated in Escherichia coli CJ236 (dut-, ung-)
(Invitrogen) using R408 helper phage. Using oligonucleotides encoding
the mutations, the following mutations were introduced: rSERT D98A,
D98E, D98G, D98N, D98T, and hNET D75A, D75E, D75G, and D75N. Identity
of the mutant cDNAs was confirmed by dideoxynucleotide sequencing using
a Sequenase-II kit (United States Biochemicals) (Sanger et al., 1977).
To verify the absence of additional mutations in the parental cDNAs,
rescue constructs of all mutations were constructed. For the SERT
mutant rescues, the XhoI/RsrII fragment containing the
various D98 mutations was removed from the mutant-containing cDNAs, and
the corresponding fragment from the wild-type rSERT cDNA was ligated
into D98 mutant cDNAs. For the hNET mutant rescues, the ScaI
fragment containing the various D75 mutations was removed from the
mutant-containing cDNA, and the corresponding fragment from the
wild-type hNET was ligated into the D75 mutant constructs. The rescue
constructs were tested for recovery of transport activity in either
[3H]5HT or [3H]NE uptake
experiments. In all cases, the rescue constructs demonstrated wild-type
rSERT or hNET transport activity (data not shown), confirming that the
disruption of transport activity observed for the rSERT D98 and hNET
D75 mutants resulted from the single mutation.
Transient expression of SERTs in mammalian cells. To
directly assess transport activity of rSERT (Blakely et al., 1991a) and the rSERT D98 mutants, as well as hNET (Pacholczyk et al., 1991) and
hNET mutants, transient expression of the transporters was achieved
using the recombinant vaccinia virus T7 expression system in HeLa cells
(Fuerst et al., 1986; Blakely et al., 1991b). The parental cDNAs, rSERT
and hNET, were previously cloned into the plasmids pBluescript II KS
and pBluescript II SK+, respectively. In each case, sense RNA is
transcribed by the plasmid-encoded T7 RNA polymerase promoter. HeLa
cells were cultured in DMEM supplemented with 10% fetal bovine
serum, 2 mM L-glutamine, and 1%
penicillin/streptomycin at 37°C in a humidified 5% CO2
incubator. Cells plated in 24-well culture plates (100,000 cells per
well) were infected with recombinant VVT7-3 vaccinia virus encoding T7
RNA polymerase at 10 plaque-forming units per cell as described (Fuerst
et al., 1986; Blakely et al., 1991b). Virus infection of the cells
proceeded for 30 min in serum-free Opti-MEM I containing 55 µM 2-mercaptoethanol at 37°C. After virus infection,
SERT or NET cDNA constructs were transfected into virus-infected HeLa
cells using liposome-mediated transfection (Lipofectin reagent) at 100 ng DNA per well at a ratio of 1 µg of DNA/3 µg of Lipofectin (mixed
in Opti-MEM I/
-mercaptoethanol).
For expression in COS-7 cells, rSERT and rSERT D98E were subcloned into
the XhoI/XbaI site of the mammalian expression
vector pcDNA3 (Invitrogen). This construct would place SERT expression under control of the cytomegalovirus promoter suitable for expression in COS-7 cells. Parental COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1%
penicillin/streptomycin at 37°C in a humidified 5% CO2
environment. For uptake experiments, 100,000 cells per well were plated
in 24-well culture plates (Falcon) and transfected with rSERT or D98E
cDNA using 100 ng/well and a 5:1 ratio of Lipofectin reagent/DNA.
Medium containing DNA and Lipofectin was removed 16-18 hr after
transfection and replaced with complete DMEM culture medium. Cells were
used in uptake assays 48 hr later. For cell-surface biotinylation
experiments, cells (200,000 cells per well) in six-well culture plates
were transfected with rSERT or D98E cDNA using 2 µg DNA/well and a
5:1 ratio of Lipofectin/DNA. Medium containing DNA and Lipofectin was
removed 16-18 hr after transfection and replaced with complete DMEM
culture medium. Cells were used in biotinylation experiments (see
below) 48 hr later.
[3H]5HT and [3H]NE
transport assays. Transfected HeLa or COS-7 cells were washed with
a Krebs'-Ringer's-HEPES (KRH) buffer (120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 10 mM HEPES, 1.2 mM
KH2PO4, 1.2 mM
MgSO4, pH 7.4). Cells were preincubated for 10 min
at 37°C in KRH containing 1.8 gm/l D-glucose, and then
transport assays with 10 nM [3H]-5HT
(~100 Ci/mmol; Amersham), 100 µM pargyline, and 100 µM L-ascorbic acid or 40 nM
[3H]NE (~50 Ci/mmol; Amersham), 100 µM pargyline, 100 µM L-ascorbic acid, and 100 µM U-0521 were performed for 10 min at
37°C. Inhibitors were added in the preincubation step. Saturation
kinetics were determined using increasing concentrations of
[3H]5HT with the specific activity diluted to
~0.1 Ci/mmol with unlabeled 5HT. Uptake was terminated by three
washes with ice-cold KRH buffer. The level of accumulated
[3H]5HT or [3H]NE was
determined either by solubilizing cells in 1% SDS and analysis by
liquid scintillation spectrometry or by solubilizing directly in
scintillant (Optiphase SuperMix) with direct counting of culture plate
in a Wallac MicroBeta plate reader. The Na+
dependence of [3H]5HT uptake was assessed in KRH
buffer using isotonic replacement of NaCl with LiCl or
N-methyl-D-glucamine (NMDG) with equivalent results, whereas the Cl dependence was determined
in KRH buffer with Cl salts replaced by sodium
gluconate, potassium gluconate, and calcium nitrate at molarities
equivalent to those in regular KRH buffer. Nonspecific
[3H]5HT transport was assessed by parallel
transfections with the host plasmid pBluescript SK II() alone and
subtracted from the total counts. Substrate Km
and antagonist Ki values were derived by
nonlinear least-square fits (Kaleidagraph, Synergy Software) using
either the Hill equation for a rectangular hyperbola or the
four-parameter logistic equation with necessary adjustments of
IC50 values for substrate concentration to determine
apparent Ki values (Cheng and Prusoff, 1973).
Experiments were performed in duplicate or triplicate and repeated in
two to three separate assays. Means were compared using two-sided
Student's t tests (GraphPad InStat for MacIntosh, v. 2.03)
or one-way ANOVA (GraphPad PRISM for Mac, v. 2.0).
[125I]RTI-55 binding. Radioligand
binding experiments using [125I]RTI-55 (NEN Life
Science Products; ~2200 Ci/mmol) were performed on wild-type and
mutant rSERTs to assess whether mutation-induced alterations in
transport correlated with changes in ligand binding. To prepare crude
membranes, HeLa cells were transfected with SERT cDNAs as described for
transport assays, except that cells were plated on 150 mM
culture dishes and used 12-16 hr after transfection to maximize SERT
expression. Cells were washed with PBS, incubated in ice-cold hypotonic
buffer (Tris-HCl 50 mM, pH 7.4, NaCl 100 mM),
detached from the plates using a cell scraper, and pelleted at
1600 × g. The resulting pellet was resuspended in
ice-cold Tris/NaCl buffer and centrifuged for 20 min at 20,000 × g. The resulting pellet was resuspended with a Brinkmann
polytron (5 sec, 25,000 rpm) and centrifuged for an additional 20 min
at 20,000 × g. The pellet was suspended in Tris/NaCl
buffer and homogenized with the polytron (10 sec, 20,000 rpm). Samples
of membrane suspensions were frozen at 80°C before use. Typically,
assays were performed using 1 µg (rSERT) and 10 µg (D98E) of
membrane protein as determined by the Bradford protein assay (Bio-Rad)
using bovine serum albumin as the standard. Saturation binding was
determined in duplicate using 0.1-20 nM
[125I]RTI-55 with 1 µM paroxetine
used to define nonspecific binding (Kd and
Bmax values: wild-type rSERT 0.37 ± 0.02 nM and 5.6 ± 0.45 × 1012
mol/mg protein; D98E 3.7 ± 0.6 nM and 5.4 ± 0.35 × 1012 mol/mg protein). In competition
binding experiments, cells were incubated with increasing
concentrations of antagonist before the addition of either 0.1 nM (wild-type rSERT) or 1.0 nM (D98E) [125I]RTI-55. Assay tubes were incubated for 1 hr
at 22°C and filtered using a Brandel harvester through Schleicher and
Schuell #32 glass fiber filters soaked in 0.5% polyethylenimine. Bound
radioactivity was measured by gamma emission spectrometry.
Ki values were derived by nonlinear least-square
fits (Kaleidagraph, Synergy Software) using a four-parameter logistic
equation with necessary adjustments of IC50 values for
radioligand concentration to determine apparent Ki values (Cheng and Prusoff, 1973).
Cell-surface biotinylation of SERT and NET proteins. To
determine cell-surface expression levels of the wild-type rSERT, rSERT D98E, rSERT D98G, wild-type hNET, hNET D75E, and hNET D75G,
cell-surface biotinylation studies using polyclonal antibodies to
either rSERT or hNET for protein detection were undertaken as described
previously (Melikian et al., 1996; Qian et al., 1997). Because of
technical limitations regarding the use of the T7-vaccinia virus
expression system for quantitative cell-surface biotinylation (Melikian
et al., 1996), all cDNAs were subcloned into the mammalian
expression vector pcDNA3 (Invitrogen) for transient expression in COS-7
cells. Forty-eight hours after transfection, cells were washed with
cold PBS/Ca-Mg (138 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 9.6 mM Na2HPO4, 1 mM
MgCl2, 0.1 mM CaCl2,
pH 7.3) and then treated with freshly prepared Sulfo-NHS-Biotin
(sulfosuccinimidobiotin; Pierce) (1.5 mg/ml in PBS/Ca-Mg) at 4°C for
30 min. Biotinylating reagents were removed by washing with 100 mM glycine in PBS/Ca-Mg, the reaction was further quenched
by incubation with 100 mM glycine for 20 min, and then
cells were washed with PBS/Ca-Mg before lysis with RIPA buffer (10 mM Tris-Base, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium
deoxycholate) supplemented with protease inhibitors (1 µg/ml
leupeptin, 1 µM pepstatin, 1 mg/ml soybean trypsin
inhibitors, 1 mM iodoacetamide, and 250 mM
PMSF) for 30 min at 4°C with constant shaking. Lysates were centrifuged at 20,000 × g for 30 min at 4°C, and
supernatants were incubated with monomeric avidin beads (175 µl bead
volume/1250 µl supernatant) for 1 hr at room temperature. Beads were
washed four times with RIPA, and adsorbed proteins were eluted with 50 µl of 2× Laemmli loading buffer (62.5 mM Tris-HCl, pH
6.8, 20% glycerol, 2% SDS, 5% -mercaptoethanol, and 5%
bromophenol blue) for 30 min at room temperature. A fraction of total
cell lysate (20 of 800 µl), lysates after incubation with avidin
beads (intracellular proteins) (20 of 800 µl), the final wash (40 of
800 µl), and the entire bead eluate (50 µl) were separated by
SDS-PAGE (10%), transferred for 16 hr at 150 mA onto polyvinylidene
difluoride membrane, blocked with 5% nonfat dry milk in PBS/0.1%
Tween-80, and immunoblotted with anti-SERT CT-2 (1:5000) (Qian et al.,
1995) or anti-NET (1:5000) (Melikian et al., 1994) antibodies using
1:5000 goat anti-rabbit HRP-conjugated secondary antibody.
Subsequently, blots were stripped (62.5 mM Tris-HCl, pH
6.8, 2% SDS, and 100 mM -mercaptoethanol) for 30 min at
50°C, washed with PBS/Tween-80 twice for 10 min, reblocked with 5%
dry milk for 1 hr, and probed with anti-calnexin antibody (StressGen)
(1:3000) followed by goat anti-rabbit HRP-conjugated secondary antibody
(1:5000). Immunoreactive bands were visualized by ECL chemiluminescent
detection (Amersham) on Hyperfilm ECL (Amersham), and scanned bands
were quantitated using ImageQuant (Molecular Dynamics). Multiple
exposures were used to insure quantitation within the linear range of
the film. Values of SERT or NET surface proteins were normalized by
levels of calnexin immunoreactivity in total cell extracts and
biotinylated (cell-surface) fractions compared with the total fractions
to control for different expression levels of each clone.
Two-electrode voltage clamp of SERT-expressing Xenopus
oocytes. The plasmids (pBluescript SK II()) containing the cDNAs
for either wild-type rSERT (Blakely et al., 1991a) or the D98E mutant were linearized with XbaI, and cRNA was transcribed using
the mMessage mMachine T7 In Vitro Transcription Kit
(Ambion). Stage V and VI defolliculated oocytes were subsequently
injected with 50-100 ng of either wild-type rSERT or D98E cRNA and
maintained at 18°C in Ca2+-Ringer's solution
(96 mM NaCl, 2 mM KCl, 5 mM
MgCl2, 5 mM HEPES, and 0.6 mM CaCl2, pH 7.6) supplemented with 1%
penicillin/streptomycin (Life Technologies/BRL) and 5% horse serum
(Life Technologies/BRL). Isotonic replacement of Na+
with NMDG was used in the Ca2+-Ringer's solution
for experiments performed in the absence of Na+.
Oocytes were used 6-8 d after cRNA injection. Resting membrane potentials for oocytes used in recording experiments ranged from 39
to 52 mV.
Two-electrode voltage-clamp was performed on SERT-expressing oocytes
using an AxoClamp 2A (Axon Instruments). Voltage-clamp glass
microelectrodes were filled with 3N KCl solutions and pulled to a
resistance of 0.5-5 M
. The recording solutions used to
perfuse the oocytes consisted of room temperature
Ca2+-Ringer's at pH 4.5, 5.0, 5.5, 6.0, or 7.6 as
indicated in the Figure legends. Perfusion was controlled by gravity at
a rate of ~6 ml/min. Data acquisition and analysis were recorded and stored digitally (MacLab 2e interface) using MacLab v. 3.4 (AD Instruments). I-V relationships were determined using
voltage-ramp protocols: oocyte membrane potential was held at 40 mV
with voltage ramp (120 to +60 mV) applied over 10 sec.
[3H]5HT uptake in SERT-expressing oocytes was
performed in Ca2+-Ringer's containing 20 nM [3H]5HT, 100 µM
pargyline, and 100 µM L-ascorbic acid for 30 min at room temperature. Nonspecific uptake was determined by assaying uninjected or water-injected oocytes in parallel with the wild-type rSERT- and D98E-injected oocytes.
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RESULTS |
The conserved Asp in TMD I of the monoamine transporters is
critical for transport activity and ion dependence
Transporter chimeras and species-scanning mutagenesis studies
(Barker and Blakely, 1996) have identified residues within SERT TMDs I
(Barker et al., 1998) and XII (Barker et al., 1994; Barker and
Blakely, 1996) that participate in competitive antagonist recognition. In evaluation of a TMD I residue (Y95 in hSERT and rSERT)
that participates in SERT antagonist recognition (Barker et al., 1998),
we recognized that this residue was one helical turn below a biogenic
amine transporter-specific acidic residue (D98 in SERTs) (Fig.
1A) that had been
implicated in substrate recognition (Kitayama et al., 1992). All other
members of the gene family (e.g., GABA, glycine, proline transporters)
possess a Gly (G) in the equivalent position, a binary option that may suggest a constrained role in transport function. Mutation of the TMD I
Asp residue in the DA transporter (DAT) leads to a disruption of DA
transport and cocaine analog recognition (Kitayama et al., 1992). On
the basis of the proposed role of a conserved Asp residue in TMD III of
adrenergic receptors for coordinating the binding of the amine side
chain of catecholamines (Strader et al., 1989, 1991) and the absolute
conservation of this residue in monoamine neurotransmitter
transporters, Kitayama et al. (1992) hypothesized that this residue
coordinates interactions with the protonated alkylamine of substrates.
Lacking from the previous studies, however, is evidence of
complementarity between sites mutated in the transporter and functional
substitutions on substrates or antagonists that can enhance arguments
for direct ligand/transporter interactions.
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Figure 1.
TMD I Asp mutants alter transport activity.
A, Amino acid sequence alignment of TMD I from various
members of the sodium-dependent transporter gene family. The position
of the Asp residue conserved in the biogenic amine transporters is
shaded. All other members of the gene family possess a
glycine in this position. rGLYT, Rat glycine
transporter; rPROT, rat proline transporter;
rGAT, rat GABA transporter; cBETAINE,
canine betaine transporter; rbCREAT, rabbit creatine
transporter; fET, frog epinephrine transporter.
B, Determination of [3H]NE uptake
activity of wild-type hNET, D75A, D75E, D75G, and D75N. Transfected
HeLa cells were incubated with 50 nM
[3H]NE for 10 min as described in Materials and
Methods. Data are presented as percentage of total uptake for wild-type
hNET. Nonspecific uptake was determined in HeLa cells transfected with
the parent vector pBluescript SK II() (pBS).
Data represent means ± SEM of three experiments performed in
triplicate. C, Determination of
[3H]5HT uptake activity of wild-type rSERT, D98A,
D98E, D98G, D98N, and D98T. Transfected HeLa cells were incubated with
50 nM [3H]5HT for 10 min as described
in Materials and Methods. Data are presented as percentage of total
uptake for wild-type rSERT. Nonspecific uptake was determined in HeLa
cells transfected with the parent vector pBluescript SK II()
(pBS). Data represent means ± SEM of three
experiments performed in triplicate. D, Saturation
kinetics for [3H]5HT uptake for wild-type rSERT
( ) and rSERT D98E mutant ( ). Transfected COS-7 cells were
incubated for 10 min with increasing concentrations of
[3H]5HT as described in Materials and Methods.
Data plotted represent means of duplicate determinations and are
representative of three separate experiments. Mean
Km and Vmax
values are given in Table 1. Saturation plots (inset)
were converted to Eadie-Hofstee transformations showing a reduced
Vmax value with no change in
Km value for the D98E mutant.
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To test whether the conserved Asp in TMD I (Fig. 1A)
plays a critical and general role in biogenic amine transporter
function, we introduced several mutations at this position in both
rSERT (D98) and hNET (D75). Analysis of radiolabeled neurotransmitter uptake after transient expression of SERT D98 or NET D75 mutants revealed that most mutations completely abolished transport activity, including the switch to Gly generated to mimic the natural deviation in
other family members (Fig. 1B,C). The more
conservative mutation D75E in hNET was also nonfunctional, but
strikingly the rSERT D98E mutant retained 35-50% of wild-type rSERT
activity. This retention of transport function allowed us to
characterize properties of the D98E mutant that were inaccessible in
the other substitutions. Thus we found that transient expression of
rSERT D98E in both HeLa (data not shown) and COS-7 cells (Fig.
1D, Table 1) reduced 5HT transport capacity (Vmax) by ~50%
with little or no change in the 5HT Km
value.
To determine whether the reduced Vmax values
obtained for the D98E mutant in COS-7 cells was caused by reduced
translation or surface expression of mutant transporters,
immunoprecipitation and cell-surface biotinylation experiments were
performed (Melikian et al., 1996; Ramamoorthy et al., 1998). All hNET
and rSERT mutants were found to be synthesized at equivalent levels as
assessed by immunoprecipitations of [35S]-labeled
cell extracts (data not shown). Direct immunoblots of rSERT-transfected
and rSERT mutant-transfected COS-7 cells validated no differences in
steady-state protein levels (Fig. 2A). SERT and
immunoreactive proteins in COS-7 cells are revealed as two bands of 95 and 105 kDa for both the wild-type and D98E construct that are absent
from vector-transfected cells. The two bands, we suspect, represent
migration attributable to heterogeneous glycosylation (Qian et al.,
1995), although we did not explore this in detail. Both isoforms were
collected from biotinylated cell fractions that are depleted of the
intracellular marker calnexin (Fig. 2A) and thus
appear to be expressed on the cell surface. When normalized to total
calnexin content, no change was evident in D98G surface levels, whereas
a small but insignificant decrease in surface SERT protein for the D98E
mutant was detected. Wild-type levels of synthesis and cell-surface
expression were also seen for the hNET D75E (Fig. 2B)
and D75G mutants (data not shown), demonstrating that a disruption of
protein stability and/or trafficking cannot account for the loss of
transport capacity observed in hNET D75 or rSERT D98 mutants.
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Figure 2.
SERT and NET TMD I Asp mutants do not alter
transporter cell-surface expression. COS-7 cells were transfected with
rSERT, D98E, D98G, or pcDNA3 vector (control), and cell-surface
biotinylation experiments were performed as described in Materials and
Methods. A, Immunoblots identifying rSERT and rSERT
mutants using SERT-specific antibody CT-2B (Qian et al., 1995).
B, Immunoblots identifying hNET and hNET D75E mutant
using NET-specific antibody (Melikian et al., 1994). After blots were
probed with the anti-SERT or anti-NET antibodies, membranes were
stripped and reprobed with anti-calnexin antibody (StressGen). Because
calnexin is a endoplasmic reticulum transmembrane protein, it is a
marker for intracellular proteins. Low levels of calnexin bands
(<0.5% of total) were detectable in the cell-surface lanes,
suggesting only minor contamination of biotinylated (cell surface)
fractions with intracellular proteins. Density of bands was determined
using scanning densitometry (ImageQuant, Molecular Dynamics) with SERT
total fractions normalized to total calnexin immunoreactivity. Data
presented are representative of three separate experiments.
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Another property of SERTs that might be compromised by D98E and explain
a loss of transport capacity is an alteration in Na+
or Cl recognition that provides for energetic
coupling to drive 5HT influx. Complete substitution for
Na+ using lithium or NMDG or for
Cl with gluconate and nitrate salts resulted in a
full loss of 5HT transport for both SERT-transfected and SERT
D98E-transfected cells (data not shown), revealing a retention of ion
dependence for amine uptake. However, when we titrated
Na+ with NMDG or Cl with
gluconate, we found that SERT D98E exhibited a markedly altered ion
dependence (Table 1). As described previously (Rudnick and Clark, 1993;
Gu et al., 1994), Na+ concentration-dependence data
for rSERT could be fit to a single hyperbolic function, consistent with
a 1 Na+/1 5HT coupling stoichiometry. In contrast,
Na+ dependence for D98E did not saturate in an
isotonic substitution range, yielding a Km value
we could only estimate as 
100 mM (vs ~20 mM
for rSERT). Cl dependence for either rSERT or
rSERT D98E could be fit to a single hyperbolic function as reported
previously (Gu et al., 1994), supportive of a 1 Cl/1 5HT+ coupling
stoichiometry. However, in rSERT D98E we found an eightfold increase in
the Cl Km value. These
findings indicate that the D98E mutation compromises utilization of
external Na+ and Cl for
catalyzing inward 5HT transport, effects that may explain the reduction
in 5HT transport capacity in the face of little or no reduction in
surface expression or alteration in 5HT Km value.
D98E differentially alters antagonist potency
Pharmacological characterization of the rSERT D98E mutant by
[3H]5HT transport inhibition experiments revealed
selective alterations in SERT antagonist potencies (Table 1). Thus,
paroxetine and mazindol potency were essentially equal at both
wild-type rSERT and D98E mutant. Cocaine and imipramine exhibited a
three- to fivefold reduction in potency. Of the SSRIs tested,
citalopram potency was most severely affected, with a 100-fold loss of
potency at the rSERT D98E mutant. We confirmed these effects using
[125I] RTI-55-labeled competition binding
experiments (Boja et al., 1992; Wall et al., 1993). RTI-55 is a
substituted phenyltropane like cocaine, and its
Kd value for binding to SERT was significantly increased at D98E (0.37 nM vs 3.7 nM) as were
the Ki values of cocaine (133 ± 35 nM vs 580 ± 160 nM) and imipramine
(11.2 ± 2 nM vs 94 ± 34 nM).
Interestingly, in these assays 5HT displayed a sixfold loss of affinity
as an inhibitor of [125I]RTI-55 binding (184 ± 40 nM vs 1160 ± 130 nM), suggesting
that 5HT recognition is subject to conformational accommodations of 5HT
or SERT or both under the conditions used for transport. Alternatively, kinetic terms besides 5HT binding that can influence the
Km value (Rudnick, 1998b) may have been
perturbed by D98E, offsetting affinity losses.
[125I]RTI-55 Bmax values
were equivalent for rSERT and rSERT D98E, consistent with no major loss
in SERT protein expression induced by the mutation.
Selective effects of D98E on substrate recognition
The previous proposal (Kitayama et al., 1992) that amine substrate
recognition involves ion pairing between the Asp side chain carboxylic
acid and the positively charged amine group on amine substrates, as in
G-protein-coupled amine receptors (Strader et al., 1989, 1991),
suggests that mutations at D98 of rSERT might be offset by specific
structural modifications of 5HT or analogs, in particular modifications
of the alkylamine chain extending from the indole ring. We identified
two 5HT derivatives, dimethyltryptamine (DMT) and gramine (Fig.
3A), that differ only in the
length of their amine-containing side chain. If the ethylamine of DMT
interacts with the carboxylic acid at D98 (Fig. 3A),
shortening the alkylamine chain by one carbon to yield gramine should
disrupt this interaction and lead to lower potency inhibition of 5HT
uptake (Fig. 3B); however, gramine should reestablish this
ion pair if the carboxylic acid at D98 is lengthened by one carbon as
in the D98E mutation (Fig. 3C). When the potencies of DMT
and gramine for inhibition of 5HT uptake were compared at the wild-type
rSERT and the D98E mutant, we found that DMT interacts with both the
wild-type and mutant transporter equivalently (Fig. 3D,
Table 2). The shorter-chain gramine is
more than one order of magnitude weaker than DMT as a 5HT uptake
inhibitor at wild-type rSERT (Fig. 3D, Table 2). Remarkably, gramine
became a more potent inhibitor of 5HT uptake when tested on SERT D98E,
a potency indistinguishable from DMT or 5HT at wild-type rSERT (Fig.
3D, Table 2). The preference of the D98E mutant for the
shorter methylamine derivative was selective in that other
substitutions on the tryptamine structure (e.g., 5'-OH, indole N,
alkylamine methylation) were insensitive to the increased chain length
associated with the D98E mutation (Table 2). Furthermore, we sought
other structures that might generalize an interaction of the alkylamine
side chain with the carboxylic acid at rSERT D98. DA and its
methylamine derivative dihydroxybenzylamine (DHBA) are weak inhibitors
of 5HT uptake relative to the tryptamines; however, they present a
similar opportunity to adjust the alkylamine side chain length by one
carbon and test for a gain of function to the D98E mutation. We found
that DHBA is more than one order of magnitude less potent than DA at
inhibiting 5HT uptake by wild-type rSERT, whereas the two compounds are
comparable in potency at the D98E mutant because of a reduced potency
of DA and a gain of function for DHBA (Table 2).
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Figure 3.
Structure-activity studies with
tryptamine derivatives and the rSERT D98E mutant implicate a direct
interaction between neurotransmitter and D98. A,
Proposed model for interactions between carboxylic acid of SERT D98 and
tryptamine alkylamine group. B, Disruption of carboxylic
acid-amine interaction by shortening DMT to the methylamine
structure gramine. C, Reestablishing carboxylic
acid-amine interactions by lengthening carboxylic acid alkyl chain by
one methyl group (D98E) combined with shorter methylamine substrate
gramine. D, Evaluation of DMT and gramine potency for
inhibition of [3H]5HT uptake at wild-type rSERT
and D98E mutant. [3H]5HT uptake assays were
performed with transiently transfected COS-7 cells as described in
Materials and Methods, with increasing concentrations of DMT or gramine
added simultaneously with the addition of 10 nM
[3H]5HT. Nonspecific uptake was determined in
COS-7 cells transfected with the parent vector pcDNA3 and subtracted
from total values. Data were plotted as percentage of specific
5HT uptake. All data plotted represent means ± SEM of triplicate
determinations and are representative of three separate experiments.
Apparent Ki values are presented in Table
2.
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rSERT D98E disrupts substrate-gated ion flow
To gain further insights into the functional impact of the D98E
mutation and its interaction with tryptamine derivatives, we examined
substrate-induced currents in voltage-clamped Xenopus oocytes. Electrophysiological measurements in transporter-expressing oocytes have demonstrated that substrates, but not antagonists, activate transporter-associated currents (Mager et al., 1994; Sonders
et al., 1997), and we used this facet of SERT behavior instead of
conventional [3H]substrate flux measurements
because of the lack of commercial availability of
[3H]DMT or [3H]gramine.
Consistent with previous reports (Mager et al., 1994), saturating
concentrations of 5HT induced an inward current at pH 7.6 in oocytes
expressing the wild-type rSERT voltage clamped at 80 mV (Fig.
4A). These currents are
blocked by imipramine and are absent from uninjected oocytes.
Surprisingly, the D98E mutant demonstrated little (<2 nA) to no
measurable 5HT-induced current at pH 7.6 (Fig. 4B).
This was remarkable because [3H]5HT uptake
activity for the D98E mutant assayed in the same batches of oocytes
used for voltage-clamp experiments was reduced only ~40% as compared
with wild-type rSERT (Fig. 4C), similar to findings in COS-7
cells. Lowering buffer pH from 7.6 enhanced both a leak current as well
as 5HT-induced currents (Cao et al., 1997). We retested rSERT and D98E
at pH 5.0 and found that both transporters exhibited increased leak
currents (see below), and we were able to significantly increase
5HT-gated currents in the wild-type transporter (Fig.
4A). Moreover, acidic medium pH allowed us to uncover
substrate-gated ion flow in the SERT D98E (Fig. 4B).
At pH 5.0, we confirmed that 5HT and DMT, as well as gramine, induce
currents in the D98E mutant, indicating that all three compounds behave
as substrates for the transporter. Consistent with this notion,
imipramine (Fig. 4A,B) blocked all substrate-induced currents in D98E-injected oocytes tested at pH 5.0 and revealed an
outward current presumably attributable to block of a
substrate-independent inward leak (Mager et al., 1994). Thus both
wild-type rSERT and rSERT D98E are expressed in the oocyte, but acidic
pH is required to reveal substrate-induced currents for D98E. This does
not occur because acidic pH allows substrates to permeate more readily
because the shift to pH 5.0 actually diminishes 5HT transport activity more for D98E than for rSERT (Fig. 4C), suggesting that D98E
perturbs the transporter's charge movement/substrate flux ratio.
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Figure 4.
rSERT D98E mutant does not demonstrate
substrate-induced current at pH 7.6. Xenopus oocytes
were injected with either wild-type rSERT (A) or
D98E mutant (B) cRNA, and two-electrode
voltage-clamp experiments were performed as described in Materials and
Methods. Oocytes were held at 80 mV and perfused with various
tryptamines as indicated. For wild-type rSERT, the 5HT-activated
current observed at pH 7.6 was augmented at pH 5.0. Also, the shift to
pH 5.0 induced an inward current in the absence of 5HT, consistent with
previously published results (Cao et al., 1997). DMT and gramine
experiments were performed at pH 5.0 to increase substrate-induced
currents. The transporter antagonist imipramine was capable of
inhibiting both 5HT- (data not shown) and gramine-induced currents. For
the D98E mutant (B), 5HT-activated current was
not detectable above background at pH 7.6. Substrate-induced currents
were observed at pH 5.0, although the magnitude of the inward current
was greatly reduced (note change in scale between A and
B). No substrate-induced currents at either pH 7.6 or
5.0 were observed for water-injected or uninjected oocytes (data not
shown). The results shown are from single oocytes expressing either
wild-type rSERT or the D98E mutant and are representative of data from
six separate oocytes. C, Comparison of
[3H]5HT uptake in Xenopus oocytes
injected with wild-type rSERT or D98E cRNA. Oocytes injected with
wild-type rSERT cRNA, D98E mutant cRNA, or water (control) were
incubated in the presence of 10 nM
[3H]5HT for 30 min in room temperature
Ca2+-Ringer's, pH 7.6 or 5.0, as described in
Materials and Methods. Data represent total
[3H]5HT uptake (cpm) (n = 9).
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The presence of DMT- and gramine-induced currents in D98E-expressing
oocytes (measured at pH 5.0) allowed us to seek evidence for a shift in
gramine Km value as a transported substrate as predicted by the 5HT uptake inhibition studies reported in COS-7 cells.
Thus we measured inward currents at pH 5.0 induced by increasing concentrations of gramine in oocytes expressing either wild-type rSERT
or the D98E mutant (Fig. 5A).
In both cases, gramine-induced currents were saturable and fit readily
with single hyperbolic functions. Analyses of these fits revealed a
sevenfold increase in the potency (decreased Km
value) for gramine-activated currents at the D98E mutant (0.3 ± 0.04 µM) as compared with wild-type rSERT (2.2 ± 0.4 µM). Thus, gramine is more potent at the D98E mutant
in both 5HT transport inhibition assays and in activation of
substrate-dependent ion flow. At saturating concentrations at pH 5, gramine and 5HT gated equivalent levels of whole-cell current
(Imax) for the D98E mutant, whereas
gramine was less efficacious than 5HT at wild-type rSERT (Fig.
5B). This indicates that gramine is not only a more potent
substrate for the D98E mutant, but also D98E shifts the relative
efficiency of gramine to gate substrate-activated currents to a level
comparable to that observed for 5HT.
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Figure 5.
Saturable transporter-associated currents in
response to increasing concentrations of gramine.
Xenopus oocytes were injected with either wild-type
rSERT or D98E mutant cRNA, and two-electrode voltage-clamp experiments
were performed as described in Materials and Methods. Oocytes were held
at 80 mV and perfused with increasing concentrations of gramine in
Ca2+-Ringer's, pH 5.0. A, Data
represent steady-state currents fit to Michaelis-Menten rectangular
hyperbole by nonlinear regression (GraphPad Prism) from representative
oocytes (n = 7). Mean apparent
Km values: wild-type rSERT (), 2.2 ± 0.4 µM; D98E mutant (), 0.3 ± 0.04 µM. B, Comparison of currents induced by
5HT (5 µM) and gramine (5 µM) in
Ca2+-Ringer's, pH 7.6 or 5.0. 5HT- and
gramine-activated currents in the D98E mutant at pH 7.6 were barely
detectable above background. Data represent percentage of maximum
5HT-induced current observed for wild-type rSERT at pH 5.0.
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A change in ion selectivity induced by rSERT D98E
The lack of detectable substrate-gated currents for the rSERT D98E
mutant at pH 7.6 (Fig. 4B) despite significant 5HT
uptake in unclamped oocytes and changes in charge/substrate flux ratios suggested a mutation-induced disruption in a specific aspect of 5HT-gated ionic permeation. Na+ is involved in
gating inward current flow (Mager et al., 1994) and has been proposed
as the major charge-carrying species in the leak pathway at neutral pH
(Lin et al., 1996). Protons appear to readily permeate biogenic amine
transporters in the absence of amine substrates (Sonders et al., 1997)
and may also contribute to the substrate-gated current at acidic
extracellular pH (Cao et al., 1997). We thus asked whether D98E alters
Na+ or pH dependence or both for leak or
substrate-activated currents. As described previously (Mager et al.,
1994; Cao et al., 1997; Galli et al., 1997), Na+
dependence of 5HT-activated inward currents was evident when NMDG was
substituted for Na+ on wild-type rSERT at pH 7. 6. At pH 5.0, where 5HT-activated currents can be detected in rSERT D98E,
we found a similar loss of currents on Na+
substitution (Fig.
6A,B). Thus, as with
5HT transport activity measured in COS-7 cells, a dependence on
extracellular Na+ for gating nonstoichiometric ion
flow is retained in the D98E mutant. 5HT application to rSERT-injected
or rSERT D98E-injected oocytes in NMDG buffer revealed outward currents
(Fig. 6A,B), consistent with
Na+-independent 5HT binding to SERTs (Mager et al.,
1994; Cao et al., 1997; Galli et al., 1997) and H+
permeation of a leak pathway that 5HT can block (Cao et al., 1997) and
that appears to be intact in the D98E mutant.
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Figure 6.
Na+ is required for SERT inward
currents at pH 5.0. Xenopus oocytes were injected with
either wild-type rSERT (A) or D98E mutant
(B) cRNA and two-electrode voltage-clamp
experiments performed as described in Materials and Methods. Oocytes
were held at 80 mV (Vm = membrane
potential) and perfused with 5HT 10 µM in the presence or
absence of Na+ at either pH 7.6 or 5.0. Na+ concentration in the
Ca2+-Ringer's buffer was isotonically replaced with
N-methyl-D-glucamine (NMDG).
The results shown are from single oocytes expressing either wild-type
rSERT or the D98E mutant and are representative of data from three
separate oocytes.
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If acidic medium is required to detect substrate-activated currents for
the D98E mutant in Na+ buffer, decreased pH may
titrate residues that restore Na+ permeation through
substrate-activated channels, or protons may simply be traversing this
pathway. To explore these possibilities, leak and 5HT-gated currents
carried by rSERT (Fig. 7A) and
rSERT D98E (Fig. 7B) were studied in Xenopus
oocytes as a function of extracellular medium pH. I-V
curves were determined by ramp protocols during sequential perfusion
with buffer (Ctrl), substrate (5HT, 5 µM), or antagonist [citalopram (Cit), 30 µM]. Initial experiments were conducted at pH 5.0 where
5HT can be shown to gate both wild-type rSERT and rSERT D98E currents
(Fig. 4A,B). The application of citalopram to
injected oocytes in the absence of 5HT (but not uninjected oocytes;
data not shown) resulted in positive (outward) currents below 30 mV.
The shapes of the subtracted I-V plots were similar
comparing mutant and wild-type SERTs (data not shown). The reversal
potential for these currents
(ICit-ICtrl) was
unmodified by the D98E mutation (wild-type rSERT 12.2 ± 5.8 mV
vs D98E 6.0 ± 3.5 mV). Similarly, the effect of pH change on
the reversal potential of the leak current was similar in both
wild-type rSERT and the D98E mutant, with slopes of 42 mV/pH unit and
46 mV/pH unit, respectively (Fig. 7C), suggesting that
protons carry a significant fraction of current in the leak pathway at
pH 5.0 and confirming that H+ permeation in the
absence of substrate (leak) is unperturbed by the D98E mutation.
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Figure 7.
Current-voltage
(I-V) relationships for wild-type rSERT
and D98E mutant conducting states. Currents were measured in a single
voltage-clamped (40 mV) oocyte during voltage-ramp protocols (120
to +60 mV in 10 sec) in the presence of either buffer (control,
Ctrl), 5HT 5 µM, or the transporter
antagonist citalopram 30 µM (Cit).
A, I-V plots for currents for wild-type
rSERT. B, I-V plots for currents for
D98E mutant. All experiments were performed at pH 5.0. Data shown are
representative of recordings from single oocytes expressing either
wild-type rSERT or D98E mutant (n = 9-12).
C, Effect of pH on reversal potential of
substrate-independent transporter-associated leak current
(ICtrl-ICit)
for rSERT ( , solid line) and D98E mutant (,
dashed line). Data plotted are means ± SEM
(n = 7-9) fit to least- square linear regression
with slopes of 42 mV/pH U (rSERT;
r = 0.99) and 46 mV/pH U (D98E;
r = 0.99). D, Effect of pH on
reversal potential of 5HT-activated transporter-specific current
(I5HT-ICit)
for rSERT (, solid line) and D98E mutant (,
dashed line). Data plotted are means ± SEM
(n = 7-12) fit to least-square linear regression
with slopes of 28 mV/pH U (rSERT;
r = 0.99) and 53 mV/pH U (D98E;
r = 0.98).
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The addition of 5HT to transporter-injected oocytes elicited inward
currents at all potentials (I5HT),
increasing in magnitude relative to ICtrl at
negative voltages for both wild-type and rSERT D98E. When normalized
for maximum current, the shape of the
I5HT-ICtrl plot was
similar, indicating a comparable overall voltage dependence to the
substrate-activated current (data not shown). However, the
I5HT profile for D98E was noticeably shifted to
the right relative to I5HT for wild-type SERT (X
intercept at 18 mV for SERT vs 0 mV for D98E), suggesting a change in
the permeant species carrying the substrate-induced current. Because I5HT never becomes outward relative to
ICtrl (Mager et al., 1994; Galli et al., 1995;
Sonders et al., 1997), this may reflect the ability of the substrate to
induce an inward current at negative potentials and block outward leak
currents at positive potentials. Regardless, a true reversal potential
for this current (plotted as
I5HT-ICtrl)
cannot be calculated. However, because antagonist blocks both leak and
substrate-activated currents, I5HT does
intersect with ICit, defining the total
composite SERT current arising from substrate occupancy. When
I5HT-ICit was plotted as
a function of pH, the reversal potential for wild-type rSERT
demonstrated a 28 mV shift per pH unit change consistent with a partial
contribution of H+ to 5HT-gated ion flow (Fig.
7D). In contrast, the D98E mutant exhibited a 53 mV shift in
reversal potential per pH unit change, a value approaching the
predicted value for a pure H+-selective current (58 mV). These data suggest that acidic medium does not restore conductance
of the same permeant species to D98E that define the
substrate-activated current in rSERT. Rather, the acidic extracellular
medium appears to provide a source of permeant ions for a channel that,
like the leak pathway, is highly permeant to H+.
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DISCUSSION |
SERTs, like other members of the Na+-dependent
transporter family, are multi-functional proteins. First identified as
mediators of 5HT uptake across brain and platelet membranes, SERTs are
now recognized as major targets for antidepressants and
psychostimulants (Barker and Blakely, 1995). In addition to these
transport-associated processes, SERTs have multiple ion channel-like
conducting states (Mager et al., 1994; Lester et al., 1996; Lin et al.,
1996; Galli et al., 1997) that are sensitive to competitive transport
antagonists. The structural requirements for substrate recognition,
antagonist selectivity and 5HT-gated ion flow are as yet largely
undefined and may involve nonoverlapping determinants. Alternatively,
neurotransmitter and ion permeation may share a common permeation
pathway. Our data indicate that D98 in TMD I of SERT influences
antagonist recognition and acts as a selectivity determinant for
substrate recognition and substrate-gated ion flow, linking TMD I to
cocaine and antidepressant-sensitive permeation pathways for both
neurotransmitter and nonstoichiometric ion flow.
Although most mutations at the Asp residue in TMD I for NET and SERT
were nonfunctional, rSERT D98E retained significant transport activity,
allowing investigation into the role of Asp98 in transporter function.
The unaffected potency of several transporter antagonists as well as a
wild-type Km value for 5HT in the D98E mutant
supports a lack of mutation-induced perturbations in SERT structure.
Our major kinetic finding was reduced 5HT transport capacity
(Vmax) that did not appear to arise from
reduced translation or surface expression, suggesting inefficient
substrate translocation. 5HT can bind to SERTs in the absence of
Na+ and Cl (Humphreys et al.,
1994), but the amine does not translocate. D98E ion dependence
demonstrated a shift of both Na+ and
Cl dependence to higher Km
values. The Na+ Km value for
D98E was out of the isotonic range, making quantitative modeling of
this perturbation on 5HT uptake difficult. However, using a
Na+ Km value of 100 mM and the derived Cl
Km value of 40 mM, and
assuming monophasic kinetics with no ion interactions, transport
capacity should drop to 47% of control in our standard assay buffer
(120 mM Na+, 129.1 mM
Cl), reasonably close to the measured reduction in
COS-7 cells. Our findings suggest that major determinants for both
Na+ and Cl coupling reside
within TMD I, although we cannot rule out a mutational effect on
critical contact sites that lie outside this region. In keeping with a
role for TMD I in ion dependence, Mager and coworkers (1996) found
disrupted Na+ transient currents from mutation of a
conserved TMD I tryptophan in GAT1 (W103 in rSERT), and Chen et al.
(1997a) noted a cation dependence to methanethiosulfonate inactivation
of rSERT C109, a residue in the extracellular loop between TMDs I and II.
Models of -adrenergic receptor interactions with catecholamines
propose a direct coordination of the positively charged alkylamine of
ligands with the negatively charged carboxylic acid of a conserved TMD
III Asp (Strader et al., 1991). To support this model, Strader and
coworkers (1991) found that catecholamine derivatives could compensate
for mutations in the -adrenergic receptor. Thus, mutation of the Asp
(D113) in TMD III to Ser (D113S), thereby replacing the carboxylic acid
with a hydroxyl group, results in mutant receptors that can be
activated by ligands possessing hydrogen bond-accepting groups in place
of the alkylamine side chain. Similar findings of complementarity
between mutant transporters and "mutant" ligands would increase the
implications that direct contacts between ligands and transporters were
being evaluated through site-directed mutagenesis. In the present
study, we found that the D98E mutant compensates for disruptions in
potency induced by alterations in substrate structure. Thus, gramine,
bearing a methylamine side chain, was significantly more potent both as
an inhibitor of 5HT uptake and at activating substrate-induced currents
at the mutant D98E as compared with wild-type rSERT. Although the
effect was not as dramatic as that seen with gramine, the
shorter-chained analog of dopamine, DHBA, showed a similar preference
for D98E, suggesting that catecholamines may be coordinated similarly
to indoleamines, at least by SERTs. Interestingly, Buck and Amara
(1994) also have implicated TMD I in substrate recognition for DATs
through chimera studies. Antagonist ([125I]RTI-55)
binding studies revealed a loss of affinity for 5HT at D98E, whereas
uptake studies revealed no change in the 5HT Km
value. The determination of the transport Km
includes steps distal to binding, including translocation and release
of substrates, and these additional steps may offset mutation-induced
effects on 5HT binding. Additionally, the conditions of transport
versus binding assays (e.g., 37° vs 4°C) may allow movements of
either the acidic side chain on the transporter or the alkylamine chain of the substrate to accommodate 5HT. Gramine and DHBA, however, may
already be in an extended conformation and require the lengthening of
the acidic side chain at D98 to recover the potency seen for the
ethylamines DMT and 5HT. Because the D98E mutant could promote favorable interactions between gramine with yet another residue, studies evaluating charge-charge interactions in support of this interaction are warranted.
Because D98 influences 5HT transport, we also determined whether
this residue represents a critical contact site for transporter antagonists. Evidence such as protection against alkylating agents argues that competitive antagonists such as imipramine and cocaine possess mutually exclusive, overlapping binding sites with 5HT on SERT
(Graham et al., 1989). However, comparisons of radioligand dissociation
rates from native SERTs suggest heterogeneity in the binding of SERT
antagonists and propose distinct ligand-specific contact sites (Plenge
et al., 1987, 1991). For the D98E mutant, we observed a loss of
affinity for cocaine as well as for the cocaine analog RTI-55, the
tricyclic imipramine, and the SSRI citalopram. These effects, however,
were not shared by all antagonists (e.g., mazindol, paroxetine),
consistent with the existence of unique determinants of antagonist
recognition. We recently linked Y95 in rSERT, located one helical turn
away from D98 in TMD I, with species-specific recognition of some but
not all SERT antagonists (Barker et al., 1998). In the context of our
present studies, these findings support a model by which competitive
antagonists can physically obstruct substrate binding sites in addition
to conformational alterations arising from ligand binding (Ferrer and
Javitch, 1998). Recently, Chen and coworkers (1997b), using SCAM
techniques, identified both I172 and Y176 of TMD III as potential shared sites for the recognition of both 5HT and cocaine, supporting a
model whereby TMDs III and I lie close together in the folded configuration of the membrane-embedded transporter.
Not only does D98 appear to dictate ligand recognition, but the D98E
mutation also compromises the SERT permeation pathway for
nonstoichiometric ion flow (Mager et al., 1994; Lin et al., 1996). No
detectable 5HT-induced current was observed for the D98E mutant at pH
7.6. However, the D98E mutant was clearly expressed because (1)
transport activity was present, (2) leak currents were intact, and (3)
a shift of external pH to pH 5.0 revealed substrate-gated currents. The
loss of detectable 5HT-activated current in the D98E mutant expressing
oocytes at pH 7.6 could be attributed to either a mutation-induced
disruption of gating mechanisms or an alteration in permeant species or
both. Because 5HT and other substrates can gate ion flow at pH 5.0 and
this activity still requires extracellular Na+,
gating of ion flow per se appears to be intact. Single-channel analysis
of SERT-conducting states has suggested that Na+ is
a major current-carrying species for both 5HT-activated and the
substrate-independent leak currents (Lin et al., 1996), although recent
studies suggest that H+ may have significant
permeability through these pathways (Cao et al., 1997; Sonders et al.,
1997). Although the Na+ and Cl
Km values were elevated and can account
for a loss of ion-coupled uptake capacity, this reduction in transport
would not account for the D98E-induced disruption on 5HT-gated currents
at neutral pH because 5HT carries a very small fraction of the total
current (Mager et al., 1994; Galli et al., 1997). Because 5HT is
essentially fully protonated at neutral pH, the shift to acidic pH, as
argued previously by Cao and coworkers (1997), must reveal currents not by titrating 5HT but by either (1) titrating a functional group on SERT
to permit cation flow disturbed by D98E or (2) increasing the driving
force for H+ flow across a permeation pathway
unaffected by the D98E mutation. Analysis of the pH sensitivity of the
reversal potential for the leak pathways of rSERT and D98E confirms
that substrate-independent ion flow at acidic pH is essentially intact
in D98E. However, although the 5HT-gated current of wild-type rSERT
contains other charge carriers besides H+ (and
probably Na+), the D98E 5HT-gated current is
essentially proton selective. This suggests that the mutation has
impacted 5HT-gated ionic selectivity rather than gating per se such
that Na+ is no longer the major current-carrying
species. We attempted to confirm a loss of Na+
influx by D98E via [22 Na] cotransport studies but
found the expression levels of SERTs inadequate to achieve this measurement.
Because the D98E mutant affects the ion coupling of 5HT transport and
eliminates a component of the substrate-activated conducting current,
both of which involve Na+, ion selectivity in SERT
channels may be determined by domains and residues that also
participate in coupling 5HT permeation to ion gradients. The simplest
model would involve substrate and ion flow, including
H+, as routed through a common pathway whereby the
D98E mutant retains the ability to pass 5HT and H+
but no other ions. As discussed by Cao and coworkers (1997), the
movement of protons through SERTs may involve conduction in a
"water-wire" mechanism that Na+ and other
cations have no access to, whereas Na+ conduction
may require distinct structural elements that impact selectivity and
conduction. Recent studies of Drosophila SERT currents (C. Petersen and L. DeFelice, unpublished observations) reveal anomalous
mole fraction effects between Na+ and
Li+ in the leak pathway that are abolished by 5HT,
suggesting that the substrate itself may modify cation interactions in
a common permeation pathway. Interestingly, GAT1 GABA transporters lack transmitter-gated nonstoichiometric ion flow (Mager et al., 1996), and
the Gly at the position homologous to D98 in GAT1 may contribute to
this biophysical distinction with biogenic amine transporters. We
cannot rule out that two pathways for nonstoichiometric ion flow could
be gated by 5HT, one involved with proton permeation and a second,
affected by the mutation, carrying other ions (and perhaps also 5HT)
that comprise the bulk of the nonstoichiometric ion flow at neutral pH.
Besides a lack of positive data to support the latter model, 5HT
appears to block rather than gate the leak pathway (when tested in the
absence of Na+). In addition, direct single-channel
recordings of substrate-independent and substrate-activated channels by
Lin and coworkers (1996) indicate a similar conductance for the leak
and 5HT-gated permeation pathways. Whichever model is correct, D98, and
by extension TMD I, clearly constrains multiple facets of SERT function
including 5HT, Na+, Cl, and
antagonist recognition as well as ion permeation, effects that must be
accommodated by models linking transporter structure to function. Given
the conservation of the TMD I Asp in biogenic amine transporters, we
suspect that our findings generalize to NETs and DATs.
| |
FOOTNOTES |
Received Jan. 29, 1999; revised March 23, 1999; accepted March 26, 1999.
This work was supported by National Institutes of Health Grants
R01-DA07390 (R.D.B.) and F32-DA05679 (E.L.B.) and a National Alliance
for Research on Schizophrenia and Depression Established Investigator
Award (R.D.B.). We acknowledge the effort of Robert Gereau and Susan
Taylor-Rouse (Emory University) for assistance in the generation of the
SERT and NET mutants. We also thank Louis DeFelice, Aurelio Galli,
Christina Petersen, and Ian Scott Ramsey for helpful discussions and
critical reviews of this manuscript.
Correspondence should be addressed to Dr. Randy D. Blakely, MRBII Room
419, Center for Molecular Neuroscience, Vanderbilt School of Medicine,
Nashville, TN 37232-6600.
Dr. Barker's and Dr. Rakhshan's present address: Department of
Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy, W. Lafayette, IN 47907.
| |
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TOP
ABSTRACT
INTRODUCTION
