 |
INTRODUCTION |
The dopamine (DA)1
transporter (DAT) in neuronal plasma membranes clears DA from the
(extra)synaptic space (1-3) by an active uptake process with
co-transport of Na+ and Cl
(for recent
reviews see Refs. 4 and 5) but probably not counter-transport of
K+ (6). In calculating the overall stoichiometry of the
neuronal DA uptake process as 2:1:1 for
Na+:Cl
:DA, the authors have combined the
evidence for co-transport of two Na+ ions and one
Cl
ion per DA molecule with the assumption that the
cationic form of DA is the substrate for uptake (7-9). DA has an amino
group that can accept a proton and a phenolic hydroxyl group that can donate a proton; the second phenolic group has a pKa value greater than 12. Therefore, except at extremely basic pH values
where both hydroxyl groups can be dissociated, DA can exist as a cation
(+H3NDOH, with D for DA skeleton), a zwitterion
(+H3NDO
), a neutral form
(H2NDOH), or an anion (H2NDO
).
With pKa1 and pKa2 values of 8.86 and 10.5, respectively (10-12) (see "Experimental Procedures"), it
can be calculated that at physiological pH, DA exists mostly as a
cation, which has prompted the assumption that this is the active form for transport (7-9). Indeed, in the analogous cases of neuronal uptake
of serotonin and norepinephrine, strong evidence for translocation of
the cationic form has been advanced (13, 14). Indirect evidence in
favor of the cation also being the active form for DA uptake comes from
site-directed mutagenesis studies (15) and molecular modeling (16)
implicating an interaction between the protonated amine group of DA and
Asp-79 of the DAT. The question of the active monoamine form for uptake
by the vesicular monoamine transporter was given a great deal of
attention more than a decade ago, with mixed conclusions. A case has
been made for the cationic (17, 18) as well as the uncharged form (for
review see Ref. 19), and to our knowledge no new information on this
issue is available.
In the present study, the question of the active form of DA for the
neuronal DAT is addressed by monitoring for the human DAT (hDAT) cloned
by Janowsky and colleagues (20, 21) and the pH dependence of (i) uptake
and binding of DA, which can be in an ionized or neutral form, and (ii)
binding of guanethidine, which is positively charged in the pH range
studied (see below), and bretylium, which is always positively charged
as a quaternary ammonium salt. Uptake was measured by monitoring the
accumulation of [3H]DA, and binding was assessed
indirectly through inhibition of high affinity binding of the cocaine
analog [3H]2
-carbomethoxy-3
-(4-fluorophenyl)tropane
(WIN 35428), which interacts with a domain on the DAT that overlaps
with the DA domain (5, 15, 16, 22-24). By comparing DA uptake and
binding, we can determine whether changes in uptake as a function of pH are solely determined by changes at the level of DA recognition, the
first step in the DA translocation cycle. In contrast to DA, which is a
dihydroxylated phenylethylamine, bretylium is a nonhydroxylated bromophenylmethylamine in which the amine is quaternary (25), and
therefore the formation of an overall neutral species is not possible
for this compound. Bretylium is usually employed for its inhibitory
effect on norepinephrine release (see Ref. 26) and monoamine oxidase
(27). In addition, it can be taken up into dopaminergic cells (28) and
interferes with DA uptake into striatal synaptosomes (29). Guanethidine
is a compound derived from guanidine. The latter is a strong base with
a pKa of 12.5 (30) and is therefore predominantly in
the positive guanidinium ion form over a wide pH range up to 11. Guanethidine has a pKa1 of 11.4 and a
pKa2 of 8.3 (31). The former pKa
value describes the ability of guanethidine, at the pH values between
6.0 and 8.2 studied here, to carry one proton at the guanidine residue,
whereas the latter pKa value represents the ability
of the guanido group to accept an additional proton as a function of pH
(at pH 8.2 in ~50% of the molecules). In a similar but not identical
manner as bretylium, guanethidine is known to interfere with
catecholaminergic transmission in mammalian (32) and invertebrate (33)
systems. Guanethidine can act as a substrate for the norepinephrine
transporter (34) and has been reported to inhibit neuronal uptake of
norepinephrine (25), but its affinity for the DAT, to our knowledge,
has not been reported.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[3H]WIN 35428 (84.5 Ci/mmol) and
[3H]dopamine (21.5 Ci/mmol) were from NEN Life Science
Products. Unlabeled WIN 35428 was from the Research Triangle Institute
(Research Triangle Park, NC). All other chemicals were from Sigma.
Glass fiber filter mats and Betaplate Scint scintillation mixture for
the binding assays were from Wallac Inc. (Gaithersburg, MD). GF/C fiber
filter mats and CytoScint ES mixture for the uptake assays were from
Brandel (Gaithersburg, MD) and ICN (Costa Mesa, CA), respectively. The
source of the DAT was the HEK-293-hDAT cell line developed by Janowsky
and co-workers (21).
[3H]WIN 35428 Binding Assay--
The general
conditions for growing the cells, working up the membrane preparations,
and conducting the binding assays were as described previously by us
(35). Briefly, after cell lysis, membranes were prepared and
homogenized with the Brinkmann Polytron (setting 6, 15 s) in
ice-cold saline. Binding assays were performed in ice-cold 30 mM sodium phosphate buffer (the result of mixing primary
and half-strength secondary sodium phosphate buffer to a selected pH
value between 6.0 and 8.2, as indicated, at room temperature) also
containing 122 mM sodium chloride, 5 mM
potassium chloride, 1.2 mM MgSO4, 10 mM glucose, 1 mM CaCl2, and 0.1 mM tropolone for catechol-O-methyltransferase
inhibition (21) ("assay buffer"). The listed pH values are those
measured at room temperature after all of the above components had been
added. In this buffer, binding assays were conducted for 10 min at
21 °C in 96-well plates with 4 nM [3H]WIN
35428. The assays contained additionally, where appropriate, nonradioactive WIN 35428 (1-300 nM), DA (0.3-100
µM), guanethidine (10-3,000 µM), or
bretylium (10-3,000 µM) (for KD or KI estimation, respectively), HEK-293-hDAT membranes (~120 µg of protein as determined by the method of Lowry; see Ref.
35) with or without 100 µM cocaine for assessment of
nonspecific binding. Assays were terminated on glass fiber filter mats
(presoaked in 0.05% (v/v) polyethyleneimine) with the MACH 3-96
Tomtec harvester (Wallac Inc.) using phosphate (10 mM)-buffered saline (pH 7.4). Filters were counted in a
Microbeta Plus liquid scintillation counter (Wallac Inc.) at an average
counting efficiency of 35%.
[3H]DA Uptake Assay--
The general conditions
for conducting the uptake assays were as described by us previously for
synaptosomal suspensions (22). Briefly, HEK-293-hDAT cells were lifted
from the culture flasks with phosphate (10 mM)-buffered
saline (pH 7.4) and centrifuged at 100 × g for 6 min.
The pellet of cells from two confluent 150-cm2 flasks was
resuspended in 6 ml of saline in a glass homogenizer with a
motor-driven Teflon pestle (clearance of approximately 0.15 mm compared
with a cell diameter of 0.01 mm). This step helped to produce even
suspensions while not causing more cell damage than resuspension
through vortexing (combined with repeated movements through a 5-ml
pipette) as judged under the microscope by the Trypan Blue dye
exclusion test. Uptake assays were conducted in a final volume of 0.4 ml of the above described assay buffer at selected pH values and
contained 50 µl of cell suspension, 20 µl of [3H]DA
stock (contributing a final 6 nM concentration of tritiated DA (NEN Life Science Products) and 46 nM unradioactive DA),
and for saturation studies 20 µl of varying amounts of unradioactive DA (for final concentrations ranging from 0 to 1000 nM).
Nonspecific uptake was defined by 100 µM cocaine. After a
preincubation of the cell suspension and assay tubes with assay buffer
for 15 min in a 21 °C water bath, 50 µl of cell suspension was
added to the tubes, which were then incubated while gently shaking in a
21 °C water bath for 8 min. The reaction was terminated with the addition of 4 ml of ice-cold wash buffer (assay buffer minus
tropolone). Subsequent filtration (within 4 min after stopping for all
tubes) was performed with a 24-pin Brandel harvester through a Whatman GF/C glass fiber filter presoaked in 0.05% (w/v)
poly-L-lysine with three additional rinses of 4 ml of
ice-cold wash buffer. In control experiments for assay mixtures with
stop buffer, after 6 min on ice no efflux of accumulated
[3H]DA occurred, as was the case for synaptosomal assays
(22). Radioactivity on the filters was measured in 5 ml of CytoScint mixture with a Beckman model LS 6000IC scintillation counter with quench correction. The average protein content of the cell suspensions was estimated to be 250 µg determined by the method of Lowry
described above.
Distribution of Neutral and Ionic Forms of DA at Varying
pH--
The pKa1 value was set to 8.86 and the
pKa2 value to 10.5, which is the average of the
values of 8.8-8.9 and 10.4-10.6, respectively, reported by Armstrong
and Barlow (10), Lewis (11), and Mack and Bönisch (12). The first pKa governs the transition from the cationic
+H3NDOH to the mixture of
+H3NDO
and H2NDOH
(both overall neutral species) rather than representing the
dissociation of the amino group only (10, 12). Similarly, the second
pKa describes the transition of the two neutral species into the anion H2NDO
and not just the
dissociation of the hydroxyl group (10, 12). This analysis does not
consider extremely basic pH conditions in which ionization could occur
in the second hydroxyl group, which has a pKa > 12.
The percent of total DA existing in the neutral form
(+H3NDO
plus H2NDOH)
as a function of pH was calculated according to Mack and Bönisch
(12) as follows: % neutral = 100/(1 + antilog(pKa1
pH) + antilog(pH
pKa2)).
The percent of total DA existing in the cationic form was calculated as
follows: % positive = 100/(1 + antilog(pH
pKa1) + antilog(2pH
pKa1
pKa2)); this was arrived at by rewriting the
equations for the two equilibria described by Armstrong and Barlow (10)
as shown for the single case of protonation of a base by Courtney and
Strichartz (36). This derivation also takes into account that
log((+H3NDO
+ H2NDOH)/(H2NDO
))
log((+H3NDOH)/(+H3NDO
+ H2NDOH)) = pKa2
pKa1 by solving both equilibrium equations for pH.
The percent of total DA existing in the anionic form was calculated as
follows: % negative = 100
% neutral
% positive.
Prediction of DA Uptake Velocity Based on the Neutral Form of DA
Being the Substrate--
DA velocity for 56 nM
[3H]DA in the assay at varying pH was predicted based on
fixed pKa1 and pKa2 values for
the case that the neutral form is the active form for translocation. The pKa2 was always 10.5 (see above), and the
pKa1 was 8.86 (theoretical value, see above) or set
at a value between 6.8 and 9.5 (see "Results" and
"Discussion"). At pH 8.2, the highest pH studied, more DA will be
in the neutral form than at a lower pH, and for each pair of
pKa1 and pKa2, the Km of the neutral form of DA was computed from the
"composite Km" according to the following:
Km(neutral) = Km(composite,pH 8.2)·0.01·% neutral
at pH 8.2. For example, if 10% of DA is neutral at pH 8.2, then the
Km for the neutral form will be 10-fold smaller than
the measured composite Km at pH 8.2 if the neutral
form is the active form. Subsequently,
Km(composite) was computed over the entire pH range from the following:
Km(composite) = Km(neutral}/(0.01·% neutral (a
different set of calculations for each pair of pKa1 and pKa2). Finally, velocity, V, at each
pH for a given set of pKa1 and
pKa2 was calculated as V = Vmax·(56/(56 + Km(composite))) in which
Vmax is the value measured across pH (1.10 pmol/mg protein/8 min) and 56 is the total composite [DA] in
nM with Km expressed also in
nM.
Data Analysis--
IC50 values for the inhibition of
[3H]WIN 35428 binding by compounds were estimated with
the ALLFIT equation (37), and KD, Km, Bmax, and
Vmax estimates were obtained with the nonlinear computer fitting program LIGAND (38) as described by us (39). IC50 values were converted to KI values
by the Cheng-Prusoff equation (40) taking into account the level of
radioligand and the KD of [3H]WIN
35428 binding at each pH. All results are expressed as means ± S.E. Statistics included one-way ANOVA followed by the
Student-Newman-Keuls multiple comparisons test or the least significant
difference multiple range test and two-way ANOVA with factors A and B
and interactions A × B. Where needed, data were log-transformed
for homogeneity of variance. The accepted level of significance was 0.05.
 |
RESULTS |
DA Uptake as a Function of pH--
The Km for
[3H]DA uptake decreased approximately 3-fold upon
increasing the pH from 6.0 to 7.4 and did not change upon further
increasing the pH to 8.2 (Fig.
1A). There were no statistically significant differences in the
Vmax values obtained in the 6.0-8.2 pH
range.

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Fig. 1.
pH dependence of DA uptake, binding, and
ionic form distribution. A, the Km
( ) and Vmax (- - -) of
[3H]DA uptake and the KI for
inhibiting [3H]WIN 35428 binding (· · ·) were
determined as described under "Experimental Procedures."
B, the velocity (V) of [3H]DA (56 nM) uptake ( ) was compared with predicted curves
(dashed and dotted lines) based on the neutral
forms of DA being the substrate at varying pKa1
values as indicated. prot, protein. C,
contribution of positive (cation) and neutral (zwitterion plus
uncharged) forms to total DA concentration at the
pKa1 values as indicated (- - - for
pKa1 = 6.8 and · · · for
pKa1 = 8.86). Experimental data are the means ± S.E. (vertical bar) of three to four independent
experiments carried out in triplicate. * indicates p < 0.05 compared with pH 6.0 (Student-Newman-Keuls multiple comparisons
test following one-way ANOVA).
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The velocity of [3H]DA uptake at a fixed concentration of
DA of 56 nM increased sharply upon increasing the pH from
6.0 to 7.0 and then remained at a plateau up to the highest pH applied, which was 8.2 (Fig. 1B).
A different pH dependence was calculated for the concentration of
either the cationic form of DA (+H3NDOH), which
at a pKa1 of 8.86 and pKa2 of 10.5 (10-12), was in the majority at all pH values studied, or the
neutral forms (zwitterion +H3NDO
plus neutral form H2NDOH), which increased approximately
10-fold for each pH unit increase (Fig. 1C). Indeed,
velocity predictions based on the neutral form being the substrate
deviated substantially from the observed pH dependence of DA uptake
(Fig. 1B, compare solid line with data points
with broken line for pKa1 = 8.86).
DA Binding as a Function of pH--
The KI
value for DA in inhibiting [3H]WIN 35428 binding
decreased approximately 3.5-fold upon increasing the pH from 6.0 to
7.4, paralleling the change in the Km for DA uptake, and showed a smaller decrease of approximately 1.5-fold upon further increasing the pH to 8.2 (Fig. 1A).
Guanethidine and Bretylium Binding as a Function of pH--
The
affinity of guanethidine for the DAT was generally lower than that of
DA (for KI values see legend to Fig.
2). However, there was a resemblance
between guanethidine and DA in the pattern of potency changes as a
function of pH (Fig. 2). Thus, the KI value for
guanethidine in inhibiting [3H]WIN 35428 binding
decreased approximately 3-fold upon increasing the pH from 6.0 to 7.4 and showed a smaller decrease of approximately 2-fold upon further
increasing the pH to 8.2 (Fig. 2). WIN 35428 itself had a higher
potency than DA or guanethidine (see legend to Fig. 2), but again the
pattern of pH dependence was similar (Fig. 2) (note the lack of a
statistically significant interaction factor in analysis of variance of
the data for DA, guanethidine, and WIN 35428). In contrast, the total
decrease in the KI of bretylium upon increasing the
pH from 6.0 to 8.2 was only 2-fold as compared with the 5-7-fold
reductions seen with DA, guanethidine, or WIN 35428 (Fig. 2) (note the
statistically significant interaction factor in analysis of variance of
the data for all compounds including bretylium).

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Fig. 2.
pH dependence of the potency of compounds in
inhibiting [3H]WIN 35428 binding.
KI values were determined from inhibition curves for
each compound as described under "Experimental Procedures." Each
point represents the mean ± S.E. (vertical
bar, shown where greater than point itself) of three independent
experiments carried out in triplicate expressed as the percent of the
value at pH 7.4. At this pH level, the KI value for
WIN 35428 was 15 nM; for DA, 6.0 µM; for
guanethidine, 327 µM; and for bretylium, 664 µM. * indicates p > 0.05 for
interaction factor (F(9, 28) = 2.08) (two-way ANOVA with pH as factor
A and inhibitors guanethidine, DA, and WIN 35428 as factor B; both
factors were highly significant with p < 0.001). $
indicates p < 0.001 for interaction factor (F(9, 28) = 6.72) (two-way ANOVA with pH as factor A and inhibitors bretylium,
guanethidine, DA, and WIN 35428 as factor B; both factors were highly
significant with p < 0.001).
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No evidence was found for a deviation from a competitive mechanism of
inhibition of [3H]WIN 35428 binding by bretylium or
guanethidine. First, the Hill numbers for the inhibition curves were
close to unity over the entire pH range. Second, a fixed concentration
of 700 µM bretylium or guanethidine caused an increase in
the KD value without changing the
Bmax studied at both pH 6.0 and 8.2 (Table
I). As expected, binding was increased at
pH 8.2 as compared with 6.0; this happened in all cases because of a
decrease in KD (Table I).
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Table I
Saturation analysis of [3H]WIN 35428 binding to HEK-293-hDAT
in the absence or presence of bretylium or guanethidine
Bretylium or guanethidine, when added, were present at a fixed
concentration of 700 µM. The results are the average ± S.E. for the number of independent experiments, each assayed in
triplicate, as indicated in parentheses.
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 |
DISCUSSION |
Predicted DA Uptake with Overall Neutral DA as the
Substrate--
If it is assumed that the increase in DA uptake with pH
is accounted for solely by the shift in the distribution of DA forms toward the overall neutral forms
(+H3NDO
plus H2NDOH),
the predicted pH dependence does not coincide with the observed pattern
at the pKa1 and pKa2 values reported for DA of 8.86 and 10.5, respectively (10-12). It could be
argued that the pKa1 value may be reduced in the
vicinity of the binding site when its environment is more hydrophobic, making it easier for the compound to loose a proton. This pattern has
been suggested for the binding of cocaine to the local anesthetic binding site in the voltage-dependent sodium channel, which
is predicted more accurately by a pKa value of 7.1 than the value of 8.4-8.7 measured in water (41). The present DA uptake data could be interpreted in such a context with a
pKa1 value of 6.8 resulting in a reasonable fit for
uptake at a fixed concentration of 56 nM
[3H]DA in the studied pH range of 6.0-8.2. Another way
the possibility of a pKa shift can be shown is
by calculating the Km or KI of
the neutral forms of DA from the observed "composite" Km,I taking into account the proportion of the
neutral forms at each pH. If the calculated values remain constant
across pH, this result would be in agreement with the concept that
pH-dependent changes in proportion solely underlie the
uptake or binding changes. At pKa1 8.86 the
Km,I of the neutral forms was not constant across
pH, but lowering the pKa1 to 6.8 gave reasonably
constant values (Table II).
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Table II
Estimates of the Km or KI of the neutral form of DA as
substrate computed from the observed "composite" Km or
KI at varying pKa1 (see text)
If the neutral forms of DA (+H3NDO plus
H2NDOH) are the substrate for the DAT and if changes in uptake
or binding with pH are only the result of changes in the concentration
of the neutral forms as a function of pH, the Km or
KI of the neutral forms can be computed from the
observed "composite" Km,I taking into
account the proportion of the neutral form. The pKa1
was set at the values shown, and the pKa2 was
taken as 10.5 (see text). The "composite" Km,I
is from three to four independent experiments assayed in triplicate.
The corrected sum of squares is the sum of all squared differences
between the mean and the individual Km,I values
divided by the mean.
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It can only be hypothesized as to how hydrophobic the environment is
around the DA binding site to substantially reduce the pKa1 value below that found in a water milieu. DA is thought to bind to a site in a central aqueous cavity surrounded by
amphipathic membrane-spanning helices (5). Transporter chimera and
truncation studies support the idea that both substrates and blockers
interact with residues in transmembrane domains lining the permeation
pathway (reviewed in Ref. 4), which may include Asp-79 in transmembrane
domain 1 (15). Prior to translocation, DA accesses its centrally
located binding site, which is facing externally; translocation occurs
through a conformational change exposing the binding site on the
internal face (5, 42). The presently observed similarity in the pH
dependence of DA uptake and binding suggests that the pH effects occur
at the level of DA recognition. It is possible that the DA binding site
in the central crevice of the DAT is accessed by DA from the aqueous side, whereas the local anesthetic site near the inactivation gate in
the Na+ channel (43) can be reached by the relatively more
lipophilic cocaine (log P of 2.4 (44) compared with
0.99 for DA (12)) through the hydrophobic pathway in the membrane lipidic environment (45). Recent evidence for the serotonin transporter, which is closely
related to the DAT, suggests binding of serotonin to Ile-72 and Tyr-176
(46). These residues lie in the
-helical structure of transmembrane
domain 3 on a vertical patch that faces toward the binding pocket
accessible to the external medium as judged from their sensitivity to
membrane-impermeant, charged
[2-(trimethylammonium)ethyl]methanethio-sulfonate in the
cysteine-scanning approach (46).
Predicted DA Uptake with Neutral, Uncharged DA as the
Substrate--
The composition of the mixture of the zwitterion
+H3NDO
and the neutral form
H2NDOH is defined by the equilibrium dissociation constant
KZ = +H3NDO
/H2NDOH, which
has been determined to be ~7.83 (the average of the range 5.8-10.0)
by various approaches (10). Thus, the neutral, uncharged form is less
predominant than the zwitterion, but its increase as a function of
rising pH is the same as for the total mix of
+H3NDO
plus H2NDOH.
Indeed, when the DA uptake velocity at 56 nM
[3H]DA was calculated taking into account KZ and
assuming the neutral, uncharged DA as the substrate rather than the
mix, the curves were identical as shown for the mix in Fig.
1B. Thus, the hypothesis that this form of DA is the
substrate also requires the assumption of a lower
pKa1 value than is measured in a water environment.
Predicted DA Uptake with Zwitterionic DA as the Substrate--
The
increase of the zwitterion as a function of rising pH is the same as
for the neutral forms of DA and again does not fit the experimental
data at pKa1 8.86. Therefore, the zwitterion is not
likely to be the sole form of DA acting as the substrate for the DAT.
If a protonated amine group of DA is involved in the interaction of DA
with Asp-79 in the DAT as suggested by Kitayama et al. (15)
and Edvardsen and Dahl (16), then the contribution of the zwitterion
and the cation may have to be considered together (see below). Because
the contribution of the zwitterion at the pH values studied here is
only a small fraction of that of the cation (see below), the difference
between predicted DA uptake (binding) values with and without
zwitterion is too small to draw conclusions regarding the comparison of
the fit of these values to the experimental data. Therefore, the
involvement of the zwitterion in addition to the cation is a
possibility that cannot be excluded based on the present data.
Predicted DA Uptake with Anionic DA as the Substrate--
If
anionic DA is the substrate, it can be calculated that at pH 8.2 with a
0.1% contribution of the anion (at pKa1 8.86 and
pKa2 10.5, see "Experimental Procedures"; at pH
7.4 the contribution of the anion is even less than 0.1%) and a
KD(composite) = 6.0 µM
(from observed inhibition by DA of [3H]WIN 35428 binding
at pH 7.4), the KD(anion) = 6 nM. Such an affinity is typically high enough to
allow measurement by radioligand binding approaches. The
k+1 for the binding of anionic DA can be
estimated from the results of Meiergerd and Schenk (47) as
kcat(anion)/0.001
Km(composite) in which
kcat(anion) is the catalytic rate
constant for overall movement of anionic DA across the membrane
assuming a 0.1% contribution of the anionic species to total DA.
Because under those conditions
kcat(anion) equals 0.001 Vmax( composite)/Td, in which
Td is the transporter density (47), we can write
k+1(anion) = (0.001 Vmax(composite}/Td)/(0.001 Km(composite)) = (Vmax(
composite)/Td)/(Km(composite)), which is exactly the same as the k+1 for
composite DA calculated by Meiergerd and Schenk (47), i.e.
of 1.4 × 106 M
1
s
1. With this value for k+1, the
k
1(anion)
(=KD·k+1) = 6 × 10
9 M (affinity at pH 7.4). 1.4 × 106 M
1 s
1 = 0.0084 s
1, indicating a t1/2 of 1.4 min. This off-rate is slow enough for detection of binding by rapid filtration, and yet [3H]DA binding to the DAT has never been
observed. Clearly, the hypothesis that anionic DA is the substrate for
the DAT is untenable.
Predicted DA Uptake with Cationic DA as the Substrate--
Most of
the DA (at pKa1 8.86 and pKa2
10.5) was in the cationic form over the pH range studied, slightly decreasing from 99.9% at pH 6.0 to 82.0% at pH 8.2. If the protonated amine group is the important determinant, one should consider the sum
of the cation and zwitterion, which stays virtually constant from
99.9% at pH 6.0 to 97.9% at pH 8.2. Therefore, if the cationic form
(with or without zwitterion) is the substrate for the DAT, the
increases in uptake with pH must result from mechanisms beyond increases in concentration of the active substrate. The parallel changes in Km for uptake and KI
for binding suggest that changes in the affinity of DA for
the DAT occur as a function of pH.
Consistent with the idea that changes occur in the interaction of
cationic DA with the DAT as a function of pH, guanethidine, which is
positively charged at the pH range of 6.0-8.2 studied here, displays
changes in affinity for the DAT that parallel the changes for DA
itself. Although bretylium is always positively charged, it shows less
dependence on pH in interacting with the DAT than guanethidine or DA,
suggesting that elements are involved outside of the overlapping
binding domains for bretylium and DA (see "pH-dependent
Changes in Interaction between DA and DAT: Ligand or Transporter?"
below). The lesser pH dependency is not explained by invoking a
distinct mechanism of inhibition for bretylium, because both bretylium
and guanethidine behave as competitive inhibitors of
[3H]WIN 35428 binding in saturation analysis, which is in
agreement with the idea that a shared domain of the DAT is involved.
Structurally, bretylium shares the phenyl ring with DA, but the carbon
chain between the phenyl ring and the nitrogen (N+) is
shorter than in DA. In contrast, this carbon chain has the same length
in guanethidine as in DA, but the ring is made up of a
heptamethylenimino rather than a phenyl structure in guanethidine. Most
likely, these differences compared with DA contribute to the relatively
lower potencies of bretylium and guanethidine at the DAT.
The observed KI of bretylium at pH 7.4 of 664 µM is appreciably higher than the IC50 value
of 149 µM reported by Kammerer et al. (29) for
inhibition of striatal synaptosomal DA uptake. The most likely reason
for this difference is that bretylium, like DA, is a substrate for the
DAT, requiring higher concentrations for saturation of binding than
uptake. The same may apply to guanethidine, which has a high
KI value of 327 µM at pH 7.4.
Comparison with Other Transporters for Monoamines--
The present
data are consonant with the conclusion that the cationic form of DA,
perhaps including the zwitterion, is the more likely substrate for the
DAT. Accordingly, the results are consistent with the assumption that
translocation of one DA molecule brings in one positive charge (9). pH
dependence studies have led to a similar conclusion for the transport
of serotonin by the plasma membrane serotonin transporter (13) and of
norepinephrine by the plasma membrane norepinephrine transporter (14).
For transport of monoamines by the vesicular monoamine transporter, the
issue is still being debated (5). Although the majority of the earlier
studies favored the idea of the neutral forms of monoamines acting as
substrates (for review see Ref. 19), the pH dependence of the uptake of
the positively charged 1-methyl-4-phenylpyridinium cast doubt on this
conclusion (18). Thus, the uptake of 1-methyl-4-phenylpyridinium was
found to have the same pH dependence as that of monoamines, even though
as a quaternary ammonium base it is always positively charged.
Wall et al. (48) have compared pH 6.5 and 8.0 for the
potency of DA in inhibiting
[3H]1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenylpropyl)piperazine
(GBR 12935) binding to rat striatal membranes. They observed a 2-fold greater potency of DA at pH 6.5 than at 8.0, which is opposite to the
present findings for [3H]WIN 35428 binding to the hDAT. A
possible explanation is that the DA interaction with the DAT was
somewhat different in the N-(2-acetomido)-2-iminodiacetate
buffer, used at pH 6.5, and the N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic
acid buffer used at pH 8.0 by Wall et al. (48) as compared
with the sodium phosphate buffer used over the entire pH range in the
present experiments. Factors that are less likely to play a role in the difference between the former findings and the present results are the
species (rat versus human) and the radioligand
([3H]GBR 12935 versus [3H]WIN 35428).
pH-dependent Changes in Interaction between DA and DAT:
Ligand or Transporter?--
pH-induced changes in the interaction
between DA and the DAT could result from changes at the level of the
ligand. For example, introduction of positive charges on the ligand by
protonation may result in conformational changes in the ligand itself.
Alternatively, changes occur at the level of the transporter. For
example, the protonated amine group of DA (and of guanethidine) could
interact with a negatively charged group in the DAT protein as
suggested for the vesicular monoamine transporter by Darchen et
al. (18). If the latter group is ionizable with a
pK ~ 6.8, it would explain the present pattern of
uptake and binding of DA (and binding of guanethidine), which shows an
increase with pH that is less than logarithmic. In this scenario,
bretylium would not interact with the same ionizable group, perhaps
because the distance between the carbon ring and the positively charged
amine is one carbon shorter than in DA and guanethidine. Indeed, on the
basis of extensive structure-activity studies on the inhibition of
neuronal norepinephrine uptake by norepinephrine congeners and
tricyclic inhibitors, Maxwell et al. (25) have postulated
the phenyl ring and the N+ as two key elements in the
binding of these compounds to the norepinephrine transporter.
Another possibility, again at the level of the DAT, is that the pH
dependence of the interaction of many compounds with the DAT resides in
an overlapping binding domain that is susceptible to pH. It may be
recalled here that there is substantial evidence implicating a shared
binding domain on the DAT for DA, cocaine-like inhibitors, and GBR
12935-like compounds (1, 5, 24, 42, 49, 50). The pH dependence observed
here for the interaction of DA, guanethidine, and WIN 35428 with hDAT
is very similar to that reported previously by us for the interaction
of WIN 35428, cocaine methiodide, and benzocaine (39) and by Andersen
for GBR 12935 (51) with rDAT. Among these compounds, cocaine methiodide is always protonated, benzocaine is always neutral, and the others are
bases with pKa values
8.4 (see Ref. 39). It
could be concluded that a common binding domain (not shared by
bretylium) confers the pH dependence, for example by ionizable groups
(with a pK ~ 6.8) in the DAT, impacting the conformation and
thereby the binding potential. In this scenario it could be considered that either cationic or neutral DA binds with similar affinity and that
the proportion at each pH determines how much of each form is
translocated. Thus, at pH 7.4 most of the DA is in the cationic form,
overwhelmingly resulting in translocation of the cationic form. The
conclusion would be that the charge translocated with each DA molecule
is a function of pH, with less charge translocated at extremely basic
pH values, adding another variable to be taken into account in addition
to membrane potential (9) in comparing observed charge transfers with
expected values based on the stoichiometry Na+:Cl
:DA+ = 2:1:1. Such a
phenomenon may be hard to establish because there is an appreciable
leak conductance associated with the DAT, which may readily carry
protons or otherwise be sensitive to pH changes, possibly by a
pH-dependent ion selectivity (9).