(Received for publication, June 2, 1995; and in revised form, August 17, 1995)
From the
To identify the residues involved in substrate recognition by
recently cloned vesicular monoamine transporters (VMAT1 and VMAT2), we
have mutagenized the conserved residues in a cytoplasmic loop between
transmembrane domains two and three of VMAT2. Although studies of
related bacterial antibiotic resistance proteins indicate an important
functional role for this region, we found no effect of these mutations
on VMAT2 activity. However, replacement of aspartate 33 in the first
predicted transmembrane domain with an asparagine (D33N) eliminates
transport. D33N shows normal levels of expression and normal binding at
equilibrium to the potent inhibitor reserpine. However, in contrast to
wild-type VMAT2, serotonin inhibits reserpine binding to D33N very
poorly, indicating a specific defect in substrate recognition.
Replacement of three serine residues in transmembrane domain three with
alanine (Stmd3A) shows a similarly selective but even more profound
defect in substrate recognition. The results suggest that by analogy to
receptors and plasma membrane transporters for monoamines, the cationic
amino group of the ligand interacts with an aspartate in the first
transmembrane domain of VMAT2 and hydroxyl groups on the catechol or
indole ring interact with a group of serines in the third transmembrane
domain. Importantly, D33N and Stmd3A retain coupling to the proton
electrochemical gradient as measured by the
µ
-induced acceleration of
reserpine binding. This indicates that substrate recognition can be
separated from coupling to the driving force.
Synaptic transmission involves the regulated release of neurotransmitter and its interaction with post-synaptic receptors to transduce the physiological signal. Regulated release generally occurs through the exocytosis of specialized secretory vesicles filled with neurotransmitter. Since neurotransmitters appear in the cytoplasm after either synthesis or reuptake from the synaptic cleft, storage in these vesicles requires transport from the cytoplasm. In addition to packaging the transmitter for regulated release, transport into vesicles lowers the cytoplasmic concentration and so promotes uptake at the plasma membrane. The lowered cytoplasmic concentration also reduces the exposure of other organelles to potential neurotoxins that are substrates for vesicular transport.
Classical studies have indicated
four distinct types of vesicular transporter activity: one for
monoamines, another for acetylcholine, a third for glutamate, and a
fourth for -aminobutyric acid and glycine (Sudhof and Jahn, 1991;
Edwards, 1992; Schuldiner et al., 1995). Mechanistic studies
have focused on the vesicular transport of monoamines since bovine
adrenal chromaffin granules provide an abundant source of this
activity. In addition, the antihypertensive drug reserpine and the
related but more centrally acting drug tetrabenazine appear to
interfere specifically with vesicular monoamine transport. Similar to
other vesicular transport activities, monoamine transport into vesicles
depends on an electrochemical gradient generated by the vacuolar
H
-ATPase (Rudnick, 1986). This ATPase pumps protons
into the lumen of the vesicle. The vesicular amine transporter then
exchanges two protons in the lumen of the vesicle for one monoamine in
the cytoplasm (Njus et al., 1986; Johnson, 1988; Kanner and
Schuldiner, 1987). While the molecular mechanism by which
pH
drives active transport has remained unknown, recent cloning of the
cDNAs encoding two vesicular monoamine transporters (VMATs)
(Liu et al., 1992b) now enables us to determine the
structural basis for transport activity.
A cDNA clone encoding
monoamine transport into chromaffin granules (VMAT1) was originally
isolated using selection in the neurotoxin 1-methyl-4-phenylpyridinium
(MPP) (Liu et al., 1992b). The transporter
protects against MPP
by sequestering the toxin within
vesicles, away from its primary site of action in mitochondria (Liu et al., 1992a). Although classical studies had suggested a
single vesicular monoamine transporter in both the adrenal gland and
the central nervous system (Henry and Scherman, 1989; Scherman, 1986),
VMAT1 sequences do not appear in the brain. Subsequent screening of a
brainstem cDNA library led to the isolation of a highly related
sequence that is expressed by multiple central monoamine populations
(VMAT2) (Liu et al., 1992a, 1992b; Erickson et al.,
1992). The nucleotide sequences of VMAT1 and VMAT2 predict proteins
with 12 transmembrane domains. In the first six predicted transmembrane
domains, VMAT1 and VMAT2 show weak sequence similarity to a class of
bacterial drug resistance transporters that includes the tetracycline
resistance genes of pBR322 and Tn10 and a bacterial multi-drug
resistance transporter (Nguyen et al., 1983; Neal and Chater,
1987). Interestingly, this class of bacterial proteins also pumps
toxins out of the cell interior and appears to act through proton
exchange (Kaneko et al., 1985). Reserpine, a potent inhibitor
of the VMATs, also inhibits the bacterial multi-drug resistance
transporter (Neyfakh et al., 1991). Thus, the weak similarity
in structure appears to reflect a definite similarity in function
between the VMATs and these bacterial proteins.
To study the
function of the vesicular monoamine transporters, we have developed
several biochemical assays. First, expression of the transporters in a
variety of heterologous cell systems confers easily detectable
transport activity (Liu et al., 1992b; Peter et al.,
1994). The transporters contain signals that direct them to an acidic
endosomal compartment (Liu et al., 1994) and the pH gradient
across the endosomal membrane apparently supports functional transport
in a preparation of membranes from the transfected cells. Using a
quantitative transport assay, we have characterized the functional
properties of VMAT1 and VMAT2, including the affinity for substrates
and potency of inhibitors, and identified several significant
differences between the two proteins. Second, the transporter expressed
in the same heterologous systems binds with high affinity to the potent
inhibitor reserpine (Schuldiner et al., 1993). Monoamines
inhibit reserpine binding with potencies similar to their affinity for
transport, strongly supporting the hypothesis that reserpine binds at
the site of substrate recognition. Furthermore, the presence of
µ
accelerates reserpine binding
in both bovine chromaffin granules and these heterologous systems,
suggesting that the drug interacts with a conformation of the
transporter that faces the outer, cytoplasmic face of the membrane
(Weaver and Dupree, 1982; Schuldiner et al., 1993). Binding to
reserpine and tetrabenazine has also enabled us to estimate transporter
number and so calculate the rate of turnover (Peter et al.,
1994). Third, we have raised antibodies to the C terminus of both
proteins that enable us to detect them by a variety of methods
including Western analysis.
Although VMAT1 and VMAT2 show little
primary sequence similarity to other mammalian proteins, the vesicular
transporters may interact with monoamine substrates through particular
residues also present in proteins that recognize monoamines such as G
protein-coupled receptors and plasma membrane transporters. Using a
combination of site-directed mutagenesis, heterologous expression, and
the biochemical assays described above, we have now identified several
residues specifically involved in substrate recognition that do not
interfere with the overall structure of the transporters, their ability
to bind drugs, or their coupling to
µ
.
Vesicular monoamine transport involves recognition of the
appropriate substrates and their translocation across the membrane in
exchange for two protons (Njus et al., 1986; Johnson, 1988).
More specifically, the transporter recognizes monoamines on the
cytoplasmic face of the membrane and undergoes a conformational change
that deposits the substrate in the lumen of the vesicle. The proton
electrochemical gradient across the vesicle membrane then appears to
restore the original, cytoplasmically oriented conformation of the
transport protein (Schuldiner et al., 1995). Thus, the
conformational changes that link substrate movement to
µ
presumably account for active
transport. Since translocation across the membrane must involve changes
in the site of substrate recognition, we have studied first the
residues in VMAT2 that interact with monoamines.
Although the vesicular monoamine transporters define a novel mammalian gene family, we have used their relationship to bacterial antibiotic resistance transporters to guide the construction of site-directed mutants. The bacterial proteins also appear to act through proton exchange to drive the extrusion of toxins (Kaneko et al., 1985). However, they translocate a set of substrates clearly distinct from monoamine transmitters. Thus, the bacterial transporters and VMATs presumably differ in the site of substrate recognition but not in other aspects of the translocation mechanism.
Figure 1: Site of mutations in VMAT2. The sites of mutations in VMAT2 are indicated on the predicted secondary structure of the first six transmembrane domains. Circles represent individual amino acids. Aspartate (D) 33 in the predicted first transmembrane domain was mutagenized to glutamate and asparagine. In the region between transmembrane domains two and three, two glycines (G) flanking the loop were mutagenized to leucine, threonine (T) 154 was replaced with alanine, and asparagine (N) 155 was changed to aspartate and glutamine. The three serine residues (S) clustered at the end of transmembrane domain three were simultaneously mutagenized to alanine, as were two pairs of serine residues in the fourth transmembrane domain.
Figure 2:
The cytoplasmic loop between transmembrane
domains two and three. Membranes prepared from COS cells transfected
with wild-type and mutant cDNAs were incubated at 29 °C in
sucrose-Hepes (SH) buffer containing 2.5 mM ATP and
20 nM [H]serotonin. Radioligand bound to
the membranes was separated from free neurotransmitter by filtration,
and uptake at 0 °C was subtracted as background. Transport was
carried out for 2 min to indicate the initial, maximal rate of uptake.
The results show that all three mutants confer activity equivalent to
wild-type.
Figure 6:
Equilibrium reserpine binding to D33N,
N155D, and Stmd3A. Membranes prepared from COS cells transfected with
mutant and wild-type cDNAs were incubated for 5 min at 29 °C in SH
buffer containing 5 mM ATP and 2 nM [H]reserpine and the bound drug separated
from free by rapid gel filtration. [
H]Reserpine
bound is expressed as the percent bound by wild-type VMAT2. The two
mutants that show no transport activity bind reserpine approximately as
well as the wild-type protein.
Directly adjacent to asparagine 155 in the same hydrophilic region of VMAT2 between predicted transmembrane domains 2 and 3, threonine 154 is a potential site for phosphorylation by protein kinase C. Phosphorylation at this site might then contribute the negative charge required for transport even in the presence of asparagine 155. If phosphorylation of this residue were critical for transport, mutation of this residue to an amino acid that cannot be phosphorylated would reduce or eliminate activity. For this reason, we changed threonine 154 to alanine (T154A) (Fig. 1). As shown in Fig. 2, transport activity remains normal, indicating that phosphorylation of this residue is not required for transport activity.
In addition to a potential role in substrate recognition, the cytoplasmic loop between transmembrane domains 2 and 3 has been suggested to play a critical role in restricting the access of the substrate to alternating sides of the membrane during the transport cycle (Yamaguchi et al., 1992b). Within the bacterial transporters, the loop motif GXXXXRXG is conserved and can also be found in the VMATs (Liu et al., 1992b; Erickson et al., 1992). Replacement of the glycines by more bulky residues abolishes the activity of the Tn10 tetracycline transporter, supporting the hypothesis that these glycines act as a hinge for the domain (Yamaguchi et al., 1992b). However, replacement of the analogous glycine residues in VMAT2 with leucines (G151L and G158L) (Fig. 1) does not affect transport (data not shown). Thus, multiple residues in this cytoplasmic loop fail to play as crucial a role in vesicular amine transport as they do in tetracycline transport encoded by the Tn10 antibiotic resistance gene.
To determine the role of aspartate 33 in the first
transmembrane domain of VMAT2, we have replaced the residue with
glutamate (D33E) and asparagine (D33N) (Fig. 1). The
conservative mutation D33E, which retains the negative charge,
significantly reduces but by no means abolishes transport activity (Fig. 3). Measurement of the K further
indicates no significant effect on apparent substrate affinity (Table 1). However, elimination of the negative charge in the
D33N mutant essentially abolishes transport ( Fig. 3and Fig. 4), indicating that negative charge at this position is
crucial for function.
Figure 3: Mutations D33N and Stmd3A eliminate transport activity. Membranes prepared from COS cells transfected with wild-type and mutant cDNAs were incubated for 2 min as described in Fig. 2. Mutants D33E and Stmd4A show activity, although substantially reduced from wild-type levels. However, D33N and Stmd3A show no appreciable transport activity.
Figure 4:
Time course of transport by wild-type and
mutant VMAT2 proteins. Membranes prepared from COS cells transfected
with wild-type and mutant cDNAs were incubated for varying intervals
with [H]serotonin as described in Fig. 2.
Membranes from cells transfected with wild-type VMAT2 show increasing
accumulation of serotonin with time. The mutants D33N and Stmd3A show
no transport activity.
, wild-type VMAT2;
, D33N;
,
Stmd3A.
To address the possibility that aspartate 33
plays a structural role in VMAT2 rather than a direct role in the
transport cycle, we have assessed the stability and general structure
of the protein by Western analysis and drug binding. Using an antibody
to the C terminus of VMAT2 (Peter et al., 1995), Western
analysis shows that COS cells expressing the mutant protein show the
same electrophoretic mobility as wild-type VMAT2 (Fig. 5).
Moreover, the level of expression appeared approximately the same as in
cells transfected with the wild-type construct. We further assessed the
overall structure of the transporter by measuring the binding to
[H]reserpine. In contrast to the antibody, which
recognizes only the primary sequence, reserpine presumably interacts
with multiple sites on the protein in a way that depends on appropriate
folding. At equilibrium, reserpine binding to membranes prepared from
cells transfected with D33N appeared normal (Fig. 6). Since
µ
accelerates reserpine binding
to the VMATs (Weaver and Dupree, 1982; Rudnick et al., 1990),
the sensitivity of reserpine binding to
µ
can be used to determine whether VMAT2 remains coupled to
the driving force. To determine whether D33N retains coupling to
pH, we therefore measured reserpine binding as a function of time
in the presence and absence of the proton ionophore
carbonylcyanide-m-chlorophenylhydrazone (CCCP). Fig. 7demonstrates that [
H]reserpine binds
to D33N with an approximately normal time course in the presence of ATP
to generate
µ
. CCCP slows the
binding essentially to background over the time course shown in Fig. 7, indicating that D33N remains coupled to
µ
. In light of these results, it
seems very unlikely that D33N disturbs some basic aspect of VMAT2
structure.
Figure 5: Western analysis of wild-type and mutant VMAT2 proteins. Approximately 20 µg of total protein from membrane preparations of COS cells transfected with wild-type VMAT2, D33N, and Stmd3A was separated by electrophoresis through 10% SDS-polyacrylamide and the proteins transferred to nitrocellulose. The blot was then stained with an antibody raised against the C terminus of VMAT2 (Peter et al., 1995), followed by a secondary anti-rabbit antibody conjugated to peroxidase, and the reaction was visualized by chemiluminescence. The VMAT2 protein appears as a doublet with a molecular mass of approximately 60 kDa. The Western blot shows equivalent expression of the mutants D33N and Stmd3A to wild-type VMAT2.
Figure 7:
D33N remains coupled to
µ
. Membranes prepared from COS
cells transfected with the D33N mutant were incubated with
[
H]reserpine as described in Fig. 6for
the times indicated and binding at zero time subtracted as background.
The mutant D33N bound to [
H]reserpine with a
normal time course in the presence of
µ
. The presence of the proton
ionophore CCCP (5 µM) reduced binding to that seen in the
presence of excess non-radioactive reserpine (2 µM). Thus,
reserpine binding to D33N remains coupled to
µ
.
,
[
H]reserpine;
, + 2 µM reserpine;
, + CCCP.
To determine more directly whether the D33N mutation
interferes with substrate recognition, we have made use of its ability
to bind reserpine. The inhibition of reserpine binding by monoamine
substrates has suggested that reserpine interacts with the site of
substrate recognition. Thus, a disturbance in monoamine recognition may
reduce the potency of monoamines to inhibit reserpine binding. Indeed, Fig. 8demonstrates that the IC for serotonin
inhibition of [
H]reserpine binding in D33N
(>500 µM) differs dramatically from the wild-type
(
2 µM). The results strongly suggest that the D33N
mutation interferes selectively with substrate recognition.
Figure 8:
Competition of
[H]reserpine binding by serotonin. Incubation of
membranes prepared from COS cells transfected with wild-type VMAT2 or
mutant cDNAs was carried out for 5 min as described in Fig. 6in
the presence of varying concentrations of serotonin, and nonspecific
binding measured in the presence of 2 µM reserpine was
subtracted as background. Wild-type VMAT2 showed an IC
for
serotonin of
2 µM. This contrasts strongly with the
IC
values observed for D33N and Stmd3A, both of which
exceed 500 µM. Interestingly, very high concentrations of
serotonin appear to inhibit [
H]reserpine binding
to the mutant D33N, whereas they have no effect on binding to Stmd3A.
, wild-type VMAT2;
, D33N;
,
Stmd3A.
A group of serine residues similar to those found in
monoamine receptors and the plasma membrane dopamine transporter occurs
in the fourth transmembrane domain of VMAT2 (Fig. 1). However,
simultaneous replacement of all these residues (serines 197, 198, 200,
and 201) with alanine (Stmd4A) does not eliminate vesicular amine
transport activity (Fig. 3). Some loss of activity does occur,
but the K is virtually identical to wild-type (Table 1), suggesting that serines located elsewhere in VMAT2
could be involved in substrate recognition.
Another group of serines
occurs in transmembrane domain three of VMAT2 (Fig. 1). To
determine their role in substrate recognition, these three residues
(serine 180, 181, and 182) were simultaneously mutagenized to alanine
(Stmd3A). In membranes prepared from cells transfected with this
mutant, transport activity did not exceed background levels ( Fig. 3and Fig. 4). Western analysis shows that the level
of Stmd3A expression in COS cells is equivalent to wild-type VMAT2 (Fig. 5). Moreover, normal levels of equilibrium
[H]reserpine binding (Fig. 6) indicated
that the disturbance in transport activity does not result from a gross
defect in the folding of VMAT2. Furthermore, a kinetic analysis of
reserpine binding (Fig. 9) in the presence and absence of CCCP
indicated that Stmd3A remains coupled to the driving force
µ
.
Figure 9:
Time
course of [H]reserpine binding by Stmd3A.
Membranes prepared from COS cells transfected with the mutant cDNA
Stmd3A were incubated as described in Fig. 6and Fig. 7.
The time course of [
H]reserpine binding appeared
normal for the mutant Stmd3A in the presence of
µ
, whereas 5 µM CCCP
essentially eliminated the binding, indicating that the Stmd3A mutant
remains coupled to the driving force.
,
[
H]reserpine;
, + cold reserpine;
, + CCCP.
Apparently normal reserpine
binding by mutant Stmd3A further enabled us to determine the effect of
the mutation on substrate recognition. Fig. 8shows that in
striking contrast to wild-type VMAT2, 500 µM serotonin
fails to inhibit [H]reserpine binding to Stmd3A.
The results indicate that serines 180-182 have a specific role in
monoamine recognition.
Comparison of the vesicular monoamine transporters to a class of bacterial antibiotic resistance proteins has suggested a number of residues that may participate in the active transport of monoamines. In particular, previous studies of the tetracycline transporter encoded by Tn10 have implicated residues in the cytoplasmic loop between transmembrane domains two and three (conserved residues are shown in boldface type).
Replacement of an aspartate in this loop of the Tn10 protein with glutamine abolishes transport activity, whereas substitution with glutamate does not eliminate function, indicating the importance of negative charge at this position (Yamaguchi et al., 1990b). Additional mutations in this region of the bacterial protein have suggested that it may act as a gate to restrict access of the substrate to alternating sides of the membrane (Yamaguchi et al., 1992b, 1992c). In contrast, mutations in the cytoplasmic loop between transmembrane domains two and three of VMAT2 indicate a less important role in transport activity. Replacement of an asparagine in this loop of VMAT2 (Asn-155) with the aspartate found in related bacterial proteins does not affect transport activity. Measurement of the apparent affinity for serotonin also shows no change from the wild-type protein. Interestingly, the vesicular acetylcholine transporters from Caenorhabditis elegans, Torpedo, rat, and humans (Alfonso et al., 1993; Roghani et al., 1994; Varoqui et al., 1994; Erickson et al., 1994; Bejanin et al., 1994) contain an aspartate at this site but the role of this difference between vesicular monoamine and acetylcholine transporters remains unclear.
Replacement of the adjacent potential phosphorylation site (Thr-154) with alanine also does not impair function, indicating that phosphorylation at this site is not required for activity. In further contrast to the related bacterial proteins, replacement of conserved glycine residues flanking this loop with more bulky leucine residues does not reduce transport.
The relationship to other mammalian
proteins that recognize monoamines has proven more useful in
identifying residues that interact with substrate. Site-directed
mutagenesis of adrenergic receptors has suggested that a conserved
aspartate in the third transmembrane domain interacts with the
protonated amino group of agonist ligands (Strader et al.,
1987, 1988). An aspartate also occurs in the first transmembrane domain
of the plasma membrane transporters for monoamines but not
-aminobutyric acid or glycine (Amara and Kuhar, 1993), and
mutagenesis of the plasma membrane dopamine transporter has indicated a
role for this residue in substrate recognition (Kitayama et
al., 1992). Interestingly, the bacterial drug resistance proteins
also contain an aspartate in the first transmembrane domain that
appears to play a critical role in transport (McMurry et al.,
1992; Yamaguchi et al., 1992a). An aspartate also occurs in
the first transmembrane domain of both VMAT1 and VMAT2. Conservative
replacement of this residue with glutamate (D33E) impairs but does not
abolish transport activity. Moreover, measurement of the K
indicates a normal apparent affinity for the
substrate. However, elimination of the negative charge by mutagenesis
of the aspartate to asparagine (D33N) abolishes the transport of
serotonin. Western analysis indicates normal levels of D33N expression,
and the measurement of binding to [
H]reserpine
suggests normal folding. Furthermore, kinetic analysis of
[
H]reserpine binding in the presence and absence
of
µ
shows that D33N remains
coupled to the driving force for transport. Interestingly, the
analogous mutation in the Tn10 tetracycline resistance protein also
eliminates transport of a positively charged metal-tetracycline complex
(Yamaguchi et al., 1990a) and also remains coupled to the
driving force (Yamaguchi et al., 1992a). Finally, the failure
of serotonin to inhibit [
H]reserpine binding by
D33N indicates a selective defect in substrate recognition. This could
result from a defect in reserpine binding that renders it less
sensitive to displacement by serotonin. Alternatively, reserpine may
bind normally yet show reduced displacement by serotonin.
In either
case, recognition of the substrate is impaired. Interestingly, the
recent mutagenesis of a conserved histidine toward the C terminus of
the VMAT1 protein eliminates transport function and coupling to
µ
as measured by the acceleration
of reserpine binding (Shirvan et al., 1995), suggesting the
converse defect. However, the extent to which a defect in energetic
coupling influences substrate recognition remains unclear.
Our
results further suggest that monoamines are transported in their
protonated state. At physiologic pH, monoamine substrates for VMAT1 and
VMAT2 exist in both neutral and protonated forms. Previous studies had
suggested that the transport protein recognizes the neutral form (Ramu et al., 1983). However, more recent work has shown the
vesicular transport of permanently charged substrates such as
MPP (Scherman et al., 1988; Daniels and
Reinhard, 1988; Moriyama et al., 1993), strongly suggesting
that the transporter recognizes the protonated form of monoamine
substrates (Schuldiner et al., 1995). The lack of activity by
the D33N mutant of VMAT2 supports the recognition of a protonated
substrate by the wild-type protein.
The -adrenergic receptor
contains serines that appear to interact through hydrogen bonds with
the hydroxyl groups of catecholamine and indoleamine substrates
(Strader et al., 1989). Analogous serine residues occur in the
plasma membrane dopamine transporter and in the fourth transmembrane
domain of VMAT1 and VMAT2 (Kitayama et al., 1992). However,
simultaneous mutagenesis of both pairs of serine residues in the fourth
transmembrane domain of VMAT2 to alanine does not eliminate transport
activity nor does it influence the apparent affinity for substrate. We
then simultaneously replaced a cluster of three serine residues in the
third transmembrane domain with alanine (Stmd3A). This mutation
eliminates all transport activity but shows no reduction in the level
of protein expression or abnormality in equilibrium binding to
[
H]reserpine. In particular, Stmd3A remains
coupled to the proton electrochemical gradient. However, serotonin
inhibits [
H]reserpine binding even less
effectively in Stmd3A than in D33N, indicating another specific defect
in substrate recognition. As in the case of D33N, we have not
determined whether the defect extends to the recognition of reserpine,
but normal equilibrium levels of drug binding and the failure of
serotonin to inhibit binding indicate a problem with substrate
recognition in either case. Interestingly, rat VMAT1 and bovine VMAT2
contain only two serine residues at this site whereas the vesicular
acetylcholine transporters show either one or none, supporting a role
for at least two of the three residues in VMAT2 in substrate
recognition.
In summary, our results show that an aspartate in the first
transmembrane domain of the vesicular monoamine transporters and that
serine residues in transmembrane domain three but not four play a
critical role in substrate recognition, presumably by interacting
respectively with the protonated amino group of the ligand and hydroxyl
groups on the catechol or indole ring. Nonetheless, other regions in
the vesicular monoamine transporters may contribute to substrate
recognition and account for observed differences in apparent affinity
(Peter et al., 1994), as shown recently for plasma membrane
amine transporters (Giros et al., 1994; Buck and Amara, 1994;
Barker et al., 1994). Importantly, changes in coupling to
µ
do not accompany the loss of
substrate recognition in the VMAT2 mutations, indicating that these two
functions can be separated.