(Received for publication, September 22, 1995; and in revised form, November 7, 1995)
From the
The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used electrophysiological, radiotracer flux, and
electron microscopic methods to characterize the biophysical
properties, kinetics, and specificity of the transporter. Uptake of
alanine was H-dependent increasing from 14
pmol/oocyte/h at 0.032 µM H
to 370
pmol/oocyte/h at 10 µM H
. AAP1 was
electrogenic; there was an amino acid-induced depolarization of the
oocyte plasma membrane and net inward currents through the transporter
due to the transport of amino acids. AAP1 transported a wide spectrum
of amino acids favoring neutral amino acids with short side chains. The
maximal current (i
) for alanine, proline,
glutamine, histidine, and glutamate was voltage- and
[H
]
-dependent.
Similarly, the i
was voltage- and [amino
acid]
-dependent. The i
for both H
and amino acid were dependent on the
concentrations of their respective cosubstrates, suggesting that both
ligands bind randomly to the transporter. The K
of the transporter for amino acids decreased as
[H
]
increased and was
lower at negative membrane potentials. The K
for
H
was relatively voltage-independent and decreased as
[amino acid]
increased. This positive
cooperativity suggests that the transporter operates via a simultaneous
mechanism. The Hill coefficients n for amino acids and
H
were >1, suggesting that the transporter has more
than one binding site for both H
and amino acid.
Freeze-fracture electron microscopy was used to estimate the number of
transporters expressed in the plasma membrane of oocytes. The density
of particles on the protoplasmic face of the plasma membrane of oocytes
expressing AAP1 increased
5-fold above water-injected controls and
corresponded to a turnover number 350 to 800 s
.
In plants, amino acid transport is essential for the
redistribution of nitrogen. Amino acids are transported within the
phloem and xylem from their site of synthesis (roots and leaves) to
organs that are net importers of nitrogen such as leaves, seeds, and
tubers. Likewise, amino acids resulting from the degradation of storage
proteins during germination are exported via the vascular system to
supply the developing plant. Studies using whole tissues, individual
cells, protoplasts, and isolated plasma membrane vesicles showed that
amino acid transport in plants is mediated by specific membrane
transport proteins that couple the electrochemical potential gradient
of H to secondary active accumulation of amino acids
(Reinhold and Kaplan, 1984; Bush and Langston-Unkefer, 1988; Li and
Bush, 1990, 1991, 1992; Williams et al., 1990, 1992; Weston et al., 1994; Bush, 1993).
In recent years, molecular biological techniques, whereby yeast amino acid transport mutants are complemented with higher plant cDNA libraries, have enabled the genes encoding amino acid transport proteins to be isolated (Frommer et al., 1993; Hsu et al., 1993; Kwart et al., 1993; Fischer et al., 1995). One amino acid transporter clone (AAP1) was isolated from Arabidopsis thaliana by complementing a yeast mutant defective in proline uptake and was characterized as a general amino acid transporter (Frommer et al., 1993). Another clone, NAT2, was isolated from Arabidopsis by Hsu et al. (1993), which has >99% identity to AAP1. Both have a predicted molecular mass of 53 kDa, are highly hydrophobic, and contain 10-12 putative membrane-spanning regions. When expressed in yeast both transporters exhibited saturable, concentration-dependent amino acid transport and had a broad substrate specificity.
Direct and
detailed analysis of the transport properties of many cloned
cotransporters has been achieved by their expression in Xenopus oocytes. These include the plant H/hexose
cotransporter (STP1) (Boorer et al., 1994), the plant
K
/H
cotransporter (HKT1) (Schachtman
and Schroeder, 1994), and the mammalian Na
/glucose
cotransporter (SGLT1) (Parent et al., 1992a, 1992b). In this
study, we have expressed AAP1 and NAT2 in Xenopus oocytes and
used electrophysiological, radiotracer flux, and morphometric methods
to characterize their kinetics and substrate specificity. We show that
AAP1 (
)has a broad substrate specificity and is electrogenic
with membrane voltage influencing the apparent K
for protons and amino acids and the maximal current generated by
the transporter. Furthermore, a freeze-fracture analysis of AAP1
expressed in the oocyte plasma membrane allowed us to determine the
turnover number of the transporter.
For kinetic analysis, the amino
acid-induced steady-state currents (i) at each test potential (V) were fitted to using ENZFITTER
software (Elsevier-Biosoft):
where S is the [amino acid]
or [H
]
,
i
is the maximal current for saturating S
, K
is the apparent K
of the substrate (S
giving half the i
), and n is
the Hill coefficient.
The results presented are representative of experiments that were repeated at least twice with oocytes from different donor frogs. All experiments were carried out at 22 °C. Amino acids were purchased from Sigma.
Fig. 1A shows membrane potential changes in
an oocyte expressing AAP1 in response to changing
[H]
and the addition of amino
acids. Addition of 1 mM alanine at 10 µM H
depolarized the oocyte membrane potential by 71
mV (-42 mV to 29 mV). The depolarization induced by 1 mM histidine was less (20 mV). These results indicated that there was
a substrate-induced net inward movement of positive charge. Decreasing
the [H
]
from 10 to 0.032
µM and removal of organic substrate from the transport
buffer restored the oocyte resting potential. The same oocyte was
clamped at -50 mV, and currents were recorded after increasing
[H
]
and adding amino acids to
the bathing medium (Fig. 1B). Addition of 1 mM alanine induced an inward current (
250 nA), which returned to
base-line levels after washing with alanine-free buffer at 0.032
µM H
. The inward current induced by
histidine was less (
45 nA). A membrane potential depolarization
(
30 mV) and inward current (
20 nA) were also observed when
the [H
]
was increased from 0.032
to 10 µM in the absence of organic substrate
indicating an uncoupled transport of H
through the
transporter. These currents were about 10% of the currents induced by 1
mM alanine at 10 µM H
. Smaller
depolarizations (
18 mV) and inward currents (
5 nA) were also
recorded from control oocytes when the
[H
]
was increased from 0.032 to
10 µM. Amino acid-induced depolarizations and currents
were not observed in water-injected oocytes. There were no
qualitative or quantitative differences in the amino acid-induced
currents when choline in the transport buffer was replaced by
Na
or K
or when Cl
was replaced by gluconate.
Figure 1:
Membrane
depolarization and inward currents induced by alanine and histidine in
an oocyte expressing AAP1. A, changes in membrane potential in
response to the addition of 1 mM alanine and 1 mM histidine to the medium. Addition of 1 mM alanine and 1
mM histidine at 10 µM H depolarized the membrane potential, which was restored after
removing amino acid and reducing the external
[H
] by washing with buffer at 0.032
µM H
. B, inward currents induced
by amino acids in an oocyte voltage-clamped at -50 mV. Addition
of 1 mM alanine and 1 mM histidine in buffer at 10
µM H
induced inward currents that were
fully reversible after washing with buffer at 0.032 µM H
.
Fig. 2shows the current
records from a cRNA-injected oocyte, which were obtained by stepping
the membrane potential from the holding potential (V = -50 mV) to between -150 and 50 mV in 20-mV
increments. At 10 µM H
and in the absence
of organic substrate, the currents consisted of an initial capacitive
transient, which relaxed to a steady-state level 2 ms after the onset
of the voltage pulse (Fig. 2A). Apart from the
capacitive transient, no presteady-state cotransporter currents were
observed. Inward steady-state currents were induced by amino acids at
all applied membrane potentials upon the addition of 10 mM alanine (Fig. 2B). The alanine-dependent
current/voltage (I/V) curve (inset) was
obtained by subtracting the steady-state currents at 40 ms in the
absence of alanine from those recorded in the presence of alanine.
Between -150 and 50 mV, the I/V curve exhibited
a supralinear dependence on voltage.
Figure 2:
Membrane current traces and steady-state I/V relationship obtained at 10 µM H before and after the addition of alanine to an oocyte injected
with AAP1 cRNA. The test potentials shown are -150, -130,
-110, -90, -70, -50, and -30 mV and are
the average of 5 sweeps. A, current traces recorded in the
absence of alanine at 10 µM H
. B, current traces recorded after the addition of 10 mM alanine at 10 µM H
. Inward currents
were induced at all applied potentials. The inset shows the
steady-state alanine-dependent I/V curve obtained at
40 ms by subtracting the steady-state currents in the absence of
alanine from those recorded in the presence of 10 mM alanine.
To demonstrate that the inward
current induced by alanine was accompanied by the uptake of alanine,
radiotracer flux experiments were carried out. Table 1shows that
the uptake of 50 µML-[H]alanine in oocytes injected
with cRNA was H
-dependent, increasing from 14 to 371
pmol/oocyte/h as [H
]
was
increased from 0.032 to 10 µM. The uptake of alanine into
water-injected oocytes was H
-independent. There was no
significant difference in the uptake of alanine between cRNA- and
water-injected oocytes at 0.032 µM H
.
The kinetics of alanine transport were studied by measuring the
steady-state alanine-induced currents (obtained 40 ms after the onset
of the voltage pulse) as a function of [alanine] and [H
]
. Fig. 3, 4,
and 5 show the data obtained from a single oocyte and are
representative of data from three oocytes. Alanine-dependent I/V curves were obtained at 1.0, 3.2, and 10
µM H
with [alanine]
between 50 µM and 20 mM, and at 0.5, 1.0,
and 10 mM alanine with [H
]
between 0.032 and 15.8 µM H
. The I/V relationships for alanine at 10 µM H
(Fig. 3A) and 10 mM alanine (Fig. 3B) showed a supralinear dependence
on voltage between 50 and -150 mV. At 10 µM H
, the inward currents increased at each membrane
potential as the [alanine]
increased. Likewise,
at 10 mM alanine
, the inward currents increased at
each membrane potential as the [H
]
increased. I/V curves obtained at 1 and 3.2
µM H
while varying
[alanine]
and at 0.5 and 1.0 mM alanine
while varying [H
]
had the same
qualitative characteristics as the I/V curves shown
in Fig. 3.
Figure 3:
Steady-state current/voltage (I/V) relationships obtained by varying
[alanine] at 10 µM H
and [H
]
at 10 mM alanine. The oocyte membrane was held at
-50 mV, and a pulse protocol was used where test potentials
ranging from 50 to -150 mV were applied for 40 ms. Currents
recorded in the absence of alanine were subtracted from those recorded
in the presence of alanine to yield the net alanine-induced
steady-state currents. A, I/V curves
obtained as a function of [alanine]
at
10 µM H
. Alanine-induced currents
increased supralinearly as the membrane potential was made more
negative and increased as [alanine]
increased. B, net alanine-dependent I/V curves obtained as a function of
[H
]
at 10 mM alanine. Alanine-induced currents increased supralinearly as the
membrane potential was made more negative and increased as
[H
]
increased.
At each membrane potential (-10 to
-150 mV) the alanine-induced currents were plotted against
[alanine] or
[H
]
, and the
current/concentration curves were fitted to . Fig. 4shows the data obtained at -150 mV. Fig. 4A shows that the H
activation
curves at 0.5, 1.0, and 10 mM alanine saturated at 10
µM H
and were sigmoidal. The alanine
activation curves at 1, 3.2, and 10 µM H
saturated between 5 and 10 mM alanine and were also
sigmoidal (Fig. 4B). The voltage dependence of the
kinetic parameters obtained from the fitted data are shown in Fig. 5.
Figure 4:
H and alanine activation
of alanine transport. At membrane potentials between -10 and
-150 mV the alanine-induced currents were plotted against
[alanine]
or
[H
]
(symbols),
and the current/concentration curves were fitted to . For
clarity of presentation, only current/concentration curves obtained at
-150 mV are shown. A, alanine-induced currents obtained
at 0.5, 1, and 10 mM alanine while varying the
[H
]
between 0.032 and
15.8 µM H
. The voltage dependence of the
kinetic parameters maximal current for H
i
, the apparent affinity for
H
, K
, and the Hill
coefficient, n for H
, are shown in Fig. 5. B, alanine-induced currents obtained at 1, 3.2,
and 10 µM H
while varying
[alanine]
between 0.1 and 20
mM. The i
,
K
, and n for alanine obtained
from the fitted data are shown in Fig. 5.
Figure 5:
Kinetics of alanine transport. These
parameters were obtained by fitting the alanine-induced currents
obtained at each membrane potential as a function of
[H]
and
[alanine]
to . A,
the i
increased supralinearly with
voltage and increased as [H
]
increased. B, the i
increased supralinearly with voltage and increased as
[alanine]
increased. C, as
[H
]
decreased,
K
increased and became more
voltage-dependent. D, K
was
relatively voltage-independent and increased as
[alanine]
decreased. E, the
Hill coefficient n for alanine was between 1 and 2. F, the n for H
was 2 and was
independent of [alanine]
and
voltage.
The maximal current induced by alanine
i exhibited a supralinear dependence on
voltage (Fig. 5A) and, as shown in Table 2,
decreased as [H
]
decreased. The
i
showed the same voltage dependence (Fig. 5B) and decreased as [alanine]
decreased (Table 2). The i
at saturating [H
]
(
1600 nA) was similar to the i
at
saturating [alanine]
(
1400 nA), suggesting a
coupling ratio of 1 H
:1 alanine. Fig. 5C shows that K
was relatively
voltage-independent at 10 µM H
, became
more voltage-dependent as the [H
]
decreased, and increased as [H
]
decreased (Table 3). However, at hyperpolarizing
potentials, K
was almost independent
of [H
]
. The
K
was slightly voltage-dependent at low
[alanine]
, was voltage-independent at 10 mM alanine, and increased as [alanine]
decreased (Fig. 5D; Table 3). Therefore,
the apparent affinity for H
and alanine depends on the
concentration of their respective cosubstrates. The apparent coupling
coefficient n for H
was >1 and was
voltage-independent (Fig. 5F). n for alanine
was voltage-independent and was >1 over the range of
[H
]
tested (Fig. 5E).
The substrate specificity of AAP1 was
determined by measuring steady-state substrate-induced currents at
-150 mV using 10 mM various organic compounds at 10
µM H. Fig. 6shows the
substrate-induced currents normalized with respect to the current
induced by 10 mM alanine. AAP1 exhibits a very broad but
stereospecific substrate specificity preferring L- to D-isomers. Although there were no inward currents induced by
lysine,
-alanine, and 2-methylamino isobutyric acid at 10
mM, 50 mM of these amino acids induced inward
currents, which were 17, 7, and 2% of the alanine-induced current,
respectively. The D-isomers of all the amino acids shown in Fig. 6were tested. Only those that induced an inward current are
shown. Other organic compounds that were not transported included
sucrose, glucose, malate, and the dipeptide glycyl-glycine.
Figure 6:
Substrate specificity of AAP1. Oocytes
were bathed in 10 mM of a variety of substrates at 10
µM H, and steady-state
substrate-dependent currents at 40 ms and -150 mV were recorded.
Substrate-induced currents are expressed as a percentage of the
alanine-induced current at -150 mV
(100%).
To
determine whether amino acids differing in their structure and net
charge in solution have different transport kinetics, we investigated
the kinetics of AAP1 using histidine, glutamate, proline, and
glutamine. Fig. 8A shows that the
i > i
> i
>
i
i
, all of which increased
supralinearly with voltage and decreased as [amino
acid]
decreased (Table 2). The i
for each amino acid decreased with decreasing
[H
]
(Table 2). The K
values for histidine, glutamate, proline, and
glutamine at 10 µM H
were
voltage-dependent and increased as the membrane potential was made more
positive (Fig. 7B) and as
[H
]
decreased (Table 3).
The K
at 10 mM of each amino acid
was voltage-independent (Fig. 7C) and except for
proline increased as [amino acid]
decreased (Table 3). The current/concentration curves for histidine,
glutamate, proline, glutamine, and H
for each amino
acid were sigmoidal (data not shown) with Hill coefficients >1.
Figure 8:
A comparison of the density of
intramembrane particles on the P face of the oolemma of oocytes
expressing AAP1, the sodium-glucose cotransporter SGLT1, and a control
water-injected oocyte. Four freeze fracture replicas were collected
from each oocyte, and the particle density was obtained from 8.22
µm of uninterrupted P face. A, particle
density in the protoplasmic fracture face (P) of the plasma
membrane from a control water-injected oocyte. The density was 212
± 43 particles/µm
. B, particle density
in the P face of an oocyte expressing AAP1. This oocyte had a density
of 1037 ± 136 particles/µm
. C, particle
density in the P face of an oocyte expressing SGLT1. The density of the
intramembrane particles was 4,121 ± 950/µm
. In
all three cases the magnification was
100,000
.
Figure 7:
Kinetics of glutamate, glutamine, proline,
and histidine transport. A, i of
alanine, glutamine, proline, glutamine, and histidine obtained from a
single oocyte. The I/V curves were obtained at
saturating [H
]
and
[amino acid]
(10 µM H
and 50 mM amino acid). B,
voltage dependence of K
for histidine,
glutamate, glutamine, and proline obtained at 10 µM H
. Steady-state amino acid-induced currents were
obtained by varying [histidine]
,
[glutamate]
,
[glutamine]
, and
[proline]
at 10 µM H
. Steady-state amino acid-induced currents at
membrane potentials between -10 and -150 mV were fitted as
a function of [amino acid]
using , and K
values for amino acids were
determined from the fitted data. C, voltage dependence of
K
obtained at 10 mM histidine,
glutamate, glutamine, and proline by varying
[H
]
at 10 mM each amino acid and fitting the data as a function of
[H
]
to .
To obtain an estimate of the number of transporters/oocyte and the
turnover number of AAP1, we examined the density of particles on the
protoplasmic face (P face) and external face of the plasma membrane of
oocytes expressing AAP1 after recording
i. Fig. 8shows the density of
particles on the P face of the plasma membrane of a water-injected
control oocyte (A), an oocyte expressing AAP1 (B),
and, for comparison, an oocyte expressing the rabbit
Na
/glucose cotransporter SGLT1 (C). The
density of particles in the P face of the control oocyte was 212
± 43 particles/µm
(Fig. 8A);
similar values were obtained for all controls. The density of particles
in the P face of the oocyte expressing AAP1 was 1037 ± 136
particles/µm
. The capacitance of this oocyte was 297
nF, which corresponds to an oocyte surface area of 2.97
10
µm
(assuming a capacitance of 1
µF/cm
) from which was calculated the total number of
particles (2.4
10
/oocyte). The turnover number of
AAP1 was calculated using the equation i
= kzeN
, where i
= maximal current induced by alanine at -150 mV
(2712 nA), k = turnover number, z =
number of charges per transport cycle (2) , e =
elementary charge, and N
= number of
transporters per oocyte. These values yield a turnover number of
350 s
. Turnover numbers of 580 and 800
s
were calculated for two other oocytes expressing
AAP1. The particle density of the oocyte expressing rabbit SGLT1 (Fig. 8C) was 4,121 ± 950/µm
,
and the i
was 515 nÅ. This
corresponds to a turnover number of
11 s
.
Expression of AAP1 and SGLT1 did not increase the density of particles
(
900/µm
) on external faces data not shown.
Expression of the H/amino acid cotransporter
(AAP1) from A. thaliana in Xenopus oocytes has
allowed us to directly determine the substrate specificity and kinetics
of the transporter using electrophysiological, radiotracer flux, and
electron microscopic methods. Amino acid transport was
H
-driven, and the electrogenicity of the transporter
was demonstrated by amino acid-induced depolarizations of the oocyte
plasma membrane and amino acid-induced inward currents, which were
accompanied by the uptake of amino acids into the oocyte.
AAP1
transports a wide spectrum of amino acids and is stereospecific,
preferring L- to D-isomers. Neutral amino acids with
short side chains were preferred by the transporter and include
cysteine, alanine, leucine, serine, and glycine. Amino acids with
longer side chains such as citrulline and methionine and those with a
-phenyl (phenylalanine),
-amide (asparagine),
-amide
(glutamine), and a
-carboxyl (glutamate) were also transported.
AAP1 did not so readily transport amino acids containing an imidazole
side chain (histidine),
-methyl groups (threonine, valine, and
isoleucine), and cyclic amino acids (proline and hydroxyproline). The
basic amino acids arginine, ornithine, and lysine, which have
-guanido,
-amino groups, and an
-amino group,
respectively, were transported poorly. The
-amino group is
important for substrate recognition;
-alanine and 2-methylamino
isobutyric acid were transported poorly, and
-aminobutyric acid
was not. These observations confirm and extend results obtained by
expression of AAP1 in yeast (Frommer et al., 1993; Hsu et
al., 1993; Kwart et al., 1993; Fischer et al.,
1995).
A detailed kinetic investigation of AAP1 was undertaken using
alanine, glutamine, glutamate, histidine, and proline, which differ
either in their structure and/or net charge in solution. All induced
inward currents through the transporter, the magnitude of which
increased as [H]
and [amino
acid]
increased and as the membrane voltage was made
more negative. This is a direct demonstration that AAP1 operates via a
H
-coupled transport mechanism. The transporter
exhibited saturation kinetics with respect to
[H
]
and [amino
acid]
but not to voltage. Amino acid-induced currents
may saturate with respect to membrane voltage in planta where
the membrane potential that the transporter senses is probably more
negative than -150 mV. For example, in a study of nitrate
transport in Arabidopsis roots, the cell-resting potential was
200 mV, and I/V curves were supralinear at
-150 mV and did not saturate until -250 mV (see Fig. 5B) (Meharg and Blatt, 1995).
The net charge of
amino acids in solution depends on the pK of their
side groups. Greater than 99% of alanine, proline, and glutamine in
solution are zwitterions over the range of
[H
]
(0.032-10
µM) used in the experiments presented here. About 95, 24,
and 9% of histidine and 0.06, 5, and 15% of glutamate are zwitterions
at 0.032, 3.2, and 10 µM H
, respectively;
the majority of the other charged species of histidine and glutamate
bear a single positive charge or a single negative charge,
respectively. In view of the structural and charge differences between
these amino acids, it is interesting that the kinetic properties of the
transporter for each amino acid were qualitatively similar. 1) The
current/voltage curves did not saturate at negative membrane
potentials. 2) The i
decreased with
decreasing [H
]
. 3) The
i
for each amino acid decreased with
decreasing [amino acid]
. 4) The
K
decreased as
[H
]
decreased. 5) The K
was voltage-dependent and decreased as the
membrane potential was made more negative. 6) Except for proline, the
K
decreased as [amino acid]
decreased. 7) At 10 mM amino acid
K
was voltage-dependent and became slightly more
voltage-dependent at lower [amino acid]
. 8) The
substrate-activation curves for both ligands (H
and
amino acid) were sigmoidal with apparent coupling coefficients >1.
These similarities suggest that the transport mechanism for each amino
acid is the same.
We propose a kinetic model of amino acid transport
by AAP1 (Fig. 9). In this mechanism, 2 H or 2
substrate molecules (S) bind to the transporter C
at the external face of the membrane to form CH
or
CS
. These intermediates then bind S
or
H
, respectively, to form the complex
CH
S
. A conformational change allows H
and S
to be transported to the cytoplasmic surface
where they dissociate. The transport cycle is completed when the empty
transporter C
undergoes another conformational
change allowing the ligand binding sites to face the external surface.
The broken lines between [CH
]
and
[CH
]
indicate possible uncoupled
transport of H
. The rationale for this mechanism is as
follows. The H
and amino acid activation curves for
all the amino acids tested were sigmoidal with Hill coefficients for
H
and amino acids >1. To simplify the transport
model, we assume that the Hill coefficients for H
and
amino acid are 2. Thus, we propose that 2 H
and 2
substrate molecules bind to the transporter per transport cycle. The
stoichiometry of transport could not be determined from reversal
potentials as the currents did not reverse over the voltage range used
in the experiments. Uncoupled transport of H
is
proposed based on the observation that increasing
[H
]
in the absence of amino
acids induced inward currents that were significantly higher than those
induced in water-injected controls; the depolarizations observed in
control oocytes are due to the H
inhibition of an
outward K
current (Burckhardt and
Frömter, 1992). However, without a specific blocker
of amino acid transport, we are unable to draw conclusions from this
observation in the present study. Uncoupled transport of Na
occurs through the Na
/glucose cotransporter
(Umbach et al., 1990; Parent et al., 1992a, 1992b)
and the serotonin transporter (Mager et al., 1994). There is
no significant leak for amino acid through AAP1; flux experiments
showed that at low [H
]
there was
no uptake of amino acid above water-injected controls.
Figure 9:
Kinetic model for H-amino
acid cotransport. An 8-state random/simultaneous mechanism of transport
for AAP1 is proposed. [H
]
and [S
]
(where S
= substrate) bind randomly to
the empty transporter [C]
to form the
intermediates [CH
]
or
[CS
]
.
[S
]
or
[H
]
then bind to
[CH
]
or
[CS
]
, respectively, to form
the fully loaded transporter complex
[CH
S
]
.
[CH
S
]
crosses
the membrane where [H
]
and
[S
]
dissociate. The empty
transporter [C]
then reorientates in the
membrane to complete the transport cycle. Possible uncoupled transport
of H
through the transporter is indicated by the dashed lines.
A random versus an ordered, and a simultaneous versus a
sequential mechanism of transport, are evaluated using the kinetic
parameters obtained with alanine. Fig. 10shows a replot of the
data presented in Fig. 6. The K decreased at all membrane potentials as
[H
]
became saturating (Fig. 10A). Likewise, the K
decreased as [alanine]
became saturating (Fig. 10B). The increase in the apparent affinity of
the transporter for H
and amino acid as the
concentration of their respective cosubstrates increased suggests
positive cooperativity between both ligands for binding to their
respective sites on the transporter. This cooperativity suggests that
both ligands must be bound to the transporter before the
transporter-H
-amino acid complex crosses the membrane, i.e. that the transporter operates via a simultaneous
mechanism (Jauch and Läuger, 1986). The
i
increased as
[H
]
increased at all membrane
potentials (Fig. 10C), and i
increased as [alanine]
increased (Fig. 10D). The i
for both
H
and amino acid was dependent on the concentrations
of their respective cosubstrates, which would suggest a random binding
of substrates to the transporter (Jauch and Läuger,
1986). Results presented in Table 2and Table 3suggest
that the same mechanism applies to all the amino acids tested. There
was no decrease in K
when
[proline]
was increased from 1 to 10 mM,
probably because 10 mM proline is not saturating.
Figure 10:
Rationale for an ordered versus random and a simultaneous versus sequential mechanism of
transport. This figure is a replot of the data shown in Fig. 5, A-D. For clarity, the error bars have been
omitted, and only three membrane voltages are shown (-30,
-70, and -110 mV). A, K decreased as [H
]
increased at each membrane potential. B,
K
decreased as [alanine]
increased. C, i
increased as [H
]
increased. D, i
increased
as [alanine]
increased.
AAP1
transports neutral, basic, and acidic amino acids. The
i > i
> i
>
i
i
and the shape of the I/V curves are the same.
This suggests that the mechanism of transport for each amino acid is
the same and that differences in i
are due to
changes in a rate-limiting step in the transport cycle, possibly the
translocation rate of the fully loaded transporter
([CH
S
]) or any of the dissociation
steps on the inside of the cell. These rates cannot be determined from
measurement of inward currents in intact oocytes. The I/V curves did not saturate between -150 and 50 mV, and this was
independent of [H
]
and
[alanine]
. Therefore, at least one rate-limiting
step in the transport mechanism is potential-dependent over this
voltage range. There is a voltage dependence of K
and K
at low concentrations
of amino acid and H
, respectively, which decreases as
the concentration of ligands increases. Therefore, part of the effect
of voltage is to enhance the affinity of the transporter for
substrates. Even at saturating [H
]
and [amino acid]
, there is a voltage
dependence of i
. Therefore, reaction steps other
than H
and amino acid binding are voltage-dependent,
possibly the conformational changes of the empty and loaded
transporters.
Previous studies have shown that neutral, basic, and
acidic amino acids are transported by different mechanisms within the
plant. For example, in Chlorella (Cho and Komor, 1983) and
sugar cane cells (Wyse and Komor, 1984), neutral amino acids were
accompanied by an influx of H and an efflux of
K
. Wyse and Komor(1984) observed that basic amino
acids were driven by the negative membrane potential and were not cotransported with H
and that acidic amino acids
were accompanied by the uptake of 2 H
and the release
of K
. Kinraide and Etherton(1980) obtained similar
results with amino acid transport in oat coleoptiles. Unlike plant
cells where the interpretation of amino acid transport data is
complicated by the presence of a variety of transport systems,
overexpression of AAP1 in oocytes shows that neutral, basic, and acidic
amino acids are transported by one protein by the same mechanism. Due
to the qualitative similarities in their transport kinetics, we have
assumed that the zwitterionic species of alanine, proline, glutamine,
histidine, and glutamate are transported. This assumption alters the K
for histidine and glutamate, but the
dependence of K
on
[H
]
remains the same. Therefore,
if 5 and 15% of glutamate is zwitterionic at 3.2 and 10 µM H
, respectively, the K
will be 0.15 and 0.12 mM instead of
3.0 and 0.8 mM. Likewise, for histidine where 24 and 9% is
zwitterionic at 3.2 and 10 µM H
, the
K
will be 2.5 and 0.6 mM instead of 10.4 and 6.8 mM.
Freeze fracture electron
microscopic methods have been used to determine the unitary functional
capacity of various transport proteins including the
Na/glucose cotransporter from rabbit small intestine
(SGLTl) and the water channels MIP and CHIP (Zampighi et al.,
1995). These investigators showed that the expression of cloned
transporters increased the density of particles in the P face but not
in the external face of the oocyte plasma membrane. Expression of AAP1
in oocytes also increased the density of particles in the protoplasmic
fracture face of the oocyte plasma membrane (4-5-fold above
water-injected controls). The turnover number of 350 to 800
s
was calculated assuming a valence of 2 (2
charges/transport cycle) and that all particles expressed in the oocyte
were transporting substrate. This turnover number is an underestimate
because the i
did not saturate with respect to
membrane voltage. The turnover number calculated for AAP1 is higher
than that obtained for other cloned cotransporters. Using electrical
methods, a turnover number of 59 s
was determined
for the H
/hexose cotransporter (STP1) (Boorer et
al., 1994), 60 s
for human SGLT1 (Loo et
al., 1993), and 25 s
for rabbit SGLT1
(Panayotova-Heiermann et al., 1994).
In conclusion,
transport by AAP1 is relatively fast compared to other cloned
cotransporters and is H-dependent and electrogenic
with membrane voltage enhancing the maximal transport rate and the
affinities for H
and amino acid. Future kinetic
studies of other members of the AAP gene family, in conjunction with
site-directed mutagenesis of AAP1, will enable us to identify the amino
acid residues involved in substrate and H
binding and
in voltage regulation.