©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetics and Specificity of a H/Amino Acid Transporter from Arabidopsis thaliana(*)

(Received for publication, September 22, 1995; and in revised form, November 7, 1995)

Kathryn J. Boorer (1)(§) Wolf B. Frommer (2) Daniel R. Bush (3) Michael Kreman (1) Donald D. F. Loo (1) Ernest M. Wright (1)

From the  (1)Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751, (2)Institut für Genbiologische Forshung, Ihnestrasse 63, 14195 Berlin, Germany, and the (3)United States Department of Agriculture, Agricultural Research Service and Department of Plant Biology, University of Illinois, 196 PABL, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(max)) for alanine, proline, glutamine, histidine, and glutamate was voltage- and [H]-dependent. Similarly, the i(max)^H was voltage- and [amino acid]-dependent. The i(max) 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(0.5) of the transporter for amino acids decreased as [H] increased and was lower at negative membrane potentials. The K(0.5) 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.


INTRODUCTION

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 (^1)has a broad substrate specificity and is electrogenic with membrane voltage influencing the apparent K(0.5) 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.


EXPERIMENTAL PROCEDURES

Molecular Biology Methods

The 3`-untranslated region of NAT2 lacks a poly(A) tail, and that of AAP1 has a poly(A) tail of only 16 nucleotides. We have found that a poly(A) tail of at least 30 nucleotides is necessary for the efficient expression of plant transport proteins in oocytes. Therefore, a poly(A) tail of 70 adenosines was introduced to the 3`-untranslated region of NAT2 and AAP1 as follows. A poly(A) tail of 70 adenosines was inserted between the HindIII and XhoI sites of pBluescript KS+ (Strategene) to yield pKJB1. The EcoRI fragments of NAT2 and AAP1 were inserted into the EcoRI site of pKJB1, and the length of the poly(A) tail was verified by sequencing the 3`-end of the construct using Sequenase version 2.0 kit (U. S. Biochemical Corp.). The resulting plasmids pKNAT2 and pKAAP1 were linearized with KpnI, and capped cRNA was transcribed in vitro using T7 RNA polymerase and an RNA transcription kit (Ambion, Austin, TX).

Preparation of Oocytes

Stage V or VI oocytes from Xenopus laevis were incubated in 5 mg/ml collagenase-B (Boehringer Mannheim) for 1 h at 22 °C and defolliculated by incubation in 100 mM K(2)HPO(4), 0.1 g/ml bovine serum albumin for 1 h with gentle agitation. Oocytes were maintained at 18 °C in Barth's medium containing (in mM) 88 NaCl, 1 KCl, 0.33 Ca(NO(3))(2), 0.41 CaCl(2), 0.82 MgSO(4), 2.4 NaHCO(3), 10 HEPES, 0.1 mg/ml gentamycin, pH 7.4, before and after injection of 50 ng (1 µg/µl) of cRNA.

Solutions

Xenopus oocytes possess endogenous amino acid transporters, which function either as facilitators or are driven by the electrochemical gradient for Na (Campa and Kilberg, 1989; Taylor et al., 1989). Although these activities are low, experiments were done in the absence of Na to avoid background activity due to endogenous Na-dependent amino acid transporters. Radiotracer uptake experiments and electrophysiological recordings were done in transport buffer containing (in mM) 100 choline chloride, 2 KCl, 1 CaCl(2), 1 MgCl(2), 10 PIPES, (^2)and 10 HOMOPIPES. The [H](o) of the bathing medium was varied between 0.032 and 15.8 µM (pH 7.5 and 4.8) using Tris base. In some experiments, NaCl was substituted for choline chloride, the impermeant anion gluconate was substituted for chloride, and extra K was added as KCl or replaced by 2 mM choline chloride.

Radiotracer Flux Analysis

Experiments were carried out 6-7 days after injection of 50 ng (1 ng/nl) of complementary RNA (cRNA) or 50 nl of water. Groups of 7-10 oocytes were equilibrated in 700 µl of transport buffer at 0.032, 0.32, 3.2, or 10 µM H (pH 7.5, 6.5, 5.5, or 5.0, respectively) for 10 min at 22 °C. They were then transferred to 700 µl of transport buffer at 0.032, 0.32, 3.2, or 10 µM H containing 50 µML-[^3H]alanine for 1 h at 22 °C. Uptake was terminated by rinsing four times in 5 ml of ice-cold buffer. Individual oocytes were lysed in 5% SDS, 2 ml of scintillation mixture was added, and the amount of radioactivity per oocyte was determined.

Electrophysiological Methods

The 2-electrode voltage-clamp method was used to measure the kinetics of H/amino acid cotransport as described previously (Loo et al., 1993; Boorer et al., 1994). Two protocols were used to measure inward currents. 1) The oocyte membrane potential was clamped at -50 mV, and currents induced in response to the addition of amino acids were continuously monitored by a chart recorder. 2) The oocyte plasma membrane was held at -50 mV, and membrane currents were measured after stepping from the holding potential (V(h)) to test potentials (V(m)) between -150 and +50 mV in 20-mV increments. Each voltage pulse was applied for 40 ms. The currents were averaged from five sweeps, filtered at 500 Hz, and digitized at 100 µs per point. Steady-state amino acid-induced currents were obtained by taking the difference between steady-state currents at 40 ms in the presence and absence of amino acid. Base-line currents recorded at 0.032 µM H (pH 7.5) in the absence of organic substrate were monitored throughout the experiments. Oocytes were equilibrated at the [H](o) of the test solution for about 2 min before exposure to amino acids. After every exposure, they were washed in amino acid-free solution at 0.032 µM H until the currents returned to base-line levels.

For kinetic analysis, the amino acid-induced steady-state currents (i) at each test potential (V(m)) were fitted to using ENZFITTER software (Elsevier-Biosoft):

where S(o) is the [amino acid](o) or [H](o), i(max)^S is the maximal current for saturating S(o), K(0.5)^S is the apparent K(0.5) of the substrate (S(o) giving half the i(max)^s), and n is the Hill coefficient.

Substrate Specificity

The substrate specificity of AAP1 was investigated by measuring steady-state substrate-dependent currents with 10 mM of various organic substrates at 10 µM H. All substrate-induced currents were normalized with respect to the alanine-induced current obtained at -150 mV, which was taken as 100%.

Electron Microscopy

Electron microscopic methods are described in detail in Zampighi et al.(1995). After measurements of i(max), the oocytes were fixed in 3-3.5% glutaraldehyde in 0.2 M sodium cacodylate buffer at pH 7.5 for 1 h and infiltrated with 25% glycerol in 0.2 M cacodylate buffer at pH 7.5 for 1 h. Pieces of the oocyte were placed on Balzer specimen holders with the vitelline membrane facing upward, frozen in liquid propane, and fractured at between -150 and -120 °C. The fractured surfaces were coated with platinum at an angle of 80° and carbon at an angle of 90°, coated with 0.5% collodion in amyl acetate, and organic material was removed by immersion in bleach. The replicas were washed in distilled water, placed on formvar-coated copper grids, and the collodion was removed by immersion in amyl acetate. The replicas were inspected in the electron microscope, and images of the protoplasmic fracture faces were digitized and analyzed as described (Zampighi et al., 1995).

Determination of Oocyte Surface Area

The oocyte surface area was obtained by measuring the oocyte membrane capacitance (C(m)). C(m) was determined from the slope of the Q (integral of the capacitive transients) versus voltage relation for 20-mV voltage pulses from V(h) (-50 mV). The surface area of oocytes was calculated by assuming a specific capacitance of 1 µF/cm^2 (Loo et al., 1993) and was verified by electron microscopy (Zampighi et al., 1995).

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.


RESULTS

Fig. 1A shows membrane potential changes in an oocyte expressing AAP1 in response to changing [H](o) 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](o) 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](o) 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](o) 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](o) 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(h) = -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-[^3H]alanine in oocytes injected with cRNA was H-dependent, increasing from 14 to 371 pmol/oocyte/h as [H](o) 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](o) and [H](o). 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](o) between 50 µM and 20 mM, and at 0.5, 1.0, and 10 mM alanine with [H](o) 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](o) increased. Likewise, at 10 mM alanine(o), the inward currents increased at each membrane potential as the [H](o) increased. I/V curves obtained at 1 and 3.2 µM H while varying [alanine](o) and at 0.5 and 1.0 mM alanine while varying [H](o) 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](o) or [H](o), 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(max)^H, the apparent affinity for H, K(0.5)^H, 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(max), K(0.5), 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(max) increased supralinearly with voltage and increased as [H] increased. B, the i(max)^H increased supralinearly with voltage and increased as [alanine] increased. C, as [H] decreased, K(0.5) increased and became more voltage-dependent. D, K(0.5)^H 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(max) exhibited a supralinear dependence on voltage (Fig. 5A) and, as shown in Table 2, decreased as [H](o) decreased. The i(max)^H showed the same voltage dependence (Fig. 5B) and decreased as [alanine](o) decreased (Table 2). The i(max) at saturating [H](o) (1600 nA) was similar to the i(max)^H at saturating [alanine](o) (1400 nA), suggesting a coupling ratio of 1 H:1 alanine. Fig. 5C shows that K(0.5) was relatively voltage-independent at 10 µM H, became more voltage-dependent as the [H](o) decreased, and increased as [H](o) decreased (Table 3). However, at hyperpolarizing potentials, K(0.5) was almost independent of [H](o). The K(0.5)^H was slightly voltage-dependent at low [alanine](o), was voltage-independent at 10 mM alanine, and increased as [alanine](o) 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](o) 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, beta-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(max) > i(max) > i(max) > i(max) geq i(max), all of which increased supralinearly with voltage and decreased as [amino acid](o) decreased (Table 2). The i(max) for each amino acid decreased with decreasing [H](o) (Table 2). The K(0.5) 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](o) decreased (Table 3). The K(0.5)^H at 10 mM of each amino acid was voltage-independent (Fig. 7C) and except for proline increased as [amino acid](o) 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^2 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^2. B, particle density in the P face of an oocyte expressing AAP1. This oocyte had a density of 1037 ± 136 particles/µm^2. C, particle density in the P face of an oocyte expressing SGLT1. The density of the intramembrane particles was 4,121 ± 950/µm^2. In all three cases the magnification was 100,000times.




Figure 7: Kinetics of glutamate, glutamine, proline, and histidine transport. A, i(max) 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(0.5) 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(0.5) values for amino acids were determined from the fitted data. C, voltage dependence of K(0.5)^H 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(max). 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^2 (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^2. The capacitance of this oocyte was 297 nF, which corresponds to an oocyte surface area of 2.97 times 10^7 µm^2 (assuming a capacitance of 1 µF/cm^2) from which was calculated the total number of particles (2.4 times 10/oocyte). The turnover number of AAP1 was calculated using the equation i(max) = kzeN(T), where i(max) = 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(T) = 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^2, and the i(max) 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^2) on external faces data not shown.


DISCUSSION

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), beta-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), beta-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 alpha-amino group is important for substrate recognition; beta-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](o) and [amino acid](o) 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](o) and [amino acid](o) 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(a) of their side groups. Greater than 99% of alanine, proline, and glutamine in solution are zwitterions over the range of [H](o) (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(max) decreased with decreasing [H](o). 3) The i(max)^H for each amino acid decreased with decreasing [amino acid](o). 4) The K(0.5) decreased as [H](o) decreased. 5) The K(0.5) was voltage-dependent and decreased as the membrane potential was made more negative. 6) Except for proline, the K(0.5)^H decreased as [amino acid](o) decreased. 7) At 10 mM amino acid(o) K(0.5)^H was voltage-dependent and became slightly more voltage-dependent at lower [amino acid](o). 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(o) at the external face of the membrane to form CH(2) or CS(2). These intermediates then bind S(2) or H(2), respectively, to form the complex CH(2)S(2). A conformational change allows H(2) and S(2) to be transported to the cytoplasmic surface where they dissociate. The transport cycle is completed when the empty transporter C(i) undergoes another conformational change allowing the ligand binding sites to face the external surface. The broken lines between [CH(2)](o) and [CH(2)](2) 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](o) 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](o) 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(2)] and [S(2)] (where S = substrate) bind randomly to the empty transporter [C] to form the intermediates [CH(2)] or [CS(2)]. [S(2)] or [H(2)] then bind to [CH(2)] or [CS(2)], respectively, to form the fully loaded transporter complex [CH(2)S(2)]. [CH(2)S(2)] crosses the membrane where [H(2)] and [S(2)] 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(0.5) decreased at all membrane potentials as [H](o) became saturating (Fig. 10A). Likewise, the K(0.5)^H decreased as [alanine](o) 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(max) increased as [H](o) increased at all membrane potentials (Fig. 10C), and i(max)^H increased as [alanine](o) increased (Fig. 10D). The i(max) 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(0.5)^H when [proline](o) 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(0.5) decreased as [H] increased at each membrane potential. B, K(0.5)^H decreased as [alanine] increased. C, i(max) increased as [H] increased. D, i(max)^H increased as [alanine] increased.



AAP1 transports neutral, basic, and acidic amino acids. The i(max) > i(max) > i(max) > i(max) geq i(max) 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(max) are due to changes in a rate-limiting step in the transport cycle, possibly the translocation rate of the fully loaded transporter ([CH(2)S(2)]) 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](o) and [alanine](o). 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(0.5)^H and K(0.5) 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](o) and [amino acid](o), there is a voltage dependence of i(max). 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(0.5) for histidine and glutamate, but the dependence of K(0.5) on [H](o) remains the same. Therefore, if 5 and 15% of glutamate is zwitterionic at 3.2 and 10 µM H, respectively, the K(0.5) 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(0.5) 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(max) 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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK 44602 and National Science Foundation Grant MCB 9520599. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physiology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1751. Tel.: 310-825-6968; Fax: 310-206-5661.

(^1)
AAP1 and NAT2 behave identically with respect to their transport kinetics and substrate specificity. Therefore, for simplicity and in view of the large size of the AAP gene family, they will be referred to as AAP1 throughout the text.

(^2)
The abbreviations used are: PIPES, 1,4-piperazinediethanesulfonic acid; HOMOPIPES, homopiperazine-N,N`-bis-2-(ethanesulfonic acid); P face, protoplasmic face.


ACKNOWLEDGEMENTS

-We thank Manoli Contreras for the preparation and injection of oocytes and Todd Chappell for quantification of P face particles.


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