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. 2000 Jul 1;526(Pt 1):35–46. doi: 10.1111/j.1469-7793.2000.00035.x

The heterodimeric amino acid transporter 4F2hc/LAT1 is associated in Xenopus oocytes with a non-selective cation channel that is regulated by the serine/threonine kinase sgk-1

Carsten A Wagner *, Angelika Bröer *, Alexandra Albers *, Nikita Gamper *,†, Florian Lang *, Stefan Bröer *
PMCID: PMC2269991  PMID: 10878097

Abstract

  1. System L is the major Na+-independent amino acid transporter of mammalian cells. It is constituted of the type II membrane protein 4F2hc (CD98) which is covalently linked to the polytopic membrane protein LAT1 via a disulfide bridge. The transporter is known to be regulated by the mineralcorticoid aldosterone in Xenopus A6 cells. To understand the regulation of the transporter, the 4F2hc/LAT1 heterodimer was functionally expressed in Xenopus laevis oocytes and its transport properties were analysed using flux measurements and the two-electrode voltage-clamp technique.

  2. Expression of 4F2hc/LAT1 resulted in a rapid increase in a Na+-independent neutral amino acid antiport activity and simultaneously gave rise to a cation conductance. The cation channel was non-rectifying and non-selective, conducting Li+ > Cs+ = Na+ > K+. After replacement of Na+ by NMDG, however, the currents were suppressed almost completely. The cation channel was not inhibited by amiloride, Ba2+, TEA, Hoe293B, flufenamic acid or substrates of the system L amino acid transporter. Significant inhibition, however, was observed in the presence of La3+, Gd3+ and quinidine. Channel activity was upregulated by coexpression of 4F2hc/LAT1 with the aldosterone-regulated protein kinase sgk-1.

  3. The cation conductance was sensitive to changes in the redox potential, being inhibited following incubation of the oocytes with DTE for 30 min. Mutation of either of the disulfide bridge-constituting cysteines to serine resulted in a loss of ion channel activity whereas amino acid transport was unaffected.

  4. It is concluded that the 4F2hc/LAT1 heterodimer regulates a closely associated cation channel or even constitutes a cation channel itself.

System L is the major Na+-independent amino acid transporter of mammalian cells (Christensen, 1990). It is constituted of the type II membrane protein 4F2hc (CD98) which is covalently linked to the polytopic membrane protein LAT1 via a disulfide bridge (Bröer et al. 1995, 1997a; Mastroberardino et al. 1998; Kanai et al. 1998). The transport pore of the transporter is most probably constituted by LAT1 (Pfeiffer et al. 1998), whereas 4F2hc is necessary for the translocation of the complex into the plasma membrane (Mastroberardino et al. 1998; Kanai et al. 1998; Nakamura et al. 1999). Western blotting and immunofluorescence studies show that 4F2hc is located in the plasma membrane in the absence of LAT1, whereas LAT1, when expressed alone, does not reach the surface of the oocyte (Mastroberardino et al. 1998; Kanai et al. 1998; Nakamura et al. 1999). The role of the 4F2hc protein in plasma membrane trafficking is further supported by its interaction with a family of amino acid transporters. In the absence of LAT1, expression in oocytes of 4F2hc alone results in the plasma membrane insertion of a number of endogenous amino acid transporters, e.g. the 2-amino-[2,2,1]heptane-2-carboxylic acid (BCH)-inhibitable system b0,+, system y+L and some as yet unidentified transporters (Chillaron et al. 1996; Bröer et al. 1998a; Estevez et al. 1998). Subsequent to the cloning of the LAT1 cDNA, additional light chains have been identified which also interact with the 4F2hc protein to form other amino acid transporters with different substrate specificities, namely LAT2, y+LAT1, y+LAT2 and xCT (Torrents et al. 1998; Pfeiffer et al. 1999; Pineda et al. 1999; Segawa et al. 1999; Sato et al. 1999).

The LAT1 homologue of Xenopus A6 cells, designated ASUR4, has been shown to be regulated by aldosterone. Aldosterone mainly regulates the reabsorption of Na+ in mammalian cells, e.g. by activation of the epithelial Na+ channel, ENaC (Chen et al. 1999). The significance of the regulation of the Na+-independent amino acid transporter ASUR4 by aldosterone has not been established.

In this report we demonstrate that expression of the 4F2hc/LAT1 heterodimer in Xenopus oocytes results in the induction of a non-selective cation conductance. The ion conductance was inhibited by treatment of the oocytes with dithioerythritol (DTE) and abolished by mutation of the cysteine residues which form the disulfide bridge between 4F2hc and LAT1.

METHODS

Materials

L-[U-14C]Isoleucine (11.4 Gbq mmol−1) and 22NaCl were purchased from Amersham Buchler, Braunschweig (Germany). The RNA cap structure analogue 7 mG(5′)ppp(5′)G was purchased from New England Biolabs, Schwalbach (Germany). Restriction enzymes, nucleotides and RNA polymerases were from Life Technologies, Eggenstein (Germany). Collagenase (EC 3.4.24.3; 0.3 U mg−1 from Clostridium histolyticum) was from Boehringer-Mannheim (Germany); lots were tested for their suitability for oocyte preparation. All other chemicals were of analytical grade and supplied by E. Merck, Darmstadt (Germany), Roth, Karlsruhe (Germany) or Boehringer-Mannheim.

All experiments were performed in accordance with German animal protection laws and were approved by the regional committee (Regierungspräsidium Tübingen, AZ 37-9185).

Expression in Xenopus laevis oocytes

For expression studies, the EcoRI fragment of rat 4F2hc (r4F2hc) in plasmid pSPORT (Bröer et al. 1995) was subcloned into plasmid pGEM-He-Juel (Bröer et al. 1997b). Alternatively, human 4F2hc (h4F2hc) cDNA in plasmid pSP65T was used (Teixeira & Kühn, 1991). The sgk-1 cDNA was isolated as described (Waldegger et al. 1997). Rat LAT1 cDNA was cloned by high-fidelity RT-PCR. mRNA was isolated from astroglia-rich primary cultures, which were prepared from humanely killed newborn rats (Bröer et al. 1999). The mRNA was reverse transcribed with Superscript II reverse transcriptase. For PCR two primers were constructed, which flanked the coding sequence of LAT1. Oligonucleotide LT1s (5′ CGG AAT TCG AGC CGG GAA CGT CGA GA 3′) corresponded to bases 44-62 of the rat LAT1 cDNA sequence (Kanai et al. 1998) fused to an EcoRI restriction site (emboldened characters). Oligonucleotide LT1a (5′ TGT CTA GAG TCA CAG TGC ACT CTC CCG 3′) corresponded to bases 1623-1640 fused to an XbaI site (emboldened characters). The cDNA was amplified in a 35 cycle PCR reaction (45 s at 98°C, 45 s at 50°C, 480 s at 72°C) using Pfu-Polymerase (Promega, Mannheim, Germany). The amplified band was extracted from the agarose gel, cut with XbaI and EcoRI, and ligated into the XbaI-EcoRI-digested oocyte expression vector pGEM-He-Juel (Bröer et al. 1997b). For in vitro transcription, plasmid DNA was linearized with NotI and transcribed in vitro with T7-RNA polymerase in the presence of a cap analogue. The protocol supplied with the polymerase was followed with the exception that all nucleotides and the cap analogue were used at twofold concentrations (1 mM) to increase the yield of complementary RNA (cRNA). Template plasmids were removed by digestion with RNase-free DNase I. The cRNA was purified by phenol-chloroform extraction followed by precipitation with 1/2 volume of 7.5 M ammonium acetate and 2 volumes of ethanol to remove unincorporated nucleotides. After determination of the amount of cRNA by measurement of the absorption at 260 nm, the integrity of the transcript was verified by denaturing agarose gel electrophoresis.

Female Xenopus laevis were purchased from the South African Xenopus facility (Knysna, Rep. South Africa). Oocytes (stages V and VI) were obtained as described (Bröer et al. 1994). Briefly, frogs were anaesthetized by immersion in 0.1% 3-aminobenzoic acid ethyl ester in water. Small pieces of ovary were removed and the incision sutured. Frogs were placed in shallow water until full recovery of reflexes, and subsequently released into the tank. The oocytes were injected on the following day with 10 nl cRNA in water at a concentration of 1 μg μl−1, using a microinjection device (Bachofer, Reutlingen, Germany). Uninjected oocytes were used as controls. At least 8 weeks were allowed between each removal of oocytes. After the final collection, the anaesthetized frogs were humanely killed.

Uptake experiments were performed as described previously (Bröer et al. 1994), with the exception that ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM Hepes; titrated with NaOH to pH 7.4) was used as the incubation buffer. The concentration of the [14C]isoleucine was taken into account for the adjustment of final substrate concentrations. For efflux experiments, oocytes were preloaded for 10 min with [14C]isoleucine (0.1 mM). Subsequently, oocytes were washed 4 times with 4 ml ice-cold ND96 buffer. To initiate efflux, the washing buffer was aspirated and replaced by ND96 buffer at room temperature, which was supplemented with 1 mM isoleucine where indicated.

Site-directed mutagenesis

Site-directed mutagenesis was performed without subcloning using the ‘QuikChange site-directed mutagenesis kit’ (Stratagene Europe, Amsterdam). Briefly, two complementary primers were constructed in which the desired mutation was flanked by 10-15 nucleotides corresponding to the h4F2hc or rLAT1 cDNA sequence. The primers were used to amplify the complete cDNA containing vector by 12 PCR cycles. Subsequently, the template DNA was removed by digestion with DpnI. The plasmid was isolated from transformed bacteria and sequenced to verify the mutation. To avoid second site mutations, which might have been introduced during the amplification reaction, the transport activity of three independent clones was determined.

The following oligonucleotides were used (only sense sequence shown in 5′ to 3′ direction, mutated base given in lower case letters; the numbering of the amino acids corresponds to the human 4F2hc sequence (Teixeira & Kühn, 1991) and rat LAT1 sequence (Kanai et al. 1998); mutants are designated: wild-type amino acid/position/mutant amino acid):

graphic file with name tjp0526-0035-mu1.jpg

Electrophysiology

Two-electrode voltage-clamp experiments were performed 15-48 h after injection of the cRNA at room temperature using a Geneclamp 500 amplifier and a MacLab D/A converter. Experiments were normally performed at a holding potential of -50 mV, unless otherwise stated. For the determination of the cation selectivity, voltage ramps between -90 and +40 mV were performed, from a holding potential of -40 mV. Data were filtered at 100 Hz. ND96 was used as the external control solution in all experiments. For some experiments, all extracellular cations were replaced with NMDG (N-methyl-D-glucamine), choline, K+, Li+ or Cs+. For some experiments, all Cl− was replaced with gluconate. In another set of experiments the influence of pH and osmolarity was examined. The pH was adjusted to the value indicated with HCl or NaOH; altered osmolarity was achieved by reducing [NaCl] to 48 mM and adding glucose to the osmolarity indicated. The flow rate of the solution was 20 ml min−1 and a complete exchange of the bath solution was reached within about 10 s. The size of the recorded currents varied significantly, depending on the time period after cRNA injection and on the batch of oocytes (from different animals). Therefore, throughout the paper, we show experimental data obtained from one specific recording.

Single-channel recording

Oocytes were devittelinised in 200 mM potassium aspartate solution. Patch-clamp recordings were performed at room temperature under continuous superfusion (1 ml min−1), applied by a dish insert that reduced the bath volume to 0.4 ml. Borosilicate glass pipettes (1.5-3 MΩ tip resistance, GC 150 TF-10, Clark Medical Instruments, Pangbourne, UK) manufactured by a microprocessor-driven DMZ puller (Zeitz, Augsburg, Germany) were used in combination with a MS 314 electrical micromanipulator (Märzhäuser, Wetzlar, Germany). Currents were recorded in the excised-patch, inside-out mode and low-pass filtered at 1 kHz using an EPC-9 amplifier (HEKA, Lambrecht, Germany) equipped with an ITC-16 interface (Instrutech, Port Washington, NY, USA). Data acquisition (10 kHz sampling rate) and analysis were run by Pulse software (HEKA). Single channels were identified in inside-out excised membrane patches using KCl external solution (100 mM KCl, 10 mM Hepes, 10 mM EGTA; pH 7.2 with KOH) and ND96 buffer as pipette solution. Single channels were recorded at holding potentials from -100 to +100 mV. Original current tracings were depicted after 500 Hz low-pass filtering.

Calculations

All data are given as means ±s.e.m. unless indicated otherwise. All measurements were performed with equal numbers of cRNA and non-injected oocytes. In experiments with labelled compounds, all values represent net uptake rates calculated as: mean uptake rate of seven cRNA-injected oocytes – mean uptake rate of seven non-injected oocytes = mean net uptake rate, using Gauss’ law of error propagation for the calculation of the final standard deviations. All data were tested for significance using Student's paired and unpaired t tests and data were considered as significant only at the P = 0.05 level. Controlling only the extracellular cations, the permeability ratio PNa/PX was computed from a modified Goldman-Hodgkin-Katz equation (Hille, 1992; Begenisich, 1998):

graphic file with name tjp0526-0035-mu2.jpg

where Vr,X and Vr,Na are the measured reversal potentials for the tested cation (X) and Na+, respectively, and F, R and T are Faraday's constant, the gas constant and absolute temperature in kelvins, respectively. The conductance (g) was calculated from voltage ramps between -90 and +40 mV and the measured current using g =I/U, where I is the current and U is the potential difference (130 mV). Each experiment (4-7 oocytes) presented was performed at least twice, with similar results.

RESULTS

Amino acid transport in oocytes expressing 4F2hc/LAT1

To investigate the function of the membrane proteins 4F2hc and LAT1, the corresponding cRNAs (5 ng of each) were injected separately or in combination into Xenopus laevis oocytes. To avoid competition between LAT1 and oocyte endogenous amino acid transporters, isoleucine transport activity was determined following an expression time of 1 day. In agreement with other studies (Mastroberardino et al. 1998; Kanai et al. 1998), we detected high Na+-independent isoleucine transport activity in oocytes expressing both 4F2hc and LAT1 together, lower activity in oocytes expressing 4F2hc alone, and no signal above the endogenous activity of non-injected oocytes in LAT1-expressing oocytes (Fig. 1A). Coinjection of 4F2hc with LAT1 resulted in a faster as well as a larger Na+-independent isoleucine transport activity compared with 4F2hc-injected oocytes. In agreement with published results (Mastroberardino et al. 1998; Kanai et al. 1998), we found that the transport activity in oocytes expressing 4F2hc/LAT1 displayed all the characteristics of system L amino acid transport. Na+-independent isoleucine transport was completely inhibited by a hundredfold excess of neutral amino acids such as leucine, histidine (neutral form) and the amino acid analogue BCH (Fig. 1B). Methylated amino acids, such as methyl-aminoisobutyric acid (MeAIB), or amino acids bearing a net charge, e.g. glutamate and arginine, were not effective as inhibitors (Fig. 1B). The absence of inhibition of Na+-independent isoleucine transport by arginine clearly showed that oocyte endogenous transporters were not present at significant levels in the plasma membrane. The oocyte endogenous isoleucine transporter, which is activated by 4F2hc, can be inhibited by neutral amino acids, including BCH, as well as by cationic amino acids (Bröer et al. 1998a).

Figure 1. Characteristics of isoleucine transport in 4F2hc- and LAT1-expressing oocytes.

Figure 1

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Oocytes were injected with 5 ng 4F2hc cRNA, 5 ng LAT1 cRNA or with 10 ng of a mixture of both cRNAs. Non-injected oocytes were used as controls. A, after 1 day, uptake of 14C-labelled isoleucine (0.1 mM) was determined in Na+-free transport buffer. The mean (±s.d.) uptake activity of seven oocytes is given for each injection. n.i., non-injected. B, uptake of 14C-labelled isoleucine (0.1 mM) was determined in Na+-free transport buffer in the presence of a hundredfold excess of unlabelled amino acid. The mean (±s.d.) uptake activity of seven oocytes is given for each experiment. The uptake activity of non-injected oocytes has been subtracted. MeAIB, methyl-aminoisobutyric acid; BCH, 2-amino-[2,2,1]heptane-2-carboxylic acid; other amino acids indicated by the single letter code; -, no addition. C, efflux of preloaded 14C-labelled isoleucine in 4F2hc/LAT1-expressing oocytes. Seven oocytes were preloaded with 100 μM [14C]isoleucine for 10 min. Subsequently, oocytes were washed four times with ice-cold amino acid free incubation buffer and finally suspended in 1 ml transport buffer with (•) or without (○) added isoleucine (1 mM). Samples (100 μl) were removed at the indicated times and radioactivity was determined. The released radioactivity was integrated over time and the values were corrected for the number of oocytes. D, pH dependence of Na+-independent [14C]isoleucine uptake in 4F2hc/LAT1-injected oocytes. Uptake of labelled isoleucine (0.1 mM) was determined in Na+-free transport buffer of different pH. The mean (±s.d.) uptake activity of seven oocytes is given for each experiment (one of two experiments shown). The uptake activity of non-injected oocytes has been subtracted.

Labelled isoleucine was seemingly accumulated in 4F2hc/Lat1-expressing oocytes. When the uptake of 100 μM 14C-labelled isoleucine was followed over 30 min, the oocytes took up more than 100 pmol oocyte−1. Using a solute accessible oocyte volume of 380 nl (Bröer et al. 1999), this corresponded to an intracellular concentration of 260 μM. In agreement with previously published data (Mastroberardino et al. 1998; Kanai et al. 1998), 4F2hc/LAT1 was found to obey an antiport mechanism. Preloaded [14C]isoleucine could not be released from oocytes in the absence of extracellular amino acids, whereas addition of 1 mM unlabelled isoleucine resulted in the rapid release of labelled isoleucine from the oocytes (Fig. 1C). The proton gradient, which has been suggested to energize amino acid transport via system L (Mitsumoto et al. 1986), did not influence amino acid transport (Fig. 1D). Variation of the extracellular pH from 6 to 8 at a constant cytosolic pH of 7.3 (Bröer et al. 1998b) did not affect isoleucine uptake into oocytes, although the H+ gradient changed from an inward to an outward direction under these conditions. Removal of Na+ from the extracellular buffer was similarly without effect on isoleucine transport (176 ± 44 pmol (10 min)−1 in the presence of Na+ and 224 ± 36 pmol (10 min)−1 in the absence of Na+). The accumulation of isoleucine in the cytosol of the oocytes, therefore, resulted from the exchange of labelled isoleucine with unlabelled system L substrates which are present in the oocyte cytosol.

Na+ transport in oocytes expressing 4F2hc/LAT1

The transcription of the Xenopus laevis LAT1 homologue ASUR4 is upregulated in A6 cells by aldosterone (Mastroberardino et al. 1998). Aldosterone mainly controls Na+ reabsorption in epithelia, e.g. by regulating the epithelial sodium channel or the Na+,K+-ATPase (Verrey, 1999). There is no obvious reason for regulation by aldosterone of the electroneutral Na+-independent antiport mechanism mediated by system L. When the expression period of 4F2hc/LAT1 was extended to more than 2 days, extrusion of egg yolk became visible in almost every oocyte, pointing to a possible osmotic imbalance. We therefore studied amino acid transport under current-clamp and voltage-clamp conditions to investigate possible ion transport in 4F2hc/LAT1-expressing oocytes. The resting potential of 4F2hc/LAT1-expressing oocytes (-22 ± 1 mV, 15 oocytes) was significantly less negative than the resting potential of non-injected (-38 ± 1 mV, 9 oocytes) or 4F2hc-injected oocytes (-37 ± 2 mV, 20 oocytes), 24 h after cRNA injection. To allow clamping of the oocytes at -50 mV, a constant inward current had to be maintained; this was -188 ± 25 nA (15 oocytes) in 4F2hc/LAT-injected oocytes compared with -13 ± 3 nA (20 oocytes) in 4F2hc-expressing oocytes and -10 ± 2 nA in control oocytes (9 oocytes). Addition of amino acids (1 mM) which were either substrates of system L or inhibitors thereof, e.g. BCH, to the extracellular solution did not induce any additional currents in voltage-clamped oocytes (1.2 ± 2.3 nA). These results are in agreement with the ion-independent antiport mechanism for neutral amino acids as described above.

In contrast, the inward currents that had to be applied to clamp the oocytes were strongly reduced on replacement of extracellular Na+ by NMDG or choline. The reduction of the conductance could be reversed by a switch back to Na+-containing solutions (Fig. 2). The conductance was selective for cations over anions as the reversal potential shifted from -22 ± 2 mV in ND96 buffer to -44 ± 1 mV when all cations were replaced by NMDG (n = 10 oocytes). These three observations, (i) extrusion of egg yolk, (ii) depolarized resting potential and (iii) outward currents elicited by removal of Na+, pointed to the presence of an influx pathway for Na+ in 4F2hc/LAT1-expressing oocytes. This conductance, although only detectable in system L-expressing oocytes, was independent of the presence or absence of substrates of the transporter. Increased permeability for Na+ was verified by uptake studies using 22NaCl (Table 1). In oocytes expressing 4F2hc/LAT1, a fivefold increase in 22NaCl uptake was observed, compared with non-injected or 4F2hc-expressing oocytes.

Figure 2. Expression of 4F2hc/LAT1 induces a cation conductance (Icat).

Figure 2

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Original tracing showing the inhibition of Icat on replacement of all cations by NMDG. The oocyte was held at a potential of -50 mV (n = 27).

Table 1.

Transport of 22Na+ and [14C]isoleucine in oocytes expressing 4F2hc and LAT1

Injected cRNA [14C]Ile uptake(pmol (10 min)−1) 22Na+ uptake(nmol (5 min)−1) Icat(nA)
4F2hc 1.3 ± 0.2 0.33 ± 0.01 7.8 ± 4.1
4F2hc/LAT1 36 ± 4 1.68 ± 0.4 215.3 ± 9.6
4F2hc/LAT1/sgk-1 38 ± 2 4.44 ± 0.7 750.1 ± 193.6
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Oocytes were injected with 5 ng of 4F2hc cRNA, 5 ng of 4F2hc and 5 ng LAT1 cRNA, or 5 ng 4F2hc and 5 ng LAT1 cRNA plus 3 ng human sgk-1 cRNA, followed by an expression period of 1 day. [14C]isoleucine uptake was determined using an incubation time of 10 min; 22Na+ uptake was followed over 5 min. Flux studies were performed in ND96 buffer containing 96 mM NaCl. Icat was determined as the difference between the current in the absence and presence of monovalent cations. The mean uptake activity of seven oocytes was determined for each measurement, one of four experiments shown.

Regulation of ion channel activity

Recent evidence (Chen et al. 1999) suggests that early aldosterone action is mediated, in part, by the volume-regulated serum and glucocorticoid-inducible kinase sgk-1 (Waldegger et al. 1997). In agreement with the notion that the 4F2hc/LAT1-induced Na+ transport might be a possible target of aldosterone regulation, it was found that coinjection of 4F2hc/LAT1 cRNA together with human sgk-1 cRNA resulted in a three- to fourfold increase in the cation conductance (Icat) and 22Na+ uptake, whereas amino acid transport remained unaffected (Table 1). This result suggests that aldosterone may have two different effects on 4F2hc/LAT1: on the expression of LAT1 and on the activity of the associated cation conductance.

Characterization of the 4F2hc/LAT1-induced cation conductance

The currents flowing through the 4F2hc/LAT1-induced cation channel showed an almost linear dependence on voltage between -90 and +40 mV (Fig. 3A), and could therefore be described as non-rectifiying. In control oocytes comparable currents could not be detected, as shown in Fig. 3B (n = 5 oocytes). The channel not only mediated Na+ flux but also allowed the passage of other cations such as K+, Li+ or Cs+ with a low selectivity (Fig. 3A) The relative ion permeability decreased in the following order: Li+ > Na+ = Cs+ > K+ with PNa/PK = 1.44 ± 0.15, PNa/PLi = 0.65 ± 0.08 and PNa/PCs = 0.97 ± 0.10 (n = 5 oocytes; Table 2). The cation conductance was not blocked by the epithelial Na+ channel blocker amiloride (10 μM), the K+ channel blocker Ba2+ (1 mM), the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 μM), niflumic acid (100 μM) and flufenamic acid (100 μM), the IKs inhibitor Hoe 293B (10 μM), or tetraethylamine (TEA, 1 mM) (Table 3). Quinidine (500 μM) partially inhibited the cation conductance. The non-specific cation channel blockers GdCl3 (300 μM) and LaCl3 (300 μM) both partially inhibited Icat. For the Gd3+- and La3+-sensitive part of Icat, IC50 values of 40.9 ± 6.3 and 20.6 ± 14.5 μM were determined for GdCl3 and LaCl3, respectively (Fig. 3C). Of all these ion channel inhibitors, treatment of oocytes with only Gd3+ and quinidine also resulted in a decrease in the amino acid transport activity (Table 3, Fig. 3C). The inhibition of amino acid transport by Gd3+ showed two components of different sensitivity. About 50 % of the amino transport activity was highly sensitive to Gd3+ (IC50 = 2.4 ± 0.4 μM; Fig. 3C), whereas the remaining transport activity was less sensitive, being completely inhibited only by addition of 300 μM GdCl3 (not shown). In contrast to Icat, amino acid transport was found to be insensitive to inhibition by La3+.

Figure 3. Cation selectivity and pharmacology of Icat.

Figure 3

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A, cation selectivity of Icat in 4F2hc/LAT1-expressing Xenopus oocytes. Voltage ramps were performed from a holding potential of -40 mV to -90 and +40 mV and cation-induced currents were measured as the difference between the current in the presence of the respective cation and the current in the absence of all cations (NMDG). B, cation currents in control oocytes. C, concentration-dependent inhibition of Icat in 4F2hc/LAT1-expressing Xenopus oocytes by LaCl3 (IC50 = 20.6 ± 14.5 μM) and GdCl3 (IC50 = 40.9 ± 6.3 μM) and of Na+-independent isoleucine transport by GdCl3 (IC50 = 2.4 ± 0.4 μM). The IC50 value was calculated by fitting the curves to the data using the equation: I =ImaxCnH/(CnH+ IC50), where nH and C are the Hill coefficient and the inhibitor concentration, respectively. Imax is the maximal extrapolated inhibition and IC50 is the concentration needed for half-maximal inhibition. The experiment was performed twice with five oocytes (or seven in the flux studies).

Table 2.

Cation selectivity and permeability

Cation Reversal potential (mV) g (μS) PNa/PX
Na+ 13.6 ± 1.2 4.6 ± 0.3 1
K+ 4.2 ± 1.8 16.5 ± 1.5 1.44 ± 0.15
Li+ 24.8 ± 2.1 2.5 ± 0.2 0.65 ± 0.08
Cs+ 14.3 ± 1.7 10.1 ± 0.4 0.97 ± 0.10
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Oocytes were injected with 5 ng 4F2hc cRNA and 5 ng LAT1 cRNA and experiments were performed 24–36 h after injection. Icat was determined as the difference between the cation current and the current in the absence of all monovalent cations (NMDG). The reversal potentials were obtained from I–V ramps performed between −90 and +40 mV, from a holding potential of −40 mV (five oocytes; two different experiments). Conductance (g) and relative permeability (P) were calculated as indicated in Methods.

Table 3.

Influence of inhibitors on amino acid transport and cation conductance

Inhibitor Na+-independent [14C]Ile uptake(% of control) Icat(% of control)
None 100 100
Amiloride (10 μM) 102 ± 8 99 ± 2
TEA (1 mM) 118 ± 17 100 ± 2
BaCl2 (1 mM) 101 ± 7 105 ± 4
LaCl3 (300 μM) 109 ± 5 52 ± 4
GdCl3 (300 μM) 43 ± 10 44 ± 4
Niflumate (100 μM) 87 ± 13 100 ± 1
Flufenamate (100 μM) 79 ± 12 96 ± 2
NPPB (100 μM) 104 ± 14 89 ± 3
Hoe 293B (10 μM) 102 ± 18 100 ± 1
Quinidine (500 μM) 58 ± 20 65 ± 11
PMA (50 nM) 61 ± 11 n.d.
Staurosporine (0.001 mM) 95 ± 4 n.d.
DTE (5 mM) 92 ± 8 21 ± 6
Mercaptoethanol (1 mM) 88 ± 14 n.d.
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Oocytes were injected with 5 ng of both 4F2hc and LAT1 cRNAs, followed by an expression period of 1–2 days. Due to the fast expression of 4F2hc/LAT1 control values ranged from 17 to 70 pmol (2.5 min)−1 depending on the expression period. Na+independent isoleucine uptake was measured with [14C]isoleucine. Icat was determined as the difference between the current in the absence and presence of Na+. Oocytes were incubated with the indicated inhibitors for 5 min prior to and during the transport experiment. A preincubation period of 1 h was used in experiments with phorbolester (PMA) and staurosporine and 30 min in the case of DTE and mercaptoethanol. Transport activity was determined using seven oocytes in two different experiments. n.d., not determined.

Single-channel recordings on 4F2hc/LAT1-expressing oocytes confirmed the presence of a non-specific cation conductance, which was not observed in non-injected oocytes. In 15 out of 22 inside-out excised patches from 4F2hc/LAT1-injected oocytes non-selective cation channels were identified using KCl bath and ND96 pipette solutions (Fig. 4A). These channels exhibited non-rectifying I-V curves with a mean conductance of 31.5 ± 0.5 pS and a reversal potential of about 0 mV (as determined by linear regression between -100 and +100 mV; 5 patches; Fig. 4B) and were not found in 12 excised patches from non-injected oocytes. Replacement of 70 mM NaCl by 70 mM sodium gluconate in the pipette solution did not result in any shift in reversal potential of these 30 pS channels, suggesting that the channels were highly selective for cations (Fig. 4B). Application of NMDG from both sides of the membrane caused a reversible flickering-type block of the channels (Fig. 4C and D).

Figure 4. Identification of single cation-selective channels in inside-out excised patches.

Figure 4

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A, original current traces recorded at different voltages (as indicated) with KCl bath and ND96 pipette solutions. B, I-V relations recorded under control conditions (as in A; ▪) and with 70 mM sodium gluconate/26 mM NaCl pipette solution (▴). Channel amplitudes represent means ±s.e.m. of five inside-out patches. C and D, inhibition of non-selective cation channels by NMDG, with transition from KCl bath solution to NMDGCl solution (C) and transition from NMDGCl to KCl solution (D). The original current traces shown were recorded at +100 mV (upper traces) and -100 mV (lower traces). Four different batches of oocytes were tested.

Functional significance of the disulfide bridge in the 4F2hc/LAT1 heterodimer

Besides the heterodimeric composition, the most unusual feature of this family of amino acid transporters is the disulfide bridge between both subunits. We therefore investigated the influence of the disulfide bridge-reducing agent DTE on both amino acid transport and cation channel activity. Addition of DTE (5 mM, 30 min) to the superfusate inhibited Icat by 79 ± 6 %, whereas amino acid transport was unaffected (Table 3).

It has previously been shown that this bridge is formed between cysteine 109 of 4F2hc and cysteine 165 of LAT1 (Pfeiffer et al. 1998). To investigate whether the inhibitory effect of DTE on the cation conductance indeed resulted from a reduction of the disulfide bridge, we constructed two mutants in which either the cysteine of 4F2hc or the cysteine of LAT1 were changed to serine. The h4F2hc(C109S)/rLAT1 and h4F2hc/rLAT1(C165S) heterodimers both displayed fully active amino acid transport, whereas the cation conductance was strongly reduced (Fig. 5). Amino acid transport activity in oocytes expressing h4F2hc(C109S)/ rLAT1 was even faster than that in oocytes expressing the wild-type heterodimer, indicating that the plasma membrane trafficking of the mutated heterodimers was unaffected (Fig. 5).

Figure 5. Amino acid transport and cation channel activity in wild-type and mutated 4F2hc/LAT1 heterodimers.

Figure 5

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Oocytes were injected with 10 ng of the following cRNA mixtures: h4F2hc/rLAT1, h4F2hc(C109S)/rLAT1 or h4F2hc/rLAT1(C165S); non-injected oocytes were used as controls. After an 8-30 h expression period groups of seven oocytes were removed to determine [14C]isoleucine uptake (A), as described in the legend to Fig. 1. Icat was determined simultaneously in oocytes of the same batch (B). Due to the differences in the activity between different experiments, the uptake activity of wild-type (wt) was set to 100 %; absolute transport activities ranged between 20 and 50 pmol (10 min)−1 oocyte−1. The mean (±s.d.) uptake activity of seven oocytes is given for each experiment (one of three experiments shown). The uptake activity of non-injected oocytes has been subtracted.

To elucidate the role of cysteines in amino acid transport and ion conductance, the effect of low HgCl2 concentrations on both activities was investigated (Fig. 6A and B). Amino acid transport was highly sensitive to inhibition by HgCl2, with an IC50 value of 0.7 ± 0.3 μM (7 oocytes per data point; Fig. 6A). The inhibition did not depend on the integrity of the disulfide bridge between the two subunits, as demonstrated by inhibition of amino acid transport mediated by the h4F2hc(C109S)/rLAT1 heterodimer with an identical IC50 value of 0.7 ± 0.3 μM (7 oocytes per data point; Fig. 6A). The cation conductance, in contrast, increased strongly following incubation of oocytes with HgCl2 (1 μM) for 15 min (Fig. 6B). This increase was not observed in non-injected control oocytes, demonstrating that HgCl2 treatment did not result in a non-specific leakage.

Figure 6. Effect of HgCl2 on amino acid transport and cation channel activity in wild-type and mutated 4F2hc/LAT1 heterodimers.

Figure 6

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Oocytes were injected with 10 ng of cRNA mixtures of r4F2hc/rLAT1 (•), h4F2hc/rLAT1 (▪) or h4F2hc(C109S)/rLAT1 (□) or were uninjected (control). A, after 1 day expression groups of seven oocytes were removed to determine [14C]isoleucine uptake in the presence of increasing concentrations of HgCl2. B, in parallel experiments oocytes were treated for 15 min with 1 μM HgCl2 and Icat was determined before (-) and after (+) incubation, in oocytes expressing h4F2hc/rLAT1 (Inline graphic), h4F2hc(C109S)/rLAT1 (□) or in uninjected control oocytes (▪). The mean (±s.d.) uptake activity of seven oocytes is given for each experiment (one of three experiments shown). The uptake activity of non-injected oocytes has been subtracted.

The close association between cation conductance and amino acid transport was corroborated by the tightly coupled time course of expression of both activities (Fig. 7). Due to the rapid expression of the 4F2hc/LAT1 heterodimer, amino acid transport and cation conductance were followed between 15 and 24 h. During this time course, both the amino acid transport and the cation conductance increased in parallel by eightfold. No time lag between the increase in either activity was observed.

Figure 7. Correlation between the increase in isoleucine uptake activity and Icat.

Figure 7

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Oocytes were injected with 10 ng of a mixture of 4F2hc and LAT1 cRNAs or were not injected (control). At the times indicated, groups of seven oocytes were removed to determine [14C]isoleucine uptake (○) as described in the legend to Fig. 1. Icat (•) was determined simultaneously in oocytes of the same batch. The mean (±s.d.) uptake activity of seven oocytes is given for isoleucine uptake (one of two different experiments shown). The uptake activity of non-injected oocytes has been subtracted.

DISCUSSION

Expression of 4F2hc/LAT1 in Xenopus laevis oocytes resulted in the induction of two transport activities. The first was an amino acid transport activity that demonstrated all the properties of system L, the second was a non-selective cation conductance. Two different models can be envisaged to explain these observations. (i) Cation conductance and amino acid transport activity are two distinct properties of the 4F2hc/LAT1 heterodimer, or (ii) the 4F2hc/LAT1 heterodimer is associated with an oocyte endogenous cation channel.

Transport-associated conductances have been described for a number of transporters, e.g. amino acid transporters of the EAAT/ASCT family (Fairman et al. 1995; Wadiche et al. 1995). In these cases it is thought that amino acids and anions pass through the same translocation pore. Three observations suggest that it is unlikely that the ions pass through the same pore as the amino acids in the 4F2hc/LAT1 heterodimer. (i) Both activities have distinct pharmacological properties, (ii) the ion conductance is not affected by the presence of amino acid substrates, and (iii) the ion channel activity is independently regulated by the serine/threonine kinase sgk-1. A very close association between both activities was, however, suggested by another series of experiments. (i) Cation conductance and amino acid transport activity increase with the same time course during expression in oocytes, (ii) treatment of the 4F2hc/LAT1 heterodimer with the reducing agent DTE abolishes the cation conductance, indicating that the redox status is important for channel function, and (iii) mutation of both C109 of h4F2hc and C165 of rLAT1 to serine results in the loss of ion channel activity without loss of amino acid transport actvity. The analysis of the two mutants clearly indicates that the disulfide bridge of the heterodimer is not necessary for amino acid transport or membrane trafficking but is necessary for the associated ion channel activity. In agreement with this observation we found that amino acid transport mediated by the 4F2hc(C109S)/rLAT1 heterodimer was as sensitive to inhibition by HgCl2 as that mediated by the wild-type heterodimer, suggesting that another cysteine of the LAT1 subunit is sensitive to inhibition by HgCl2. These results are at variance with data on the interaction of the C109S mutant with a related oocyte endogenous light chain, which resulted in a partial protection against inactivation by HgCl2 (Estevez et al. 1998). In contrast to the inhibitory effect of HgCl2 on amino acid transport, we found that the ion conductance strongly increased after the incubation period. The increase in conductance of both the wild-type and the mutated transporter was similar following treatment with HgCl2. This suggests that the channel is still associated with the mutated heterodimer but is not in an open state. Since the disulfide bridge connects 4F2hc with LAT1 and substitution of serine on either side affects ion channel activity, it is likely that the disulfide bridge plays a crucial role in the conformation of the ion channel. Whether the heterodimer itself constitutes the ion channel or whether it is associated with oocyte endogenous channels is at present unclear. It has indeed been shown that 4F2hc is associated with other plasma membrane proteins, such as lymphocyte surface antigens, integrins and virus receptors (Ohgimoto et al. 1995; Cerny et al. 1996; Fenczik et al. 1997). Xenopus oocytes have been shown to express a non-selective cation channel, the properties of which partially resemble those described here (Bielfeld-Ackermann et al. 1998). However, the pharmacology differs in so far as flufenamic acid did not inhibit Icat. The patch-clamp studies confirm the presence of an ion channel in 4F2hc/LAT1-expressing oocytes that is not observed in non-injected oocytes, the properties of which are in agreement with whole-cell recordings in oocytes under voltage-clamp conditions. Xenopus oocytes also possess a K+ conductance induced by the similarly heterodimeric XKvLQT1-IsK channel complex but this conductance would have been blocked by the IKs blocker Hoe 293B (Busch et al. 1996). Taken together it seems likely that the 4F2hc/LAT1 heterodimer at least modulates an ion channel activity.

The 4F2hc protein interacts with oocyte endogenous amino acid transporters, which are similar to corresponding transporters in mammalian cells. By analogy, an interaction between the 4F2hc/LAT1 heterodimer and a non-selective cation channel might occur in mammalian cells as well. In mammalian cells, non-selective cation channels have been described which are activated by hypertonicity and might be involved in volume regulation (Volk et al. 1995; Koch & Korbmacher, 1999). It is not known whether the activity of these channels is regulated by aldosterone. Similar to the channel described here, the non-selective cation channels of mammalian cells are not inhibited by conventional blockers of cation channels such as amiloride, TEA or Ba2+. In Xenopus A6 cells, a basolateral potassium conductance has been described, which is induced by aldosterone and inhibited by quinidine (Broillet et al. 1993), which also resembles the cation conductance described here. Although the current elicited by extracellular KCl was larger than that elicited by NaCl in 4F2hc/LAT1-expressing oocytes, evaluation of the reversal potential suggests that the cation channel is slightly selective for Na+ over K+. These observations are not necessarily mutually exclusive. Stronger binding of Na+ to residues in a channel might result in lower rate constants for the movement of the ion through the channel. If the differences between the binding constants are larger than the differences between the rate constants for movement through the channel, a selectivity for Na+ might coincide with larger currents in the presence of K+. Similar effects have been observed with voltage-gated calcium or sodium channels (e.g. Friel & Tsien, 1989; Hille, 1992).

In summary, we found that the disulfide bridge of the heterodimeric amino acid transporter 4F2hc/LAT1 is not necessary for amino acid transport activity or for trafficking of LAT1 to the plasma membrane but that it is necessary for the functioning of a cation channel, the characteristics of which partially depend on the heterodimer itself.

Acknowledgments

This work was supported by grants from the Deutsche Forschungsgemeinschaft to S.B. (Br1318/2-3) and F.L. (La315/4-4). C.A.W. was supported by a fellowship of the Interdisciplinary Centre for Clinical Research, University of Tübingen. N.G. was supported by a joint grant from the DFG (436 RUS 113/488/0(R)) and RFRB (98-04-04125).

Carston A. Wagner and Angelika Bröer contributed equally to this publication.

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