Biochemical Journal

Research article

The C-terminal domain of the neutral amino acid transporter SNAT2 regulates transport activity through voltage-dependent processes

Zhou Zhang, Catherine B. Zander, Christof Grewer

Abstract

SNAT (sodium-coupled neutral amino acid transporter) 2 belongs to the SLC38 (solute carrier 38) family of solute transporters. Transport of one amino acid molecule into the cell is driven by the co-transport of one Na+ ion. The functional significance of the C-terminus of SNAT2, which is predicted to be located in the extracellular space, is currently unknown. In the present paper, we removed 13 amino acid residues from the SNAT2 C-terminus and studied the effect of this deletion on transporter function. The truncation abolished amino acid transport currents at negative membrane potentials (<0 mV), as well as substrate uptake. However, transport currents were observed at positive membrane potentials demonstrating that transport was accelerated while the driving force decreased. Membrane expression levels were normal in the truncated transporter. SNAT2Del C-ter (13 residues deleted from the C-terminus) showed 3-fold higher apparent affinity for alanine, and 2-fold higher Na+ affinity compared with wild-type SNAT2, suggesting that the C-terminus is not required for high-affinity substrate and Na+ interaction with SNAT2. The pH sensitivity of amino acid transport was retained partially after the truncation. In contrast with the truncation after TM (transmembrane domain) 11, the deletion of TM11 resulted in an inactive transporter, most probably due to a defect in cell surface expression. Taken together, the results demonstrate that the C-terminal domain of SNAT2 is an important voltage regulator that is required for a normal amino acid translocation process at physiological membrane potentials. However, the C-terminus appears not to be involved in the regulation of membrane expression.

  • amino acid transport
  • C-terminus
  • sodium-coupled neutral amino acid transporter (SNAT)
  • solute carrier 38 (SLC38)
  • substrate uptake
  • transport mechanism

INTRODUCTION

SNATs (sodium-coupled neutral amino acid transporters) play an important role in transporting small neutral amino acids, such as glutamine and alanine, across cellular membranes. SNATs belong to the SLC38 (solute carrier 38) family. The family member SNAT2 has been cloned [13] and is found in every tissue tested using Northern analysis [25]. The stoichiometry of the coupling of SNAT2 amino acid transport is proposed to be one neutral amino acid to one Na+ ion. Therefore, one net positive charge is transported into the cell for each transported neutral amino acid [6,7], rendering the process electrogenic. SNAT2 also mediates an anion leak conductance [8]. SNAT2 may play an important role in the brain, where it is believed to be involved in the process of transporting glutamine from astrocytes to neurons via the glutamate–glutamine cycle [9].

Membrane proteins are complex proteins with distinct structural domains that mediate divergent functions. Recent research shows that the C-terminus of membrane proteins can play important roles in the structure, function and regulation of expression of these proteins, as indicated by the following three examples: (i) large movements in the C-terminus of CLC-0 chloride channels are associated with a slow gating process [10]; (ii) the cytoplasmic C-terminal domain of vAChT (vesicular acetylcholine transporter) contains signals targeting this transporter to SVs (synaptic vesicles) [11,12]; and (iii) the C-terminus of the L-type voltage-gated calcium channel CaV1.2 encodes a transcription factor that regulates the expression of a wide variety of endogenous genes important for neuronal signalling and excitability [13]. Based on hydropathy analysis [14] and experimental evidence from related plant transporters [1517], the predicted topology of SNAT2 shows that it includes 11 TMs (transmembrane domains) with an intracellular N-terminus and a relatively short extracellular C-terminus (Figure 1A), as shown previously by Hyde et al. [18] using an epitope-tagged transporter. This predicted topology is supported by recent evidence suggesting structural homology with a large class of amino acid transporters of known structure [19,20]. It has been reported that a highly conserved C-terminal histidine residue (His504) contributes to the pH sensitivity of SNAT2 [21]. However, the overall effect of the C-terminus on the function of SNAT2 is understood poorly.

Figure 1 Deletion of the C-terminus of SNAT2 inhibits transport current and amino acid uptake

(A) Typical transport currents induced by the application of 10 mM alanine to non-transfected (control), SNAT2Del C-ter- and SNAT2WT-expressing cells. The bath solution contained 140 mM NaMes, and the pipette solution contained 140 mM KMes at 0 mV transmembrane potential. (B) Average alanine-induced transport currents (grey bars, left-hand axis) and MeAIB uptake (black bars, right-hand axis) in HEK-293 cells transiently transfected with vector pBK-CMV [Δ(1098–1300)], SNAT2Del C-ter 9 and SNAT2WT cDNA. The inset shows the predicted SNAT2 transmembrane topology and the truncation site. aa, amino acid.

To identify the importance of the C-terminus of SNAT2, in the present study we deleted 13 amino acid residues from its C-terminus (Del C-ter), as well as the TM11 (Figures 1 and 6), and determined the function of the truncated SNAT2s. Our results show that transporters with the C-terminal deletions express normally in the membrane of cells, but that transport is absent after TM11 deletion and is inhibited strongly after the deletion of the extracellular C-terminus. The apparent affinities for both amino acid and Na+ are not impaired by the C-terminus deletion. Furthermore, pH dependence of transport is reduced, but not eliminated, by the deletion. Taken together, the results suggest that the C-terminus of SNAT2 plays an important role for amino acid translocation and its voltage dependence, but not amino acid, Na+ or proton binding, by the transporter.

EXPERIMENTAL

Molecular biology and transient expression

The cDNA coding for rat SNAT2, which was kindly provided by Dr Helene Varoqui, Louisiana State University, New Orleans, U.S.A., was subcloned into the SacI and NheI sites of a modified pBK-CMV (cytomegalovirus) vector Δ(1098–1300) (Stratagene), containing the CMV promoter for mammalian expression. The 13 amino acid residues of the SNAT2 C-terminus (VLDWVHDASAGGH) and the 54 amino acid residues (from positions 451–503) of the SNAT2 TM11 C-terminus fragment (LPSAFYIKLVKKEPMRSVQKIGALCFLLSGVVVMIGSMGLIVLDWVHDASAGGH) were removed by site-directed mutagenesis, according to the QuikChange® protocol (Stratagene) as described by the supplier. The primers for mutation experiments were obtained from the DNA core laboratory, Department of Biochemistry at the University of Miami Miller School of Medicine. The complete coding sequences of mutated SNAT2 clones were sequenced subsequently. WT (wild-type) and mutant transporter constructs were used for transient transfection of sub-confluent HEK (human embryonic kidney) cell cultures (293T/17, A.T.C.C. number CRL 11268) using the FuGENE® 6 Transfection Reagent (Roche) according to the instructions of the supplier. The 293T/17 cell line is a derivative of the 293T cell line into which the gene for SV40 (simian virus 40) T-antigen was inserted. Electrophysiological recordings were performed between 1 and 2 days post-transfection.

Electrophysiology

SNAT2-mediated currents were recorded with an Adams & List EPC7 amplifier (Heka Elektronik) under voltage-clamp conditions in the whole-cell current-recording configuration. The typical resistance of the recording electrode was 2–3 MΩ; the series resistance was 5–8 MΩ. Because the currents induced by substrate, anion or cation application were small (typically <500 pA), Rs (series resistance) compensation had a negligible effect on the magnitude of the observed currents (<4% error). Therefore Rs was not compensated for. The extracellular bath buffer solution contained 140 mM NaMes (sodium methanesulfonate), 2 mM MgGlu2 (magnesium gluconate), 2 mM CaGlu2 (calcium gluconate) and 30 mM Tris pH 8.0. Except for the pH-dependent experiments, all other experiments were conducted at an extracellular pH of 8.0 because amino acid transport by SNAT2 is pH dependent and the transport rate is maximal at this pH. For testing the Na+-dependence of the currents, Na+ in the extracellular solution was replaced with NMG+ (N-methylglucamine). The pipette solution contained 140 mM KMes or KSCN, 2 mM MgGlu2, 10 mM EGTA and 30 mM Hepes, pH 7.3. For determining the voltage dependence of SNAT2 alanine transport, a combined voltage ramp/solution exchange protocol was used. In this protocol, the cell membrane was held initially at 0 mV, before ramping the voltage up to its final value (−90–+60 mV) within 2 s. Two seconds after establishing the new voltage, the extracellular solution was changed from no alanine to the final concentration of alanine, followed by ramping the voltage back to 0 mV. The currents were low-pass filtered at 1–10 kHz (Krohn-Hite 3200) and digitized with a digitizer board (Axon Instruments Digidata 1200) at a sampling rate of 10–15 kHz, which was controlled by the manufacturer's software (Axon PClamp). All the experiments were performed at room temperature (22 °C). Solutions were applied to the cells as described previously [8,20,22].

Amino acid uptake assay

HEK-293 cells were plated on collagen-coated 12-well plates (1×105 cells/well) in Dulbecco's modified Eagle's medium containing 10% (w/v) fetal bovine serum, penicillin (100 units/ml), streptomycin (100 mg/ml) and glutamine (4 mM). 48 h after transfection with the vector, WT SNAT2 or truncated SNAT2 cDNA, the cells were washed two times with the uptake buffer. The uptake buffer contained 140 mM NaMes, 2 mM MgMes2, 2 mM CaGlu2, 30 mM Tris/Mes pH 8.0 and 5 mM glucose. The cells were then pre-incubated in the same buffer for 5 min at 37 °C before the buffer was replaced with fresh buffer containing unlabelled MeAIB (N-methylaminoisobutyric acid) and 0.4 μCi of [14C]MeAIB (PerkinElmer Life Sciences; total concentration, 40 μM). After 1 min of incubation at room temperature, the uptake was terminated by washing twice with 1 ml of uptake buffer on ice (after 1 min uptake was in the linear range, as determined by quantifying the time dependence of uptake for times up to 5 min). The cells were then solubilized in 0.5 ml of 1% SDS, and radioactivity was measured by scintillation counting in 3 ml of scintillation fluid (Ecoscint, National Diagnostics). The MeAIB uptake measurements were performed in duplicate.

Data analysis

Nonlinear regression fits of the experimental data were performed with Origin (Microcal Software) or Clampfit (pClamp8 software, Axon Instruments). The dose response relationships of currents were fitted with a Michaelis-Menten-like equation, yielding Km (Michaelis constant) and Imax (maximum current). Endogenous alanine transport activity in HEK-293T cells, as measured by the current recording from non-transfected cells, is minimal (see the Results section). For the Na+-concentration-dependence of the leak current, the dose-response data was corrected by subtraction of the nonspecific component of the current, which increases linearly with increasing Na+. The non-specific component was determined from non-transfected HEK-293 cells. Each experiment was repeated at least three times with at least two different cells. The error bars represent mean±S.D., unless stated otherwise. SNAT2 Imax vary approx. by a factor of 3 between different cells, depending on the expression levels of each individual cell. Such changes in expression levels did not affect the Km for the amino acid. The Imax values were obtained by averaging the Imax values from these individual cells.

Confocal microscopy

Confocal microscopy was performed after expressing an AcGFP [Aequorea coerulescens GFP (green fluorescent protein)]–SNAT2 fusion construct in HEK-293 cells. Rat SNAT2 cDNA was subcloned from the pBK-CMV vector into a pAcGFP1-C In-Fusion Ready Vector (Clontech) (primers: sense 5′-AAGGCCTCTGTCGACACACCGTTCCTCCGGATCAGC-3′, antisense - 5′-AGAATTCGCAAGCTTTCTGAGCGAGTTGAGTGGACCCAA-3′), to add a GFP protein sequence in frame to SNAT2's N-terminus. After a 30 h incubation post-transfection, the AcGFP–SNAT2 expressing HEK-293T cells were wet mounted and visualized using an Axiovert inverted microscope (Carl Zeiss) using a ×40 oil-immersion objective lens. Single optical sections were taken and recorded digitally using a Zeiss LSM 510 META confocal imaging system. The images were taken upon excitation with an argon laser. Images of non-transfected HEK-293 cells were also recorded in bright field mode.

RESULTS

Amino acid transport is impaired after deletion of the SNAT2 C-terminus

Application of 10 mM alanine to SNAT2WT-expressing cells resulted in inwardly directed transport currents (Figure 1A) in the presence of 140 mM Na+ at the extracellular side of the membrane [V (voltage)=0 mV, average current of −127±30 pA, n=28; grey bars in Figure 1B). In contrast, application of 10 mM alanine to SNAT2Del C-ter resulted in a small inward current (Figure 1A), which was on average −18±6 pA (n=7) and 11% of the SNAT2WT response (corrected for nonspecific currents, Figure 1B). These results suggest that deletion of the SNAT2 C-terminus strongly interferes with the ability of SNAT2 to catalyse alanine transport current. Although the current carried by SNAT2Del C-ter was small, it was significantly larger (4.5-fold) than that observed in non-transfected cells (−4±2 pA, n=19, Figure 1B).

The lack of alanine-induced transport current for SNAT2Del C-ter may be caused by reduced expression. To test this possibility, we determined subcellular localization by using confocal microscopy with an AcGFP-tagged transporter (Figure 2). The current-recording experiments show that the N-terminal AcGFP tag does not affect the function of the transporter (results not shown). Non-transfected cells showed no significant fluorescence (Figures 2D and 2E), whereas both SNAT2WT- and SNAT2Del C-ter-expressing cells exhibited intracellular fluorescence, as well as significant fluorescence at the cell boundaries (Figures 2A and 2B, white arrows). Although small differences in the expression levels of SNAT2WT and SNAT2Del C-ter cannot be quantified using this method, these results indicate that the deletion of the C-terminus of SNAT2 does not affect the transporter localization in the membrane in a major way. This result is consistent with transport current measurements at +60 mV (Figures 4A and 4C), which indicate clearly that SNAT2Del C-ter is expressed in the membrane.

Figure 2 Confocal microscopy images testing for cellular localization of truncated SNAT2-AcGFP fusion constructs

SNAT2WT (A), SNAT2Del C-ter (B), SNAT2Del TM11 (C), control non-transfected cells (D) and the bright-field control image (E) showing that cells were present. The arrows indicate regions of intense cell boundary fluorescence. AcGFP was attached in frame to the N-terminus of SNAT2. Scale bars, 50 μm.

In order to test directly whether amino acid transport by SNAT2Del C-ter is impaired, we performed amino acid uptake experiments. SNAT2WT-transfected cells showed significant specific MeAIB (a specific SNAT substrate) uptake activity (7-fold higher than vector-transfected cells), whereas uptake was insignificant in SNAT2Del C-ter- and vector-transfected cells (Figure 1B). These results support the electrophysiological analysis of SNAT2Del C-ter, indicating that deletion of the SNAT2 C-terminus results in a loss of electrogenic transport current, as well as uptake activity.

The loss of alanine transport activity of SNAT2Del C-ter may be caused by impaired binding of alanine to the truncated transporter. However, alanine application induced large outward currents in the presence of intracellular KSCN for SNAT2Del C-ter (Figure 5), which allowed us to determine the apparent affinity of SNAT2Del C-ter for alanine (internal KSCN generates an inward current by moving outward through the uncoupled leak anion conductance, thus generating alanine-dependent current in the absence of electrogenic transport current). The apparent alanine Km values were 175±64 μM for SNAT2WT (Figure 3B, n=4) and 59±6 μM for SNAT2Del C-ter (Figure 3A, n=4). This result indicates that removal of the SNAT2 C-terminus does not interfere with alanine binding. Therefore the impairment of alanine transport activity cannot be caused by an increase in the Km for this amino acid.

Figure 3 The C-terminal deletion does not interfere with substrate binding

The apparent affinities for the substrate L-alanine of SNAT2WT (A) and SNAT2Del C-ter (B) were determined by recording substrate-inhibited anion leak currents as a function of alanine at 0 mV in the presence of 140 mM intracellular KSCN (SNAT2Del C-ter) and 140 mM extracellular NaSCN (SNAT2WT).; Imax, maximum current; Km, Michaelis constant. Values are means ± S.D. for at least three independent experiments.

The C-terminus is involved in controlling the voltage dependence of amino acid transport

To test whether transport activity of SNAT2Del C-ter is restored by an increase in the electrical driving force, we determined the voltage dependence of L-alanine-induced transport currents. As shown previously, application of L-alanine (10 mM) evoked inward transport currents in SNAT2WT. These transport currents showed a relatively small dependence on the membrane potential from −60 mV to +60 mV, but increased steeply at a potential of −90 mV (1.95 times the current at 0 mV; Figures 4B and 4C). The reason for this steep current increase at −90 mV is not known, but may be related to a shift in the rate limiting step at very negative potentials. The voltage dependence of alanine-induced (10 mM) transport currents mediated by SNAT2Del C-ter was the opposite to that of SNAT2WT. Transport currents were only observed at positive membrane potentials, but not at potentials more negative that 0 mV (Figures 4A and 4C). The transport current was restored to the same level as SNAT2WT at +60 mV (−150±30 pA for SNAT2Del C-ter and −132±30 pA for SNAT2WT). These results indicate that the SNAT2 C-terminus plays an important role in regulating the voltage-dependent processes in the transport cycle.

Figure 4 The deletion of the C-terminus affects the voltage dependence of substrate transport

I-V relationships of L-alanine-induced transport currents at a concentration of 10 mM for SNAT2Del C-ter (A) and SNAT2WT (B). (C) The average current-voltage relationships of alanine-induced transport currents in non-transfected cells (control, open circles), SNAT2Del C-ter (solid squares) and SNAT2WT (solid triangles). aa, amino acid. Values are means ± S.D. for at least three independent experiments.

Anion leak current is unaffected by the C-terminal truncation

In a previous publication [8], we reported that SNAT2 mediates an anion leak conductance, which is inhibited by the transported substrate. In order to test if deletion of the C-terminus of SNAT2 affects this anion leak conductance, we performed experiments with the highly permeant anion SCN on the intracellular side of the membrane. Application of 10 mM L-alanine induced outwardly directed currents in SNAT2WT at negative membrane potentials (Figure 5B), which is caused by inhibition of a tonic inward anion current carried by SCN leaving the cell. As expected, the outward currents increased at more negative transmembrane potentials, which increased the driving force for SCN efflux. The total current was a sum of transport current and anion leak current with a reversal potential for SNAT2WT of −1±4 mV (Figure 5C, n=6), as reported previously [8]. Application of 10 mM alanine also induced outwardly directed currents in SNAT2Del C-ter with a similar magnitude to that of the WT transporter at −90 mV (+360±130 pA for SNAT2WT and +290±60 pA for SNAT2Del C-ter). The reversal potential for SNAT2Del C-ter current was shifted to a positive value (+46±2 mV, Figure 5C, n=7). This result is consistent with the idea that the alanine-induced current is dominated by the anion component in SNAT2Del C-ter, due to the small transport current at voltages more negative than +30 mV.

Figure 5 Deletion of the C-terminus decreases, but does not eliminate, anion leak current

Voltage dependence of alanine-sensitive currents mediated by SNAT2Del C-ter (A) and SNAT2WT (B), in the presence of 140 mM intracellular KSCN and 140 mM extracellular NaMes. 10 mM L-alanine was applied at zero time, as indicated by the grey bar. (C) The current–voltage relationships of 10 mM L-alanine-sensitive currents in non-transfected cells (open circles), SNAT2Del C-ter- (open triangles) and SNAT2WT- (filled squares) expressing cells in the presence of 140 mM intracellular KSCN. Values are means ± S.D. for at least three independent experiments.

Na+ binding to transporters is unaltered by the C-terminal truncation

Na+ activates an anion leak conductance in SNAT2, which we used as a tool to determine the apparent affinity of the WT and truncated transporters for Na+ [8,20,22]. Upon application of extracellular Na+ in the presence of intracellular SCN, large inward currents were observed in HEK-293T cells expressing SNAT2WT (Figures 6A and 6B, −98±16 pA, n=8, 0 mV) or SNAT2Del C-ter (Figures 6A and 6B, −130±40 pA, n=7, 0 mV, in the absence of amino acid; original traces are shown in Figure 6A). Non-transfected control cells showed only small current responses to Na+ jumps under the same conditions (Figures 6A and 6B, −18±2 pA, n=10), and the current evoked by SNAT2WT was 5-fold higher than control cells. Within experimental error, the current evoked by SNAT2Del C-ter was in the same range as that of SNAT2WT. The leak current was Na+ concentration dependent (Figures 6C and 6D). Kinetic analysis indicated that the apparent affinity of the transporters for Na+ was 2-fold higher for SNAT2Del C-ter (Km=43±9 mM, n=8) compared with SNAT2WT (Km=100±7 mM, n=5) at pH 8.0. This result indicates that Na+ affinity is not reduced by deletion of the C-terminus, excluding the possibility that the loss of transport activity of SNAT2Del C-ter is caused by a decrease of the Na+ Km of the transporter.

Figure 6 SNAT2Del C-ter retains a normal apparent affinity for Na+

Whole-cell current recordings were performed with a KSCN-based pipette solution (140 mM) and at 0 mV transmembrane potential. (A) Comparison of typical leak anion currents induced by the application of 140 mM extracellular Na+ among non-transfected cells (control), SNAT2Del C-ter- and SNATWT-expressing cells, as indicated by the grey bars. (B) Statistical analysis of average Na+-induced currents shown in (A). Leak anion currents (solid squares) as a function of extracellular Na+ for SNAT2Del C-ter (C) and SNATWT (D), after subtracting the unspecific currents (open circles) determined from non-transfected cells. The open squares are the results from the original experiments before subtraction of the unspecific leak currents determined in non-transfected control cells. Values are means ± S.D. for at least three independent experiments.

In the absence of amino acid substrate, SNAT2WT induces transient currents in response to step changes of the transmembrane potential, which are believed to be caused by electrogenic Na+ binding to and dissociation from the transporters [22]. As shown in Supplementary Figure S1A (at http://www.BiochemJ.org/bj/434/bj4340287add.htm), SNAT2Del C-ter exhibited similar transient currents in response to voltage jumps as SNATWT, although the transient currents evoked by SNAT2Del C-ter were smaller in magnitude (Supplementary Figures S1C and S1D). Both WT and SNAT2Del C-ter transient currents exceeded significantly the non-transfected control current levels (Supplementary Figure S1B). The charge movement Q, obtained by integrating the on and the off response of these currents, were similar to each other in WT and truncated transporters within experimental error (Supplementary Figures S1E and S1F), suggesting that this charge movement is capacitive in nature. The voltage dependence of the charge movement was unchanged by the truncation (Supplementary Figure S1). Overall, these results are consistent with the data from Na+-induced leak currents, indicating that removal of the SNAT2 C-terminus does not interfere with Na+ binding.

The effect of extracellular pH

As demonstrated in the literature, amino acid transport by SNAT2 is highly pH dependent [3,23], with a maximum transport rate observed around pH 8.0 (+60 mV, Supplementary Figure S2 at http://www.BiochemJ.org/bj/434/bj4340287add.htm). Because a histidine residue in the C-terminus (H504A) was proposed to be involved in this pH effect [21], we tested the pH dependence of alanine transport currents of SNAT2Del C-ter at +60 mV, where transport current is substantial. As shown in Supplementary Figure S2, SNAT2Del C-ter transport current was activated maximally at pH 8.0, in analogy to the WT transporter. However, the pH dependence was somewhat reduced, but not eliminated, compared with that of SNAT2WT. This result supports the previously published data on the H504A mutant transporter, which has a decreased pH sensitivity [21].

Truncation before TM11 results in a non-functional transporter

The crystal structures of transporters thought to be homologous with SNAT2 suggest that TM11 is not an integral part of the transport pathway [24]. In fact members of the SGP (spore germinating protein) family, which are distant relatives of SNAT2, have only 10 TM domains, but still bind amino acid [25]. Therefore, we generated and tested a transporter that was C-terminally truncated after leucine 451, which deleted TM11 (SNAT2Del TM11). The transport current induced by SNAT2Del TM11 in the presence of 10 mM of alanine was −7±3 pA at a membrane potential of 0 mV (Figure 7A, n=5). This is, within experimental error, the same as that in non-transfected cells (−4±4 pA, n=10, Figure 7A) suggesting when taken together with the MeAIB uptake data (Figure 7A) that the amino acid transport function was eliminated by removing TM11. Transport cannot be restored by an increase of the electrical driving force (Figure 7B). Alanine-inhibited anion leak currents (Figure 7C), as well as Na+-induced anion leak currents, were not significantly larger than those in control cells (n=6, Figure 7D). All of these results indicate that SNAT2Del TM11 lost transport function. The cell surface expression of SNAT2Del TM11 transporter was reduced significantly (Figure 2C), compared with SNAT2WT (Figure 2A). Therefore the functional impairment of the SNAT2Del TM11 transporter is most probably caused by a defect in the cell surface targeting of the transporter.

Figure 7 Deletion of TM11 results in a non-functional transporter

(A) Transport currents induced by non-transfected cells, SNAT2WT- and SNAT2Del TM11-expressing cells at 10 mM of alanine and MeAIB uptake by vector [pBK-CMV Δ(1098–13000], SNAT2Del C-ter and SNAT2WT in transiently transfected HEK293 cells. Transport of [14C]MeAIB (40 μM) was measured at 1 min in NaMes-containing buffer. (B) Voltage-dependence of transport currents induced by SNAT2Del TM11 in the presence of 10 mM of alanine in non-tranfected cells (open circles), SNAT2WT- (squares) and SNAT2Del TM11-expressing cells (triangles). (C) Average current–voltage relationships of anion leak current in the presence of 10 mM of alanine among non-transfected cells (control, open circles), SNAT2WT- (squares) and SNAT2Del TM11-expressing cells (triangles). (D) Anion leak currents induced by 140 mM of extracellular Na+. The open triangles (SNAT2WT) and open circles (SNAT2Del TM11) are the currents from the original experiments before the subtraction of the unspecific leak currents (open squares, control, non-transfected cells). The solid circles are the anion leak currents induced by SNAT2Del TM11 after subtracting the unspecific currents (open circles). The inset shows the truncation site. Values are means ± S.D.

DISCUSSION

The sodium-dependent neutral amino acid transporter SNAT2, the ubiquitous member of the SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues [13]. Based on the predicted topology of SNAT2 from related plant transporters [1517], and a homology model published previously [20], SNAT2 includes 11 TMs with an intracellular N-terminus and an extracellular C-terminus (Figure 1). For most sodium-dependent amino acid transporters, such as the glutamate transporting EAATs (excitatory amino acid transporters) [26], LeuT (the leucine transporter) [24] and the GABA (γ-aminobutyric acid) transporter [26,27], the C-terminus is located in the cytoplasm. Studies on various members of the Na+-dependent neurotransmitter transporter family regarding the cellular mechanisms of their functional expression have highlighted that the C-terminus is critical for the regulation of trafficking to the plasma membrane [28,29]. However, the C-terminus of SNAT2 is extracellular, which probably precludes it from playing a role in the mechanisms that regulate membrane expression and/or targeting. Consistent with this idea, truncation of the C-terminus did not cause a defect in expression of the transporter in the plasma membrane, suggesting a lack of involvement of the C-terminus in processes that regulate intracellular trafficking.

The two main findings of the present study are: (i) the C-terminus is critical for the amino acid transport function of the transporter, as demonstrated by the defective alanine transport at voltages ≤0 mV, as well as defective MeAIB uptake; and (ii) the C-terminus contributes to controlling the voltage dependence of amino acid transport. At positive voltages, alanine-induced transport current was observed, indicating a dramatic change in the rate limitation by voltage-dependent steps in the transport cycle. In SNAT2Del C-ter, the transport rate may be limited by a step that is accelerated at positive membrane potentials. Alternatively, negative membrane potential may accelerate transition into an inactive state that does not allow amino acid transport. We cannot currently differentiate between these possibilities. Taken together, these results suggest that the C-terminal region plays an important role in the amino acid translocation process, or relocation of the empty transporter. A similar behaviour was reported for hNET1 [human noradrenaline (norepinephrine) transporter], in which deletion of the last seven amino acids of the C-terminal region did not affect cell surface expression, but severely affected the uptake of [3H]noradrenalin [28]. Overall, the strong influence of the C-terminal domain on the transport rate and its voltage dependence is somewhat surprising, because this domain is thought to be outside of the core membrane domain of the transporter on which the membrane potential is expected to have its major effect. Therefore it is probable that the C-terminal domain affects substrate transport and its voltage dependence through a regulatory, possibly allosteric, process. This interpretation is consistent with results from a transporter with the C-terminal H504A mutation, for which an allosteric regulation of the transport rate by protonation was proposed [21].

In addition to His504, the C-terminus of SNAT2 contains two potentially charged residues Asp494 and Asp498, which may contribute to regulation of the voltage dependence of amino acid transport. To test this possibility, we neutralized both aspartate residues by replacement with alanine (Supplementary Figure S3 at http://www.BiochemJ.org/bj/434/bj4340287add.htm). However, within experimental error, the mutant transporters displayed the same alanine transport activity as the WT transporter. Therefore, these two acidic residues are unlikely to play a major part in the modulatory process. We hypothesize that regulation of transport by the C-terminus is not caused by electrostatic interaction. The identification of other C-terminal amino acid residues that do contribute to the modulatory effect awaits further experimentation.

In contrast to the amino acid transport activity, anion leak currents, induced by Na+ application or inhibited by alanine application, were little affected by the C-terminal truncation. These results suggests that the anion leak pathway, which most probably is associated with the core translocation domain of the transporter, is not modulated by the C-terminal domain. Interestingly, this points to the possibility of modulating the transport activity and the anion leak activity independently from each other, as has been shown previously for the anion conductance of the structurally different transporters from the EAAT family [3032]. The results also suggest that amino acid translocation is not required for the anion conductance to work.

SNAT2Del C-ter exhibits approx. 3-fold higher apparent alanine affinity (Figure 3) and 2-fold higher apparent Na+ affinity (Figure 6) compared with the WT transporter. Voltage-jump-induced transient currents, which are caused by the binding/dissociation of Na+ to/from the transporter, are also not affected by the truncation. These results indicate that the C-terminal truncation does not disrupt the structure of the amino acid- and Na+-binding sites, and does not interfere with Na+ access to its binding site. These binding sites are most probably deeply buried in the membrane, as suggested by the structural homology model that we developed on the basis of the crystal structures of several bacterial amino acid transporters [20]. In fact, the Na+-binding site is composed of TM1 and TM8, which are predicted to be located on the side opposite from the C-terminal domain of the transporter [20].

The pH dependence of amino acid transport by SLC38 members is clearly established [23,3335]. Whereas SNAT3 and SNAT5 couple amino acid movement to proton transport [33,36,37], the effect of H+ on SNAT1 and SNAT2 is modulatory, inhibiting transport at pH<8.0 [3,38,39]. Truncation of the C-terminus results in a decrease in sensitivity of SNAT2 to pH, although inhibition of transport at pH 7.0 and 6.0 is still observed. These results are consistent with a previous report that linked the pH sensitivity to histidine 504 in the C-terminal region of SNAT2 [21]. This histidine is deleted in the truncated transporter in the present study. Although a modulatory role of His504, depending on its protonation state, is probable, this cannot account for the total pH sensitivity since the transporter remains somewhat pH sensitive even after C-terminal truncation. Therefore it is probable that other protonation sites exist that contribute to the modulation of the amino acid transport rate by the external pH.

We also tested the properties of a construct in which the C-terminus was removed after TM10. TM11 is thought to be removed from the catalytic core of the transport machinery. Despite this fact, the deletion of TM11 rendered the transporter non-functional in any of the aspects tested. Because the deletion of TM11 resulted in a defect in cell surface expression, it is probable that membrane targeting is disrupted in the absence of TM11.

In conclusion, in the present study, we have demonstrated that the C-terminal domain of SNAT2 is not essential for trafficking and recruitment to the plasma membrane. However, removal of the C-terminal region impairs severely amino acid transport, suggesting that the C-terminal domain affects the functional states of SNAT2, which switches between outward- and inward-facing conformations during the transport cycle. Therefore, the C-terminal domain of SNAT2 probably plays an important role in modulating the rate of translocation, as well as the voltage dependence of this rate, most probably through an allosteric mechanism.

AUTHOR CONTRIBUTION

Zhou Zhang, Catherine Zander and Christof Grewer designed the experiments. Zhou Zhang and Catherine Zander performed the experiments. Zhou Zhang, Catherine Zander and Christof Grewer analysed the experiments and wrote the manuscript.

FUNDING

This work was supported by a grant from the NIH [grant number 7R01NS049335-05] awarded to C.G., by grants from the NSFC [grant number 30870560], the Innovation Program of Shanghai Municipal Education Commission [grant number 09ZZ139], the Shanghai Normal University [grant numbers DZL808, DYL810 and DRL804] and the Shanghai Leading Academic Discipline [grant number S30406] to Z.Z.

Acknowledgments

We thank Dr Ana Diez-Sampedro for help with the MeAIB uptake and Dr Dennis McGee for help with confocal microscopy.

Abbreviations: CaGlu2, calcium gluconate; CMV, cytomegalovirus; Del, C-ter, 13 residues deleted from the C-terminus; EAAT, excitatory amino acid transporter; GFP, green fluorescent protein; AcGFP, Aequorea coerulescens GFP; HEK, human embryonic kidney; MgGlu2, magnesium gluconate; MeAIB, N-methylaminoisobutyric acid; NaMes, sodium methanesulfonate; Rs, series resistance; SLC, solute carrier; SNAT, sodium-coupled neutral amino acid transporter; TM, transmembrane domain; WT, wild-type

References

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