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Originally published In Press as doi:10.1074/jbc.M212718200 on April 8, 2003

J. Biol. Chem., Vol. 278, Issue 26, 23720-23730, June 27, 2003
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Functional Properties and Cellular Distribution of the System A Glutamine Transporter SNAT1 Support Specialized Roles in Central Neurons*

Bryan Mackenzie {ddagger}, Martin K.-H. Schäfer §, Jeffrey D. Erickson ¶ || {ddagger}{ddagger}, Matthias A. Hediger {ddagger}, Eberhard Weihe § and Hélène Varoqui ¶ ** {ddagger}{ddagger}

From the {ddagger}Membrane Biology Program and Renal Division, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, the §Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps University Marburg, D-35033 Marburg, Germany, and the Neuroscience Center and the Departments of ||Pharmacology and **Ophthalmology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112

Received for publication, December 13, 2002 , and in revised form, March 19, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K0.5 {approx} 0.3 mM), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na+ electrochemical gradient. The voltage-dependent binding of Na+ precedes that of the amino acid in a simultaneous transport mechanism. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SNAT1 mediates Na+-dependent presteady-state currents (Qmax {approx} 9 nC) and a nonsaturable cation leak with selectivity Na+, Li+ » H+, K+. Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na+ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via {alpha}-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutamine uptake is crucial to neurotransmission in central excitatory and inhibitory neurons. Neuronal glutamine is derived from astrocytes and is the major precursor for neurotransmitter glutamate and {gamma}-aminobutyric acid (GABA)1 (1–4). Glutamine uptake into neurons is mediated largely by a System A-like transporter (5) and is a crucial step in the "glutamate-glutamine cycle" (6–8). The neuronal enzyme phosphate-activated glutaminase (PAG) catalyzes the hydrolysis of glutamine to glutamate (9), which can also serve as a direct GABA precursor (10). Specific vesicular transporters package glutamate or GABA for synaptic release. Extrasynaptic glutamate is cleared by specific transporters (EAATs) found on astrocytes and at postsynaptic neuronal targets (11, 12). The predominance of astrocytic rather than neuronal glutamate uptake systems (12) and the lack of EAAT expression on presynaptic membranes highlight a requirement for a glutamate-glutamine cycle. However, GABA inactivation transporters are also present on presynaptic membranes (13). In astrocytes, the predominant fate of glutamate is conversion to glutamine by the astrocyte-specific glutamine synthetase. Completing the cycle, glutamine export from astrocytes is likely to be mediated by the System N or ASCT2 transporters (8, 14, 15). 15N/13C NMR studies in vivo have demonstrated that the fluxes through glutamine synthesis and glutamate/GABA-glutamine cycling are both substantial and necessary to replenish neurotransmitter pools. Furthermore, these fluxes represent the major fraction of glucose utilization and are central to the normal energetics of the brain (16, 17). Glutamine may also constitute a significant fuel for many neuronal populations, with glutamate being oxidized to {alpha}-ketoglutarate, which enters a truncated tricarboxylic acid (TCA) cycle (18, 19).

The sodium-coupled neutral amino acid transporter SNAT1, which was renamed as proposed in a recent review (20), was the first member of the System A amino acid transporter subfamily to be identified at the molecular level (21). The rat, human, and mouse cDNAs have each been isolated (21–24) and were formerly referred to as ATA1, SA2, SAT1, NAT2, or GlnT. SNAT1 mediates the Na+-dependent transport of glutamine and is highly enriched in neurons (21). In the present study we explored the functional properties of SNAT1 by applying voltage-clamp and radiotracer techniques in cRNA-injected Xenopus oocytes. Using confocal laser-scanning double-immunofluorescence microscopy, we examined the cellular localization of SNAT1 and the phenotypes of the neuronal populations in which SNAT1 is expressed. We find that the functional properties and cellular distribution of SNAT1 support a key role within the glutamate/GABA-glutamine cycle, and consider additional roles for SNAT1 in amino acid metabolism in the brain.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Oocytes were isolated from Xenopus laevis (under 2-aminoethylbenzoate anesthesia), treated with collagenase A (Roche Diagnostics) and stored at 18 °C in modified Barths' medium (25). SNAT1 (Slc38a1) cDNA was subcloned into the pTLNii vector under SP6, and SNAT1 cRNA was synthesized in vitro with the use of the Ambion mMESSAGE mMACHINE kit. SNAT2 cRNA was prepared as described (26). (The product of the Slc38a2 gene, SNAT2 was previously named SA1, ATA2, or SAT2.) Oocytes were injected with {approx}50 ng of cRNA and incubated 2–3 days before radiotracer or voltage-clamp experiments were performed at 22 to 23 °C (except as noted in Fig. 3H). Standard Na+ uptake medium comprised 100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES (pH 7.5 with Tris base). For low pH media, we lowered the HEPES concentration to 5 mM and added 5 mM MES. For Na+-free or low Na+ medium, NaCl was replaced by equimolar choline chloride (ChoCl). For low Cl– medium, Cl– was replaced by equimolar gluconate. Solutions containing niflumic acid (where specified) also contained 0.1% dimethyl sulfoxide.



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  		FIG. 3.
Currents associated with amino acid transport in oocytes expressing SNAT1. A, current/voltage relationship of the currents evoked by 10 mM L-alanine or L-glutamine in 100 mM NaCl medium (pH 7.5). B–G, saturation kinetics and voltage dependence of the L-alanine currents activated by Na+ (filled circles) or Li+ (empty circles) at pH 7.5. Data in B–D were for a single oocyte and those in E–G for a second oocyte from the same batch. Error bars represent the error in the estimate of kinetic parameters according to Equation 1. B, C, and D, Saturation kinetics of the L-alanine-evoked currents were determined over the Vm range –150 to +50 mV by applying L-alanine at 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mM. At –70 mV (B), in Na+, ; whereas in Li+, . The Hill coefficient (nH) for L-alanine did not differ from 1 at any Vm (data not shown). C, voltage dependence of . D, voltage dependence of . E–G, cation dependence of the currents evoked by L-alanine presented at a subsaturating concentration. E, I/V relationships for 0.2 mM L-alanine at 2, 10, and 100 mM Na+. F and G, Na+ and Li+ saturation kinetics were determined from the currents evoked by 0.2 mM L-alanine at 1, 2, 5, 10, 20, 35, 50, 75, and 100 mM Na+ or Li+. At Vm = –70 mV (F), the derived kinetic parameters for Na+-activated currents were ; whereas for Li+, . The value of nH for either cation did not vary with Vm (data not shown). G, voltage dependence of the half-maximal cation concentrations. H, temperature dependence of the currents associated with the Na+/L-alanine cotransport and Na+ leak activities mediated by SNAT1 at –70 mV. Cotransport (Na+/L-Ala, filled circles) is given as the current evoked by 10 mM L-alanine in 100 mM NaCl (pH 7.5). Na+ leak (inverted gray triangles) is given as the current induced by switching from Na+-free (100 mM ChoCl) medium to 100 mM Na+ (pH 7.5) in the absence of amino acid. Data were fitted to the Arrhenius equation (Equation 2) with activation energy (Ea) of 21.8 ± 2.0 kcal·mol–1 for Na+/L-alanine cotransport (r2 = 0.97, ln A = 43 ± 4) and 7.8 ± 0.7 kcal·mol–1 for the Na+ leak (r2 = 0.97, ln A = 18 ± 1).

 

A two-microelectrode voltage clamp was used to measure currents in control oocytes and oocytes expressing SNAT1 or SNAT2. Microelectrodes (resistance 0.5–5 M{Omega}) were filled with 3 M KCl. Voltage-clamp experiments comprised three protocols: (i) Continuous current recordings were made at a holding potential (Vh) of –70 mV, –50 mV, or +10 mV, low-pass filtered at 20 Hz, and digitized at 20 Hz. (ii) Oocytes were clamped at Vh = –50 mV, and step changes in membrane potential (Vm) were applied (from +50 mV to –150 mV in 20-mV increments), each for a duration of 100 ms, before and after the addition of substrate. Current was low-pass filtered at 500 Hz and digitized at 5 kHz. Steady-state data were obtained by averaging the points over the final 16.7 ms at each Vm step. (iii) A 1-s voltage ramp was applied from –100 mV to +60 mV, preceded by a 20-ms settling phase at –100 mV. Current was filtered at 500 Hz and digitized at 5 kHz. Test solutions were washed out with substrate-free medium (100 mM ChoCl, pH 7.5) for at least 2 min. Steady-state data from protocols (i) or (ii) were fitted to a modified Hill relationship (Equation 1) for which I is the evoked current, Imax the derived current maximum, S the concentration of substrate S (Na+, Li+ or amino acid), the substrate concentration at which current was half-maximal, and nH the Hill coefficient for S.

(Eq. 1)

Currents obtained over the range of temperatures 21–33 °C (Fig. 3H) were fitted to an integrated Arrhenius function (Equation 2), for which Ea is the Arrhenius activation energy, A the y-intercept, R the universal gas constant (1.987 cal·mol–1·K), T the absolute temperature, and I the current evoked by 100 mM Na+ or the 10 mM L-alanine-evoked current in Na+.

(Eq. 2)

Radiotracers were obtained from PerkinElmer Life Science Products: L-[3-3H]alanine and L-[3,4(N)-3H]glutamine were used at final specific activity of 0.15–2 GBq·mmol–1 and 2-methylamino-[14C]isobutyrate (MeAIB), at 4 MBq·mmol–1. At the end of the uptake period, oocytes were rinsed with ice-cold ChoCl medium and solubilized with 5% SDS before 3H or 14C contents were assayed by liquid scintillation counting. Na+/amino acid coupling stoichiometry was determined by direct comparison of net inward charge with radiotracer amino acid (L-[3H]alanine or L-[3H]glutamine) accumulation in individual oocytes under voltage clamp (25–27). The amino acid-evoked current was integrated with time to obtain the amino acid-dependent charge (QAla or QGln) and converted to a molar equivalent (assuming monovalency) using the Faraday.

Following step-changes in Vm using protocol ii, we obtained pre-steady-state currents in oocytes expressing SNAT1. These were isolated from capacitive transients (which decayed with half times of 0.5–0.9 ms) by subtracting the currents obtained for the same oocyte in the presence of 10 mM L-alanine and subtracting the final steady-state level. The compensated currents thus obtained were integrated with time to obtain charge movement (Q) and fitted to the Boltzmann relationship (Equation 3) for which maximal charge Qmax = Qdep – Qhyp (where Qdep and Qhyp represent the charge at depolarizing and hyperpolarizing limits), V0.5 is the Vm at the midpoint of charge transfer, z is the apparent valence of the movable charge, and F, R, and T have their usual thermodynamic meanings.

(Eq. 3)

Confocal laser-scanning double-immunofluorescence microscopy was performed as previously described (28, 29). In brief, deparaffinized frontal serial sections of adult rat brain fixed in Bouin-Hollande solution were incubated overnight at room temperature with the polyclonal rabbit antibody to SNAT1 (1:400 dilution) together with one of the following antibodies as markers: polyclonal guinea pig antibody to VGLUT1 (1:800 dilution), polyclonal guinea pig antibody to VGLUT2 (1:800 dilution), monoclonal mouse antibody KLON SY 38 (2 µg·ml–1; Roche Applied Science) to the synaptic vesicle marker synaptophysin, or a monoclonal mouse antibody MAB3418 (25 µg·ml–1; Chemicon International) to microtubule-associated protein-2 (MAP-2), an established marker of neuronal dendrites. Antibodies to VGLUT1 and VGLUT2 were raised against the same fusion proteins as our rabbit antisera to VGLUT1 and VGLUT2 (29). Immunoreactivities were visualized with indocarbocyanine(Cy3)-conjugated species-specific secondary antibodies diluted 1:200 and applied for 45 min at 37 °C, resulting in a red-orange fluorescence labeling, or with biotinylated IgG (Dianova) diluted 1:200 and applied for 45 min at 37 °C, followed by incubation with Alexis 488-conjugated streptavidin (MoBiTec) for 2 h at 37 °C, resulting in a green fluorescence. Sections were analyzed with the Olympus Fluoview confocal laser-scanning microscope (Olympus Optical) and documented as false color confocal images.

Somatodendritic labeling by SNAT1 antibody in all areas of the brain was blocked in the presence of 10 µM GST-SNAT1 fusion protein but not by GST protein alone (data not shown). Our SNAT1 antibody was previously characterized in heterologous expression systems and cerebellar granule neuronal cultures and by Western analysis of brain homogenates and cultured cells (21). Our VGLUT1 and VGLUT2 antibodies were previously characterized by double-fluorescence deconvolution microscopy and Western blot analysis (30). Fluorescence labeling was blocked by preadsorption of the antisera with 10 µM GST fusion proteins, and immunoreactive bands were absent following preadsorotion with 1 µM GST fusion proteins (data not shown).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SNAT1 Is a Na+-dependent, Neutral Amino Acid Transporter—We explored the characteristics of SNAT1 by measuring radiotracer uptake and currents associated with the expression of SNAT1 in Xenopus oocytes. We found that SNAT1-mediated amino acid transport was saturable, Na+-dependent, and rheogenic. SNAT1 stimulated the uptake of 100 µM [14C]MeAIB, a characteristic inhibitor of System A, in Na+ but not in choline. The Na+-dependent component of MeAIB transport mediated by SNAT1 was 44-fold that in control oocytes (Fig. 1A). A range of amino acids evoked large inward currents of up to –2,000 nA in voltage-clamped oocytes expressing SNAT1, whereas amino acid-evoked currents in control oocytes were < –3 nA (see also Ref. 31). Typical current recordings are shown at a holding potential (Vh) of –70 mV (Fig. 1B). The addition of Na+ and subsequently 10 mM L-alanine had negligible effects in control oocytes, but both Na+ alone and Na+ plus L-alanine evoked inward currents in oocytes expressing SNAT1. Similar currents were obtained previously for SNAT2 except that the currents evoked by Na+ alone were significantly smaller for SNAT2 (26).



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  		FIG. 1.
SNAT1 mediates Na+-dependent amino acid transport. A, uptake of 100 µM [14C]MeAIB in control oocytes and oocytes expressing SNAT1, measured over 20 min at 23 °C in 100 mM NaCl or choline (Cho) chloride medium (pH 7.5). Data are mean ± S.E. for 15–16 oocytes in each group. B, typical continuous current recordings in a control oocyte and an oocyte expressing SNAT1. Oocytes were clamped at –70 mV and superfused with 100 mM ChoCl (blank boxes), then NaCl medium (hatched boxes), each at pH 7.5. 10 mM L-alanine was applied as indicated by the black boxes before washing out with ChoCl.

 

Substrate Profile of the System A Transporters SNAT1 and SNAT2—To examine the substrate profile of SNAT1 we measured the currents evoked by a range of amino acids (each at 10 mM) at –70 mV (Fig. 2A). Currents were normalized to those evoked by L-alanine for which the half-maximal concentration was 0.3 ± 0.02 mM (see Fig. 3B). SNAT1 preferred L-alanine over the D-isomer, primarily due to reduced apparent affinity (data not shown). SNAT1 transported small, neutral aliphatic amino acids that were ranked (based on absolute currents): L-cysteine, L-alanine, L-serine, L-asparagine, L-glutamine, L-histidine, glycine, L-methionine, L-threonine, MeAIB, and the imino acid L-proline. The 24 ± 5% reduction in current evoked by L-glutamine compared with L-alanine was a Vmax effect and not due to a reduction in apparent affinity (, data not shown). However, the ratio Imax/K0.5 as an index of substrate selectivity was similar for L-alanine and L-glutamine (Fig. 2B). L-Threonine, an efficient substrate for System ASC, did not rank highly among SNAT1 substrates (Fig. 2A). SNAT1 did not exhibit significant reactivity with the charged amino acids L-arginine, L-aspartate, and L-lysine. L-Glutamate evoked very small currents but the very low affinity (, data not shown) and selectivity (Fig. 2B) indicate that SNAT1 is unlikely to represent a low affinity glutamate transporter described in the literature (12). SNAT1 essentially excluded the branched-chain amino acids (isoleucine, L-leucine, and L-valine, although L-valine showed marginal reactivity), L-phenylalanine, L-tryptophan, and 2-aminobicyclo (2,2,1)heptane-carboxylate (BCH), a characterizing analogue for the System L and System B0+ amino acid transporters (31–33).



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  		FIG. 2.
Substrate profile of the System A transporters SNAT1 and SNAT2. A, currents (mean ± S.E., n = 3–9 oocytes) evoked by a range of amino acids (10 mM) at –70 mV were normalized to the current evoked by L-alanine for SNAT1 (black bars, –368 ± 43 nA, n = 9) and for SNAT2 (hatched bars, –608 ± 75 nA, n = 7). Except where indicated for D-alanine, all test substrates were L-isomers; BCH, 2-aminobicyclo (2,2,1)heptane-carboxylate; MeAIB, 2-(methylamino)isobutyrate. Mean currents were compared between SNAT1 and SNAT2 with Student's t test: *, p < 0.05; ***, p < 0.001. B, Imax/K0.5 (normalized to that for L-alanine) as an index of substrate selectivity.

 

We compared the substrate profile of SNAT1 with that of SNAT2 (26), a second System A subtype of the SLC23 gene family, exhibiting apparent affinities (e.g. ) similar to those for SNAT1. We found only subtle differences between SNAT1 and SNAT2 in terms of substrate profile (Fig. 2A). SNAT2 displayed greater stereospecificity for the L-isomer (at least for alanine) than did SNAT1 but transported L-glutamine and MeAIB with less efficiency. The imino acid L-proline however is a better substrate for SNAT2 than for SNAT1 (see also Refs. 21, 26, and 34). L-Leucine is not a substrate for SNAT1 but displayed marginal reactivity with SNAT2.

Uptake of [14C]MeAIB (Fig. 1A) was only about one-tenth of the uptake of L-[3H]alanine (data not shown) and MeAIB always evoked a current much smaller than that evoked by L-alanine in oocytes expressing SNAT1 or SNAT2 (Fig. 2A). This was primarily a result of a reduction in Vmax, since 10 mM MeAIB was effectively saturating despite a modest reduction in apparent affinity (, data not shown). Selectivity for MeAIB was low compared with L-alanine or L-glutamine (Fig. 2B). Our data suggest that MeAIB binds to the System A transporters with reasonable specificity (see also Ref. 26) but that architectural constraints significantly reduce the velocity at which MeAIB is transported. Whereas inhibition of radiotracer uptake by MeAIB (when used at appropriate concentrations) is a specific test for System A, our data demonstrate that MeAIB is not itself a model or paradigm substrate per se for System A since it is poorly translocated.

Saturation Kinetics and Voltage Dependence of SNAT1-mediated Amino Acid Transport—We examined the voltage dependence and concentration dependence of the amino acid-evoked currents mediated by SNAT1 (Fig. 3). Amino acid-evoked currents are voltage-dependent, as illustrated for L-alanine and L-glutamine (Fig. 3A). Evoked currents varied with Vm in a linear fashion between –150 mV and –50 mV. Further slight reductions in current were observed at depolarized Vm (–30 mV, –10 mV), but currents persisted at positive Vm (up to +50 mV). Whereas L-glutamine is likely to be the principal substrate for the neuronal SNAT1 in vivo, we chose to conduct our subsequent experiments with L-alanine because of the appreciably larger currents obtained with L-alanine and its relative stability in aqueous solution.

Alanine-evoked currents were saturable and followed Michaelis-Menten-type kinetics, with half-maximal concentration (, derived from Equation 1) around 0.3 mM in the presence of Na+ at –70 mV (Fig. 3B). The Hill coefficient for alanine () was 1 and did not differ with in Na+ was independent of Vm in the range –150 mV to –50 mV (Fig. 3C). The increase in beginning at –30 mV and rising to 1.3 ± 0.2 mM by +50 mV should be expected since 100 mM Na+ was no longer sufficient to activate the system maximally at these Vm (see Fig. 3G). As for SNAT2 (data not shown), Li+ also supported alanine-evoked currents in the absence of Na+ but resulted in a reduction in maximal current () of 45 ± 4% at –70 mV (Fig. 3B). Although the was also higher in Li+ than in Na+ at –70 mV, it again reflected the subsaturating cation concentration (see Fig. 3G), whereas the in Li+ did not differ from that in Na+ between –90 mV and –150 mV (Fig. 3C). We conclude that alanine binding per se is independent of voltage and independent of the driving ion (Na+ or Li+).

The relationship of to Vm in Na+ medium was linear between –150 mV and –30 mV and linear again between –10 mV and +50 mV (Fig. 3D). For any given Vm, the alanine-evoked currents were greater at higher Na+ concentrations and the current/voltage relationships were shifted to the right (Fig. 3E). Amino acid transport mediated by SNAT1 is therefore driven by the Na+ electrochemical gradient. Alanine-evoked currents were saturable with Na+ concentration (Fig. 3F), and the Hill coefficient for Na+ () was 1, regardless of Vm. SNAT1 displayed extremely high apparent affinity for Na+. was exquisitely voltage-dependent, rising steeply from 2 ± 0.1 mM at –150 mV to almost 30 mM at –10 mV (indeterminable at positive Vm). We conclude that Na+ binding is voltage-dependent.

Although the K0.5 for Li+ was even more steeply dependent upon Vm than was (Fig. 3G), the K0.5 for Li+ approached that for Na+ at hyperpolarized Vm and was not significantly different at –150 mV. We also noted that, at any given Vm, was always lower in Li+ medium than in Na+ (Fig. 3D). ( did not saturate at hyperpolarized potentials in Na+ but did in Li+, even at potentials at which Li+ is in excess (–150 mV to –110 mV). We conclude that the Vmax of the transporter is significantly limited when Li+ is the driving ion compared with Na+.

SNAT1 is pH-sensitive, with amino acid transport being greatly inhibited at low pH (21, 22, 34). We found that alanine evoked smaller currents at pH 5.5 than at pH 7.5 and no currents in the absence of Na+ at either pH (data not shown) indicating that H+ cannot activate amino acid transport in SNAT1. Consistent with this observation, SNAT1-mediated glutamine transport, although pH-sensitive, was independent of the H+ electrochemical gradient, as demonstrated by the application of a proton ionophore in transfected CV1 fibroblasts (21).

SNAT1 Transports Na+ and Amino Acid by a Simultaneous, Ordered Mechanism—The current evoked by 10 mM alanine in oocytes expressing SNAT1 (at –70 mV) was markedly temperature-dependent, ranging from –312 nA at {approx} 21 °C to –1387 nA at {approx} 33 °C. Arrhenius transformation of the temperature dependence data (Fig. 3H) yielded an activation energy (Ea) of 21.8 ± 2.0 kcal·mol–1, slightly higher than estimates of System A activation energy (15–16 kcal·mol–1) derived from other cell preparations (35, 36), although it is not known which SNAT isoforms were represented in those preparations. The high Ea of {approx} 22 kcal·mol–1 for SNAT1 is consistent with a "carrier" model in which a series of ligand-induced conformational changes execute Na+/alanine cotransport (Fig. 9).



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  		FIG. 9.
Transport activities associated with overexpression of SNAT1 in Xenopus oocytes. Our data are consistent with an ordered, simultaneous transport model for SNAT1 in which Na+/amino acid cotransport is coupled 1:1 and described by a series of temperature-dependent, ligand-induced conformational changes. Our data revealed that Na+ binding is voltage-dependent (an ion well effect) and precedes the binding of the amino acid. The order of dissociation at the internal face is assumed. In this six-state model, the rate kxy describes step x -> y, and at least the rates k61, k12, k21, and k16 should include voltage-dependent terms. We also observed significant uncoupled ion fluxes including a Na+ leak (gNa) that displayed properties of a channel and appears to be discrete from the Na+ flux through the cotransport pathway. SNAT1 expression was also associated with stimulation of endogenous Cl– channel activity (gCl) by an unknown mechanism (? dotted arrow) that may involve Ca2+ activation.

 

The mechanism of cotransport and the order of substrate binding was determined by analyzing the saturation kinetics for both Na+ and alanine as a function of the cosubstrate concentrations (Fig. 4). Our data indicated that both substrates are transported simultaneously, since the half-maximal concentrations (K0.5) for both substrates (Na+ and alanine) rose substantially at low cosubstrate concentrations and were minimal when cosubstrate concentration was saturating (Fig. 4, A and B). In contrast, for a consecutive carrier model, the K0.5 is expected to rise as cosubstrate concentration is increased, since both substrates are effectively competing for the available carriers (37). Imax for Na+ () was exquisitely dependent upon the cosubstrate (alanine) concentration whereas Imax for alanine () did not substantially differ with Na+ concentration (Fig. 4, C and D), i.e. the presence of saturating alanine was always sufficient to drive the transporter at maximal velocity regardless of Na+ concentration, whereas was always limited by the available alanine. Considering a carrier model in dynamic equilibrium (Fig. 9), these data demonstrate that alanine binds after the Na+ binding event.



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  		FIG. 4.
Saturation kinetics as a function of the cosubstrate concentration. Both the L-alanine saturation kinetics as a function of [Na+] and the Na+ saturation kinetics as a function of the L-alanine concentration were determined by measuring the currents evoked by 0.02–10 mM L-alanine at 1–100 mM Na+ (pH 7.5) and fitting data to Equation 1 (r2 always exceeded 0.95). All data in A–D are for a single oocyte at Vm = –70 mV.

 

SNAT1 Presteady-state Currents Are Observed in the Absence of Amino Acid—After step changes in Vm were applied in the absence of amino acid, we observed presteady-state currents in oocytes expressing SNAT1, but not in control oocytes. The compensated presteady-state currents (Fig. 5, inset) were integrated with time, and the relationship of charge (Q) to Vm could be described by a single Boltzmann function with apparent valence (z) of –0.7 and maximal charge (Qmax) {approx} 9 nC (Fig. 5). We did not observe presteady-state currents associated with SNAT1 in the absence of Na+ (data not shown). Our observation of Na+-dependent presteady-state currents in the absence of amino acid is consistent with our conclusions that voltage-dependent Na+ binding precedes alanine binding in SNAT1. SNAT1 presteady-state currents decayed with half-time ({tau}) of 4 –5 ms (data not shown); {tau} was distributed over Vm with maximum {tau} of 4.6 ± 0.1 ms at –26 ± 8 mV.



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  		FIG. 5.
Presteady-state currents associated with SNAT1. Compensated currents (inset, shown from 3 ms after the onset of the change in Vm, protocol ii) associated with SNAT1 (at 100 mM Na+ and in the absence of amino acid) were integrated with time and fitted to the Boltzmann relationship (Equation 3) for which Qmax was 8.8 ± 0.3 nC, V0.5 was –45 ± 2 mV and apparent valence (z) was –0.7 ± 0.1. The turnover rate for alanine transport was determined as Imax/Qmax from the currents evoked by 10 mM L-alanine in the same oocyte and ranged from 203 s–1 at –150 mV and 98 s–1 at –70 mV to 24 s–1 at +50 mV. (Solutions contained 100 µM niflumic acid.)

 

SNAT1 Mediates a Discrete Cation Leak Pathway—SNAT1 exhibited an uncoupled cation transport pathway (leak) in the absence of amino acid substrate. Its presence was first apparent from the large inward currents (I{Delta}Na) observed in oocytes, expressing SNAT1 upon switching from Na+-free to 100 mM NaCl in the absence of amino acid (see Fig. 1B). At –50 mV, I{Delta}Na in oocytes expressing SNAT1 was 22-fold that observed for control oocytes whereas I{Delta}Na was only doubled for SNAT2 (Fig. 6A). We found that I{Delta}Na was concentration-dependent but did not saturate at physiological Na+ concentration (Fig. 6B). To determine the ionic species underlying the leak current in SNAT1, we applied a voltage-ramp protocol to measure reversal potentials (Vr) under varying ionic conditions (Fig. 6C). At 100 mM Na+, we observed inward currents at polarized potentials and outward currents at positive potentials, with Vr of –25 mV. Reducing the Na+ concentration reduced the magnitude of the inward current and shifted Vr to more polarized potentials. The Nernstian shift in Vr of +55.4 ± 4.0 mV per 10-fold increase in Na+ concentration indicated that the leak current was a Na+ conductance. Currents in Li+ were identical to those we we obtained for Na+ (Fig. 6C). Increasing K+ concentration from 2 mM to 10 mM (by equimolar exchange with choline in the presence of 100 mM Na+) or reducing pH from 7.5 to 5.5 (in the absence of Na+) had no significant effect upon Vr (data not shown), indicating that neither K+ nor H+ are permeant.



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  		FIG. 6.
Characteristics of a cation leak pathway mediated by SNAT1. A, the shift in current resulting from switching from Na+-free medium to 100 mM NaCl (I{Delta}Na) at –50 mV was taken as an index of the magnitude of the leak current (mean ± S.E.) in oocytes expressing SNAT1 (n = 57) or SNAT2 (n = 16) and in control oocytes (n = 14). B, Na+ concentration dependence of the SNAT1-mediated leak current at –70 mV in the presence of 100 µM niflumic acid. The currents appeared nonsaturable (whereas a hypothetical fit to Equation 1 yielded ). C, Vm ramps were used to determine the magnitudes and reversal potentials (Vr) of the SNAT1-mediated leak under various conditions in the presence of 100 µM niflumic acid. Typical records are shown for one oocyte expressing SNAT1. For clarity, only the records at 10, 35, and 100 mM NaCl and 100 mM LiCl are shown. (Control currents were omitted for clarity; the filtered control current at 100 mM Na+ ranged between –23 and +56 mV, with Vr {approx} –54 mV.) Inset, Vr for SNAT1 as a function of [Na+] on a log10 scale. Linear regression (r2 = 0.97) yielded a Nernstian shift in Vr of +55.4 ± 4.0 mV per 10-fold increase in [Na+] in the presence of 100 µM niflumic acid. (Currents shown in the main figure are from a second oocyte, for which the regression (r2 = 0.98) returned +48.4 ± 3.5 mV per 10-fold increase in [Na+]).

 

These maneuvers were performed in the presence of 100 µM niflumic acid in order to block currents mediated by endogenous Cl– channels (38, 39). In preliminary investigations with SNAT1 in the absence of niflumic acid, varying the Na+ concentration resulted in non-Nernstian shifts in Vr of about only +20 mV/decade, as observed by Chaudhry et al. (22). This result was due to activation of the endogenous Cl– channel (blocked by niflumic acid) by an undetermined mechanism, as previously observed in oocytes expressing any one of several transporters or channels (40). The narrow selectivity of the cation current mediated by SNAT1 distinguishes it from the nonselective endogenous cation current (for which K+ was permeant) observed in the latter study. Whereas cotransport was very temperature-dependent, the SNAT1 Na+ leak displayed only modest temperature dependence: the low Ea of 7.8 ± 0.7 kcal·mol–1 (Fig. 3H) is consistent with channel activity. That the SNAT1 Na+ leak differs so much from cotransport in terms of saturability and temperature dependence suggests that the Na+ leak is mediated by a pathway that (i) possesses properties of a channel and (ii) is physically discrete from the cotransport pathway.

Na+/Amino Acid Cotransport Is Coupled 1:1—Net charge uptake (the integral of the alanine-evoked current over 5 min) was directly proportional to 500 µM L-[3H]alanine uptake (in 100 mM Na+) at either –70 mV or +10 mV (Fig. 7), validating our use of the amino acid-evoked current as a direct linear index of amino acid transport mediated by SNAT1, as was the case for SNAT2 (26). From the molar ratio of net charge uptake to [3H]amino acid accumulation, we derived a Na+/amino acid coupling coefficient (n) of 1.4 ± 0.2 at –70 mV (Fig. 7) and concluded that coupling is 1 Na+ per 1 amino acid in tandem with uncoupled leaks. Hill coefficients (nH) for the driving ion (Na+ or Li+) were always 1, whereas nH somewhat in excess of 1 might be expected in the case of 2 Na+ per 1 amino acid coupling stoichiometry, depending on the degree of cooperativity between cation-binding sites. That the coupling coefficient (n) significantly exceeded 1 was not due to an amino acid-stimulated increase in endogenous Cl– channel activity, since this was blocked in these experiments by 100 µM niflumic acid. Similar values were obtained in the absence of niflumic acid, and the current/voltage relationship for L-alanine did not appreciably differ in the presence or absence of niflumic acid (data not shown). The value of n also did not differ: (i) over a range of alanine concentrations (200 µM to 1 mM), (ii) at either 5 or 100 mM Na+, or (iii) when L-[3H]glutamine was used in place of alanine (data not shown).



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  		FIG. 7.
SNAT1 Na+/L-alanine coupling coefficient determined by L-[3H]alanine uptake under voltage clamp. Oocytes were clamped at –70 mV or +10 mV and superfused with 500 µM L-[3H]alanine for 5 min. The alanine-dependent charge (QAla) was measured as the integral of the alanine-evoked current (using trapezoidal rule). QAla was taken as an index of alanine-dependent Na+ transport (by converting to pmol using the Faraday) and compared with tracer accumulation over the same period in oocytes expressing SNAT1 at –70 mV (filled circles, 8 oocytes) or +10 mV (filled triangles, 5 oocytes). Charge and tracer accumulation in control oocytes (mean ± bidirectional S.E.) at –70 mV (open circle, 3 oocytes) or +10 mV (open triangle, 3 oocytes) was first subtracted. QAla was proportional (solid lines) to L-[3H]alanine uptake both at –70 mV (r2 = 0.92) and at +10 mV (r2 = 1.00). Comparison of Na+ and L-alanine transport provided the Na+/L-alanine coupling coefficient (n) of 1.4 ± 0.2 (mean ± propagated S.E.) at –70 mV and 1.4 ± 0.3 at +10 mV (corresponding to the dashed line and dotted line, respectively).

 

We considered the possibility that n > 1 reflected thermodynamic coupling of 2 Na+ per 1 amino acid. Increasing the amino acid concentration will increase the rate k23 describing the step 2 -> 3 in our model (Fig. 9). If the Na+ leak were accounted for by Na+ uniport activity (a direct pathway between states 2 and 5), the addition of amino acid might then block the uncoupled Na+ leak by driving the carrier in favor of Na+/amino acid cotransport, such that n derived from this experimental procedure would underestimate the real coupling stoichiometry by the extent to which the Na+ uniport was inhibited by the addition of amino acid. By this reasoning, a coupling stoichiometry of 2 Na+ per 1 glucose was inferred for SGLT1 from n = 1.6 at –70 mV (27). In the case of SGLT1 however n varied with voltage, since the current/voltage relationship of the phlorizin-inhibitable Na+ uniport differed from that of the sugar-evoked current (27). In contrast, we found that the SNAT1 Na+/amino acid coupling coefficient was no different (n = 1.4 ± 0.3) at +10 mV (Fig. 7). The Na+ leak is reduced at +10 mV significantly more than is the amino acid-evoked current (see Figs. 3 and 4). Therefore, determination of n appears to be unaffected by the Na+ leak in SNAT1 and the channel-like properties of the SNAT1 Na+ leak distinguish it from the uniport activity observed in other Na+-dependent transporters such as SGLT1. We conclude that Na+ and amino acid are thermodynamically coupled 1:1 and that a Na+ leak activity, with channel-like properties, proceeds independently of cotransport. The activation of an additional, uncoupled leak in the presence of amino acid (accounting for n > 1) warrants further investigation.

Cellular Distribution of SNAT1 in the Central Nervous System—We compared SNAT1 immunoreactivity patterns with those of several cellular markers in order to determine the cellular distribution of SNAT1 and the neurotransmitter phenotypes of neurons in which SNAT1 is expressed in adult rat brain. Confocal laser-scanning double-immunofluorescence microscopy localized SNAT1 protein to many presumed glutamatergic and GABAergic neurons of the adult rat brain (Fig. 8). We did not find SNAT1 in astrocytes. SNAT1 staining was absent from those cells that were labeled with glial fibrillary acidic protein (GFAP) in the hippocampal formation or thalamus (Fig. 8, A and B) and SNAT1 also did not colocalize with GFAP in cerebellar granule cell cultures (21). However SNAT1 expression in brain was not confined to neurons, since SNAT1 was also abundantly expressed in the ependymal cells lining the ventricles (Fig. 8, A and C). We observed polarized expression of SNAT1 in ependymal cells, with SNAT1 being localized to the luminal plasma membrane (Fig. 8C). SNAT1 (with K0.5 for glutamine of 0.3 mM) is well suited to a role in the ependymal uptake of glutamine from ventricular CSF, the glutamine concentration of which is 0.4–0.5 mM (41–43). SNAT1 acting in concert with SNAT3 (SN1), which appears to be present on the luminal and abluminal membranes of these cells (14), may account for glutamine transport between CSF and brain compartments.



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  		FIG. 8.
Confocal laser-scanning double-immunofluorescence microscopy in rat brain for SNAT1 with neuronal and astrocytic markers. SNAT1 immunoreactivity is shown throughout in green and compared with that of other markers shown in red (as labeled): GFAP, glial fibrillary acidic protein; MAP2, the dendritic marker microtubule-associated protein 2; VGLUT1, VGLUT2, vesicular glutamate transporter isoform 1 and 2; Synap, synaptophysin; TH, the dopaminergic marker tyrosine hydroxylase. Colocalization appears as yellow. A, hippocampus and thalamus, indicating the CA1 and CA3 regions, dentate gyrus (DG), thalamus (Th), and ependyma (arrow). B, high power magnification of the hippocampal CA3 region. C, ependyma and subependymal layer; D, hippocampus, CA3 region; E, cerebral cortex; F and I, globus pallidus; G, pontine nuclei; H, trigeminal mesencephalic nucleus; K, reticular thalamus. J, cerebellum, indicating the granular layer (g), Purkinje cell layer (p) and molecular layer (m); L, spinal cord, ventral horn motoneurons; M, substantia nigra region, indicating the ventral tegmental area (VTA), red nucleus (Rn), substantia nigra pars compacta (SNC), and pars reticulata (SNR). N, high power magnification of the lateral substantia nigra pars compacta and adjacent pars reticula. O, brainstem structure, indicating the noradrenergic A1 cell group and gigantocellular reticular nucleus (Gi).

 

SNAT1 Is Localized to the Cell Bodies and Proximal Dendrites of Glutamatergic and GABAergic Neurons—We observed moderate levels of SNAT1 immunoreactivity in hippocampus, enriched in the CA3 layer and dentate gyrus granule cells (Fig. 8, A and B). Higher power magnification of the CA3 region revealed SNAT1 immunoreactivity in pyramidal neurons known to be glutamatergic (Fig. 8B). SNAT1 was detected previously in developing primary cultures of hippocampal pyramidal neurons (44). Higher power magnification of the molecular layer of the hippocampus revealed SNAT1 immunoreactivity in GABAergic interneurons (data not shown).

For the most part, we found that neuronal expression of SNAT1 was confined to cell bodies and proximal dendrites. SNAT1 was not detectable at the axon nerve terminal in paraffin-embedded sections. This was readily apparent for glutamatergic pyramidal neurons of the CA3 region of the hippocampus (Fig. 8D) and in the cerebral cortex (Fig. 8E). There, dendrites of SNAT1-positive neurons were identified with the dendritic marker MAP2, which also weakly stains cell bodies. We observed in the pallidum abundant SNAT1 immunoreactivity (Fig. 8F) that was confined to the cell bodies and proximal dendrites of the large cells, which are known to be GABAergic (45). SNAT1 immunoreactivity did not colocalize with the synaptic vesicle markers VGLUT1 (Fig. 8, G and L), VGLUT2 (Fig. 8, H, J, and K), or synaptophysin (Fig. 8I) indicating that little or no SNAT1 is present in nerve endings in vivo under these conditions, despite contrasting findings in vitro. For example, preparations of rat brain synaptosomes, which are considered to be pinched-off nerve endings, contained significant amounts of SNAT1 protein according to Western blot analysis (21), and SNAT1 was also detected at the tips of growing axons and at sites of synaptic contacts in cultured hippocampal nerve preparations (44).

In many neurons SNAT1 immunoreactivity appeared punctate (Fig. 8, E, F, I, J, L, and N), indicating the presence of large intracellular pools of SNAT1. In the cerebral cortex (Fig. 8E) and pallidum (Fig. 8, F and I), fine puncta filled the cell body and extended to the proximal dendrites. This may explain the abundance of SNAT1 protein in microsomal fractions from rat brain preparations (21). In other neurons, such as those of the trigeminal mesencephalic nucleus, SNAT1 immunoreactivity clearly delineated the plasma membrane (Fig. 8H). However, discrete areas of SNAT1 immunoreactivity were apparent within the cell bodies of these primary sensory neurons, possibly representing sites of synthesis or regulation such as the trans-Golgi network or endosomal recycling centers. These mesencephalic neurons, known to be the primary afferents supplying the masticatory muscles, receive abundant input from VGLUT2-expressing neurons and may be part of a very active excitatory VGLUT2-encoded pathway. Our data indicated that the cellular expression of SNAT1 within the cell body differs among various neuronal populations and suggest that trafficking of the SNAT1 protein between intracellular pools and the cell surface may be regulated.

SNAT1 protein was abundantly expressed in other presumed glutamatergic neurons, such as those dominating the pontine nuclei and gigantocellular reticular nucleus (Fig. 8, G and O). These neurons contain VGLUT1 mRNA (46, 47) and in part account for the presence of mossy fiber terminal-like VGLUT1 immunoreactivity in the granule layer of the cerebellum (48) and for VGLUT1-encoded innervation of motoneurons in the spinal cord (Fig. 8L) (49). SNAT1 was abundantly expressed in the red nucleus (Fig. 8M), an excitatory projection to the spinal cord, and in the thalamus (Fig. 8A), where both glutamatergic and GABAergic cell bodies are numerous. SNAT1-immunoreactive cell bodies were observed in the reticular thalamus where GABAergic neurons predominate (Fig. 8K). We observed moderate VGLUT2 innervation of this region, possibly on the dendritic portions of these SNAT1-positive neurons. In cerebellum, SNAT1 protein was abundant in GABAergic Purkinje cell bodies (Fig. 8J) and SNAT1 mRNA was detectable in the Purkinje cell layer (21, 22). We detected SNAT1 immunoreactivity in interneurons, probably GABAergic, of the molecular layer of the cerebellum (Fig. 8J). SNAT1 immunoreactivity did not colocalize with the VGLUT2 immunoreactivity observed in a subset of mossy fiber terminals in the granule layer and the climbing fiber terminals that line the dendrites of the Purkinje neurons. The glutamatergic granular cell layer exhibited lower levels of SNAT1 immunoreactivity than did the GABAergic cell populations in the cerebellum. Whereas SNAT1 mRNA was detected in the granular layer of the cerebellum (21, 22), SNAT2 mRNA was the dominant isoform expressed in cerebellar granule interneurons (22, 26, 50). Whereas SNAT1 expression in the granule neurons in vivo appeared low, cultured cerebellar granule cells (day 20) expressed high levels of SNAT1 protein (21).

SNAT1 Is Expressed in Subsets of Cholinergic and Dopaminergic Neurons—SNAT1 was not restricted to glutamatergic and GABAergic neurons of the central nervous system but was also found in subsets of cholinergic and dopaminergic neurons. We found abundant expression in the cholinergic motoneurons of the spinal cord (Fig. 8L), a major target for VGLUT2-expressing glutamatergic neurons (29). SNAT2 mRNA is also abundantly expressed in these cells (26). Here, VGLUT1-positive synaptic terminals abut dendritic fields and cell bodies that are strongly SNAT1-positive (Fig. 8L). The high levels of SNAT1 expression in motoneurons may account for the very high glutamate content of motoneuron cell bodies and nerve terminals (51–53). Double immunostaining with the vesicular acetylcholine transporter VAChT (which stains cholinergic cell bodies and nerve terminals) revealed that SNAT1 was only present at low levels in most other cholinergic cells in the brain, including those of the striatum, globus pallidus, and basal forebrain systems (data not shown).

Catecholaminergic neurons of the A1 cell group of the brainstem (Fig. 8O) and the olfactory bulb (data not shown) did not express SNAT1. However, we identified expression of SNAT1 in subsets of dopaminergic cells of the substantia nigra, identified by tyrosine hydroxylase immunoreactivity (Fig. 8, M and N). No appreciable SNAT1 expression was detected in the dopaminergic neurons of the ventral tegmentum or medial portion of the substantia nigra pars compacta. SNAT1 was evident, in some cases at very high levels, in dopaminergic neurons in the ventral and lateral portions of the pars compacta and the pars reticulata (Fig. 8N). The expression of SNAT1 in ventral midbrain dopaminergic neurons is consistent with the high levels of glutamate and PAG activity detected in the substantia nigra pars compacta (54, 55) and raises the possibility that these neurons synthesize and secrete neurotransmitter glutamate. Cultured dopamine midbrain neurons exhibit a mixed dopaminergic-glutamatergic phenotype (56, 57), and single dopaminergic nigrostriatal neurons may form two chemically distinct synaptic types (58). Notably, SNAT1 and tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta colocalized in those lateral and ventral neurons that are most susceptible in Parkinson's disease (59). The neurotoxic effects of glutamate (during impaired neuronal energy metabolism) are known to play a role in the etiology of neurodegeneration in Parkinson's disease (60), as well as in amyotrophic lateral sclerosis (ALS) (61, 62).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mechanisms of SNAT1-mediated Amino Acid Transport— Coupling of the amino acid flux through SNAT1 to the Na+ electrochemical gradient ensures unidirectional inward transport of amino acid at resting potential, and no reversal was observed at the Vm expected during the action potential (see Fig. 3A). The SNAT1 transport cycle does not require any other ionic species; however, Li+ can substitute for Na+ in driving transport and (contrary to common thinking) classic System A is tolerant of a Li+ for Na+ substitution (63). However, we propose that Li+ tolerance is not in itself a useful criterion with which to classify amino acid transporters, since our data for SNAT1 illustrated that: (i) Li+ tolerance is incomplete and (ii) the apparent effectiveness of Li+ as a replacement for Na+ depends both on membrane potential and on the parameters (e.g. K0.5, Imax,or absolute current) that are used in comparisons (Fig. 3). Our data are consistent with a carrier model describing SNAT1-mediated amino acid transport as a series of temperature-dependent, ligand-induced conformational changes (Fig. 9). Na+ binding is voltage-dependent and precedes binding of the amino acid and its simultaneous translocation. SNAT1 exhibited presteady-state currents, which can be used to estimate transporter density (64). Using the relationship Qmax = NT·z·e (for which e is the elemental charge), we estimate the number of functional transporters (NT) in the plasma membrane at {approx} 1011 per oocyte. In analogy to other Na+- or H+-dependent transporters (65–68), we attribute presteady-state currents for SNAT1 to two steps within the transporter cycle: (i) translocation of the empty, charged carrier and (ii) binding/dissociation of Na+ within the membrane electric field. Presteady-state currents decayed with half times ({tau}) of 4–5 ms, whereas the turnover rate for L-alanine transport was up to 200 s–1 at –150 mV. On the basis of our model (Fig. 9), we therefore anticipate that carrier reorientation (step 6 -> 1) and/or Na+ binding (step 1 -> 2) may be rate-determining at saturating voltage. However, another step in the transport cycle must be rate-determining at depolarized Vm.

Heterologous expression of SNAT1 in oocytes (Fig. 9) was also associated with stimulation of an endogenous chloride channel activity (that was blocked by niflumic acid) and a cation conductance selective for Na+ and Li+. The exclusion of protons from both the leak and cotransport pathways in the System A transporters distinguishes this branch of the SLC38 gene family from the System N subtypes, which mediate the exchange of H+ for Na+ plus glutamine (7, 69–71). Our conclusion that Na+/amino acid coupling stoichiometry is 1:1 relies upon our finding that the Na+ leak is discrete from the Na+ flux through the cotransport pathway. Evidence to this effect includes the observation of channel-like properties of the Na+ leak (i.e. nonsaturability and low temperature dependence) that distinguish it from Na+ uniport. Further supporting the view that the Na+ leak is discrete from the cotransport pathway is our observation of a substantial Na+ leak in an SNAT1 mutant (H391A) in which amino acid transport is severely impaired; of particular note, addition of amino acid did not inhibit the Na+ leak in H391A-SNAT1 even though was unaffected by the mutation.2 Our model and the Na+/amino acid coupling stoichiometry of SNAT1 could be further resolved pharmacologically if inhibitors were available to block either the leak or the cotransport pathways. For example, our model would predict that the Na+ leak will escape inhibition by a high affinity, non-transported amino acid analogue (antagonist) that can inhibit Na+/amino acid cotransport. Meanwhile, the differences in substrate selectivity between SNAT1 and SNAT2 may permit the development of specific blockers to distinguish between the two isoforms, and specific blockers could be advantageous in investigating glutamate-GABA/glutamine cycling with NMR or immunocytochemical approaches in vivo.

We have concluded that the cation leak observed in oocytes expressing SNAT1 is a function of the SNAT1 protein (Fig. 9) since the cation selectivity (Na+, Li+ » H+, K+) of the leak: (i) exactly matches the ion dependence of amino acid cotransport and (ii) differs from the predominant endogenous cation channel, which is also permeant to K+ (40). To explore the possibility that the cation leak was an artifact of overexpression in the oocyte, we monitored the time course and cRNA-concentration dependence of the Na+ leak and cotransport currents, since differences may be expected if the cation leak were as a result of saturation of some component or subunit endogenous to the oocyte. The Na+ leak and cotransport currents each began to rise significantly at 12–24 h, peaked at 48 h, and remained unchanged at 60 h (data not shown). Also, expression of both components showed comparable dependence on the amount of cRNA injected (5, 10, 20, or 50 ng) (data not shown). The parallel expression of the Na+ leak and amino acid cotransport components suggests that the cation conductance associated with SNAT1 expression in oocytes is in fact a property of SNAT1 and not an artifact of the oocyte expression system. However, until the cation leak can be demonstrated in neurons, its physiological significance will remain unknown. Nevertheless, we speculate that the presence of SNAT1 at the neuronal plasma membrane and resultant Na+ fluxes (whether via the leak or cotransport pathways) will place a significant energy burden on the cell and may increase the vulnerability of neurons that are metabolically compromised. This highlights the importance of the possible regulation of protein synthesis and trafficking between intracellular compartments and the plasma membrane, as illustrated for the peripheral System A isoform SNAT2 (72). Little is known about the regulation of SNAT1 in brain; however System A activity in cultured cerebellar granule cells (5) did not display the responsiveness to insulin observed for SNAT2 elsewhere (73, 74). The abundance of SNAT1 within intracellular compartments of some cells (e.g. cerebral cortex and pallidum) but at the plasma membrane of others (e.g. mesencephelon and ependyma) may reflect protein synthesis de novo, plasma membrane recruitment, or recycling of SNAT1 according to the demand for glutamine, as for SNAT2 (72). Long term sensitization of glutamatergic neurotransmission in Aplysia is accompanied by increased neuronal glutamine uptake (75), suggesting that glutamatergic activity may play a role in regulation of glutamine uptake. The enrichment of SNAT1 in the cell body and proximal dendrites as opposed to the axon terminal could also be expected to contribute to the acute regulation of electrical activity at the soma and neuronal excitability as a result of the depolarizing action of the SNAT1-mediated Na+ fluxes.

A Specialized Role for SNAT1 in the Glutamate/GABA-Glutamine Cycle—Whereas low-level expression of SNAT1 was detected in several tissues (21, 22, 73), SNAT1 was highly enriched in specific neuronal populations within the CNS, but SNAT1 was not present in astrocytes. We expect L-glutamine to be the principal substrate for the neuronal SNAT1 in vivo since its interstitial concentration in the brain, estimated at 0.2–1.0 mM (7, 43, 77), significantly exceeds that of any other amino acid. SNAT1 efficiently transports L-glutamine, and its of {approx} 0.3 mM is well suited to the role of glutamine uptake from the neuronal environment. These considerations, and the localization of SNAT1 almost exclusively to neuronal populations of known glutamatergic or GABAergic phenotypes, support a specialized role for SNAT1 in the glutamate/GABA-glutamine cycle. Although we observed lower levels of SNAT1 in glutamatergic granule cells of the cerebellum, these cells may upregulate SNAT1 when cultured in vitro under depolarized conditions (21) and the often punctate appearance of SNAT1 immunoreactivity in neurons in vivo implicates a role for regulated transporter recruitment to the plasma membrane. The highest levels of SNAT1 immunoreactivity were observed in the cell bodies and proximal dendrites of neurons, whereas axon terminals in adult rat appeared to lack SNAT1. Therefore the punctate SNAT1 immunoreactivity observed in most perikarya is probably not destined for nerve terminals, and the soma and proximal dendrites may represent the primary site of SNAT1-mediated glutamine uptake in these neurons. It is possible that the epitope recognized by our antibody against SNAT1 is masked at the nerve terminal in the adult brain in vivo. However, the polyclonal SNAT1 antibody was raised against a fusion protein containing 63 amino acids of the SNAT1 sequence. SNAT1 immunoreactivity in hippocampal neuronal projections in vitro (44) may be a property unique to primary culture of differentiating neurons. The abundance of SNAT1 in microsomal fractions from rat brain synaptosomes (21) is consistent with the SNAT1 immunoreactivity pattern in situ observed in this study since synaptosomal preparations include cleft and postsynaptic components (including somatodendritic synapses) in addition to presynaptic terminals (78). We do not consider expression at the nerve terminal prerequisite to a specialized role for SNAT1 in replenishing the glutamate and GABA neurotransmitter pools. That SNAT1 expressed at the cell body and proximal dendrites could fulfill this role is supported by the observations that: (i) cell bodies in many putative glutamatergic neurons possess high activity of the phosphate-activated glutaminase required for glutamate synthesis (79), (ii) the liver-type glutaminase is also localized to neuronal nuclei (80), and (iii) glutamate or GABA concentrations are uniformly high throughout the cytoplasm of glutamatergic or GABAergic neurons (54, 81) suggesting that rapid diffusion occurs. A role in the glutamate/GABA-glutamine cycle would make a SNAT1 a potential target for modulating presynaptic glutamatergic/GABAergic function.

Alanine, although present in brain interstitial fluid at only one fifth the concentration of glutamine (43, 77), is also a very efficient substrate of SNAT1. Modest uptake of alanine into glutamatergic neurons may also contribute to maintaining neurotransmitter glutamate pools, especially during the recovery from ischemia/hypoxia, when alanine concentration rises and glutamate concentration falls (82), since transamination of {alpha}-ketoglutarate plus alanine can replenish the glutamate pool released during potassium-induced depolarization (i.e. loss of neurotransmitter glutamate) in cerebellar granule cells (83).

Additional Roles for Neuronal SNAT1—The recent localization of liver-type glutaminase in neuronal nuclei (80) prompts us to consider additional neuronal glutamine requirements with discrete control pathways, and alternative roles for neuronal SNAT1. The expression of SNAT1 in ventral midbrain neurons considered to be dopaminergic may well be explained by the existence of a mixed glutamatergic-dopaminergic phenotype in those cells, as supported by studies in vitro (56–58). A mixed phenotype has also been considered for cholinergic motoneurons (52). Alternatively, the expression of SNAT1 in these neuronal populations may point to additional roles for SNAT1-mediated uptake, not only of glutamine but also of other amino acids efficiently transported by SNAT1. For example, cysteine and glycine, together with glutamate, are required for neuronal synthesis of glutathione, a major antioxidant responsible for the detoxification of reactive oxygen species readily generated within the brain (84, 85). Locally high concentrations of cysteine and glycine may result from cleavage of astrocyte-derived dipeptides (e.g. CysGly) by the ectopeptidase aminopeptidase N, which, like SNAT1, is present on the neuronal cell body (86). SNAT1 could therefore supply neurons with the components of glutathione synthesis. In fact glutamine, through this pathway, may play an important role in regulation of oxidative metabolism in brain (87). Notably, the ventral midbrain neurons that express SNAT1 are known to be particularly susceptible to oxidative stress resulting from autoxidation of dopamine. Finally, SNAT1 may also serve neurons with an elevated demand for glutamine as a metabolic fuel, possibly via {alpha}-ketoglutarate, which may then enter the tricarboxylic acid cycle (18, 19). It is of note that there is also significant expression of SNAT1 in the heart (22, 23, 73), where glutamine metabolism appears to be crucial to postischemic recovery (76).

The functional characterization of SNAT1 in terms of substrate profile, ion dependence, thermodynamics, and kinetics provides the foundation for structure-function analysis of SNAT1 and the development of specific inhibitors, and a knowledge of the properties and cellular distribution of SNAT1 in the brain provides clues to understanding the physiological roles of SNAT1 in central neurons.


   FOOTNOTES
 
* This research was supported by Grants NSF/LEQSF (2001-04)-RII-01 (to H. V.) from the National Science Foundation, 1P29RR16816 (to H. V.), and NS36936 (to J. D. E.) from the National Institutes of Health, and SFB 297 and BMB+F (to E. W. and M. K.-H. S.) from the German Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} To whom correspondence may be addressed: Neuroscience Center, University of Louisiana Health Sciences Center, 2020 Gravier St., Suite D, New Orleans, LA 70112. E-mail: hvaroq{at}lsuhsc.edu or jerick{at}lsuhsc.edu.

1 The abbreviations used are: GABA, {gamma}-aminobutyrate; BCH, 2-aminobicyclo[2,2,1]heptane-carboxylate; ChoCl, choline chloride; GFAP, glial fibrillary acidic protein; MeAIB, 2-(methylamino)isobutyrate; PAG, phosphate-activated glutaminase; SNAT1, sodium-coupled neutral amino acid (System N/A) transporter 1 (formerly SAT1, ATA1, SA2, NAT2, GlnT); SNAT2, sodium-coupled neutral amino acid (System N/A) transporter 2 (formerly SAT2, ATA2, SA1); VGLUT1 (or VGLUT2), vesicular glutamate transporter 1 (or 2); MES, 4-morpholineethanesulfonic acid; GST, glutathione S-transferase. Back

2 B. Mackenzie, D. Yao, M. E. K. Morris, H. Varoqui, M. A. Hediger, and J. D. Erickson, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank J. Sabbagh, M. Zibuschka, E. Rodenberg, P. Sack, and H. Schneider for assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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