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J. Biol. Chem., Vol. 282, Issue 19, 14447-14453, May 11, 2007

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Molecular Basis for Substrate Discrimination by Glycine Transporters^*

From the Department of Pharmacology, Institute for Biomedical Research, University of Sydney, New South Wales 2006, Australia

Received for publication, September 27, 2006 , and in revised form, February 19, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts on the N-methyl-D-aspartate family of receptors. There are two Na⁺/Cl^--dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations and these transporters show differences in substrate selectivity and blocker sensitivity. A bacterial Na⁺-dependent leucine transporter (LeuT_Aa) has recently been crystallized and its structure determined. When the amino acid residues within the leucine binding site of LeuT_Aa are aligned with residues of the two glycine transporters there are a number of identical residues and also some key differences. In this report, we demonstrate that the LeuT_Aa structure represents a good working model of the Na⁺/Cl^--dependent neurotransmitters and that differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors (1), and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts as a coagonist with L-glutamate on the N-methyl-D-aspartate subtypes of glutamate receptors (2, 3). Glycine transporters regulate glycine concentrations within both inhibitory and excitatory synapses. The GLYT1 subtypes of glycine transporters are expressed in glial cells surrounding both excitatory and inhibitory synapses (4), whereas the GLYT2 subtypes of glycine transporters are expressed in presynaptic inhibitory glycinergic neurons (4).

The differential expression patterns and physiological roles of the glycine transporter subtypes have recently been exploited in the development of novel transport inhibitors to treat schizophrenia (5-7) (GLYT1 inhibitors). GLYT1 transports glycine and also the N-methyl derivative of glycine, sarcosine (8), whereas GLYT2 only transports glycine (9). The GLYT1 inhibitor, N[3-(4-fluorophenyl)-3-(4'-phenylphenoxy)propylsarcosine (NFPS)³ contains the sarcosine moiety and it is tempting to speculate that the specificity of NFPS for GLYT1 over GLYT2 may be due to the sarcosine moiety. However, in a previous study on the mechanism of action of NFPS on GLYT1 we were unable to demonstrate that NFPS competed with sarcosine or glycine in binding to GLYT1, which implied that the inhibitor and substrates do not share a common binding site (5).

The crystal structure of a bacterial Na⁺-dependent leucine transporter has recently been determined (10), and this protein shows remarkable amino acid sequence similarity with the mammalian family of Na⁺/Cl^--dependent neurotransmitter transporters, which include transporters for glycine, -aminobutyric acid, dopamine, serotonin, and norepinephrine (10, 11). The structure consists of 12 TM domains with TM1-5 and TM6-10 arranged in a pseudo-symmetric structure. The substrate binding site of LeuT_Aa is formed at the junction between the two regions and is composed of amino acid residues from TM1 and TM6. Both of these two transmembrane domains contain an unwound segment and many of the substrate contact sites are with the main chain atoms of these unwound segments (Fig. 1). Given the high degree of sequence similarity, Yamashita et al. (10) predicted that mammalian neurotransmitter transporters have very similar structures and that the neurotransmitter binding sites are located within analogous regions of their respective transporters. When the amino acid residues within the leucine binding site of LeuT_Aa are aligned with residues of the two glycine transporters there are a number of identical residues and also some key differences (Fig. 1). In this report we test the hypothesis that the LeuT_Aa structure represents a good working model of the Na⁺/Cl^--dependent glycine transporters and demonstrate that differences in substrate selectivity between GLYT1 and GLYT2 can be attributed to a single amino acid difference within the glycine recognition site. However, this difference cannot explain the differences in blocker selectivity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemicals—All chemicals were obtained from Sigma (Sydney, Australia) unless otherwise stated. NFPS was obtained from Tocris and dilutions were made from a stock solution of 100 mM in 50% Me₂SO. ALX1393 was diluted from a stock solution of 740 µM in 30% Me₂SO. The highest concentration of Me₂SO applied to the cells was 0.04%, which did not cause a measurable effect.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Substrate recognition site of LeuTAa. A, amino acid sequence alignment of GLYT1, GLYT2, and LeuTAa in the vicinity of the leucine binding site of LeuTAa. Residues with a triangle in LeuTAa interact with leucine. B, the structure of the leucine binding site of LeuTAa. The dotted line represents hydrogen bonding between the amino group of leucine and the hydroxyl group of Ser-256. The helix backbone is depicted in green, carbon in white, nitrogen in blue, oxygen in red.

Oocytes were harvested from X. laevis, as described previously (15) with all procedures in accordance with the Australian National Health and Medical Reseach Council guidelines for the prevention of cruelty to animals. 50 nl of cRNA was injected into defoliculated, stage V oocytes, and incubated at 16 °C in standard frog ringers solution (ND96; 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.55), supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 µg/ml gentamicin. 2-5 days later, current recordings were made at -60 mV using the two-electrode voltage clamp technique with a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA) interfaced with a MacLab 2e chart recorder (ADI Instruments, Sydney, Australia) using the Chart software (ADI Instruments). The expression of GLYT1 and GLYT1 mutants at the oocytes cell surface was checked using a cell surface protein biotinylation procedure (Pierce). Protein were labeled according to the manufacture's instructions and then oocytes were vortexed in a cell lysis buffer containing 1% Triton X-100, 0.1 M NaCl, 20 mM Tris-HCl pH 7.6, 0.5 mg/ml 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride. After purification of biotinylated proteins, the samples were analyzed by SDS-PAGE followed by Western blotting. A rabbit anti-GLYT1 antibody was obtained from David Pow (University of Newcastle, Australia) and used at a dilution of 1: 1000. Goat anti-rabbit secondary antibodies tagged with alkaline phosphatase were used to detect GLYT1.

Charge-to-flux Ratios—Uptake of [³H]glycine (Amersham Biosciences, Sydney, Australia) was measured under voltage-clamp in oocytes expressing wild type and mutant transporters. Oocytes were voltage-clamped at -60 mV and 30 µM [³H]glycine was applied for 1 min and the transport current was recorded. This was followed by a 3-min washout and oocytes were removed from the bath and lysed in 50 mM NaOH, and scintillation counting was performed. Nonspecific [³H]glycine uptake was estimated by measuring [³H]glycine uptake by uninjected oocytes and was subtracted from uptake by oocytes expressing the glycine transporters. The amount of charge transfer associated with [³H]glycine uptake was determined by integrating the current measurement for the time of [³H]glycine application.

Data Analysis—The analysis of kinetic data were carried out using the Kaleidagraph Software version 3.1. Current (I) as a function of substrate concentration ([S]) was fitted by least squares analysis to: I/Imax = [S]/(EC₅₀ + [S]) (Equation 1), where I_max is the maximal current and EC₅₀ is the concentration of glycine that generates a half-maximal current. Data for sarcosine-elicited currents were normalized to the maximal current generated by glycine for each cell.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the crystal structure of the bacterial leucine transporter, LeuT_Aa, the amino group of leucine is hydrogen-bonded to the main chain carbonyls of Phe-253 and Thr-254 and the hydroxyl group of the side chain of Ser-256. We have focused on the role of the corresponding residues in GLYT1 and GLYT2 in an attempt to explain why GLYT1 can transport the N-methyl derivative of glycine, sarcosine, while the GLYT2 subtype does not bind or transport sarcosine. An alignment of the amino acid sequences of GLYT1, GLYT2, and LeuT_Aa in the region proposed to bind substrate (10) is presented in Fig. 1. In GLYT1, the residue corresponding to the Ser-256 of LeuT_Aa that forms a hydrogen bond with the amino group of the substrate leucine is a Gly residue, whereas in GLYT2 the residue is a Ser. Furthermore, the neighboring residues are not conserved among the three transporters. These observations lead us to investigate whether the three differences highlighted in Fig. 1 are responsible for the differences in substrate and blocker selectivity between GLYT1 and GLYT2.

Applications of glycine to oocytes expressing either wild type GLYT1b or GLYT2a generate concentration-dependent inward currents (Fig. 2) that are due to the coupled transport of Na⁺/Cl^- and glycine (14, 16, 17). Sarcosine also generates concentration-dependent currents for GLYT1b but not GLYT2a (Fig. 2), which is indicative of sarcosine being a substrate for GLYT1b but not GLYT2a.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Glycine and sarcosine transport by wild type and mutant glycine transporters. A, representative current traces from oocytes expressing wild type and mutant glycine transporters perfused with 30 µM sarcosine (filled bars) or 30 µM glycine (open bars). For the GLYT2a-S481A mutant, 300 µM sarcosine and 300 µM glycine were used. B, concentration response curves for glycine (open circles) and sarcosine (filled circles) transport by wild type and mutant glycine transporters. Currents were normalized to the maximal current generated by glycine and fitted to Equation 1. Data represent mean values ± S.E. from n = 4-6 oocytes.

Glycine generates inward currents in cells expressing the GLYT2a-A482C mutant, but in contrast to the S481G mutant, the substrate selectivity of this mutant is similar to the wild type GLYT2a. Applications of high doses of sarcosine (300 µM to 1 mM) did generate small currents (1% of the maximal glycine current) with this mutant but it was not possible to reliably analyze these results. We also investigated whether sarcosine could bind to this transporter and inhibit glycine transport. However, doses of sarcosine up to 1000 µM have minimal effect on the amplitude of the current generated by 30 µM glycine (99 ± 2% of control glycine currents, n = 5 cells, data not shown), which suggests that sarcosine does not compete with glycine for occupancy of the transporter. The reverse mutation in GLYT1b, C306A, resulted in a wild type GLYT1b phenotype. Both glycine and sarcosine are substrates with EC₅₀ values that are comparable with wild type GLYT1b (Fig. 2 and Table 1). These observations suggest that this residue difference between the two transporter subtypes does not play a role in determining the differences in substrate selectivity. Application of glycine to oocytes expressing the double mutant GLYT1b-G305S,C306A generated small inward currents (I_max 6.1 ± 1.2 nA, n = 6). Cell surface biotinylation assays demonstrated that the transporter was expressed at the surface but at substantially reduced levels compared with wild type, which could explain the small currents observed.

The EC₅₀ for glycine is 140 ± 40 µM (n = 6), but in contrast to wild type GLYT1b, sarcosine at concentrations up to 1 mM did not generate inward currents. Thus, we conclude that as the GLYT1b-C306A mutant did not cause any substantial change in the GLYT1b substrate selectivity, then the differences in substrate selectivity observed for the double mutant can be attributable to the Gly to Ser change.

To investigate further the role of the amino acid side chain differences in GLYT1b and GLYT2a at this site we constructed GLYT1b-G305A and GLYT2a-S481A. Application of glycine and sarcosine to GLYT1b-G305A generates concentration-dependent inward currents with similar EC₅₀ values to that of wild type GLYT1b (11 ± 2 and 14 ± 3, respectively, for the mutant), with a maximal sarcosine-induced current of 110 ± 4% of the maximal glycine-induced current. Thus, either a Gly or an Ala residue are sufficient to allow sarcosine transport by GLYT1b. The cell surface expression levels of GLYT1b-G305A were 60% of that for wild type GLYT1b. Glycine and sarcosine also generated inward transport currents when applied to oocytes expressing GLYT2a-S481A. The amplitude of the glycine transport currents were similar to that of wild type GLYT2a (84 ± 7 nA, n = 5), but the EC₅₀ values for glycine and also sarcosine were greater than the EC₅₀ for glycine transport by the wild type GLYT2a (1070 ± 80 µM for glycine (n = 5) and 590 ± 50 µM for sarcosine (n = 5) transport by GLYT2a-S481A compared with 12 ± 1 µM for glycine transport by GLYT2a). The maximal current generated by sarcosine was 70 ± 3% (n = 5) of that for glycine, which is comparable with that of wild type GLYT1b (87 ± 1%). Thus, for GLYT2a, sarcosine can be transported if an Ala or a Gly, but not a Ser residue, are present at this site.

View larger version (10K): [in this window] [in a new window]   FIGURE 3. ALX1393 blocks a leak current in wild type GLYT2a and GLYT2a-S481G. Representative current traces for ALX1393 block of glycine transport and block of a leak current in GLYT2a and GLYT2a-S481G. 1 µM ALX1393 (filled bar) blocks a leak current in both the wild type and the mutant, but the amplitude of this leak current is greater in the mutant. Applications of 30 µM glycine are indicated by the open bar.

GLYT2a-F478Y and the reverse mutation in GLYT1b,Y302F were generated, but application of glycine or sarcosine to the GLYT2a-F478Y mutant did not generate any currents. Application of both glycine and sarcosine to the GLYT1b-Y302F mutant did generate transport currents that were considerably smaller than wild type GLYT1b. The maximal current generated by glycine was 6.9 ± 1.2 nA, with an EC₅₀ of 1.1 ± 0.2 µM and for sarcosine the maximal current was 77 ± 4% of that for glycine with an EC₅₀ of 1.8 ± 0.7 µM (Fig. 2 and Table 1). The cell surface expression of the GLYT1b-Y302F mutant was 60% of that for wild type GLYT1b. These results demonstrate that the Y302F mutant is expressed at the cell surface and does alter the function of the transporter, but it does not appear to change the relative substrate selectivity.

Blocker Sensitivities of Mutant Transporters—NFPS is an irreversible blocker of GLYT1 and has no apparent effect on GLYT2a (5, 6). Co-application of 30 µM glycine with 1 µM NFPS to oocytes expressing GLYT1b, GLYT1b-G305A, or GLYT1b-C306A caused slow onset reductions in the current. After 40 s exposure to 1 µM NFPS and 30 µM glycine, the glycine evoked current decayed by 43 ± 3% for wild type GLYT1b, 31 ± 7% for GLYT1b-G305A, and 52 ± 3% for GLYT1b-C306A (Table 1). Co-application of 30 µM glycine with up to 10 µM NFPS to oocytes expressing GLYT2a, GLYT2a-S481G, and GLYT2a-A482C generated stable inward currents, which were of similar magnitude to glycine alone (data not shown).

ALX1393 is a GLYT2a selective transport blocker (18), and we also investigated whether the GLYT2a mutants showed any changes in sensitivity to this blocker. Co-application of 1 µM ALX1393 and 30 µM glycine to oocytes expressing the wild type GLYT2a caused a gradual decline in the transport current and after 40 s the current was reduced to 72 ± 5% of the current measured in the absence of ALX1393 (Table 1). ALX1393 caused similar reductions in the glycine transport currents for the GLYT2a mutants compared with the wild type GLYT2a (Table 1). Thus, the GLYT2a-S481G and GLYT2a-S481A mutations, which affect substrate selectivity, do not prevent ALX1393 from binding to the transporter. It is interesting to note that ALX1393 applied alone to oocytes expressing GLYT2a-S481G generated an outward current at -60 mV (18 ± 3% of the amplitude of the 30 µM glycine current, Fig. 3), which is also apparent in the wild type transporter, but to a much lesser extent (7 ± 1% of the amplitude of the 30 µM glycine current, Fig. 3). Based on observations of the effects of transport blockers on other neurotransmitter transporters (19-21), the most likely explanation for these outward currents is that ALX1393 blocks a substrate-independent leak current and that the amplitude of the leak current is greater in the mutant than the wild type transporter.

Glycine transport by GLYT1 is coupled to the co-transport of 2Na⁺ ions and 1 Cl^- ion (17), which generates a charge-to-flux ratio of 1. In contrast, transport by GLYT2a is coupled to the co-transport of 3 Na⁺ ions and 1 Cl^- generating a charge to flux ratio of 2. We measured the charge to flux ratios of GLYT1b, GLYT2a, and the GLYT2a mutant S481G to see if the change in substrate selectivity of the mutant also affected the ion coupling stoichiometry (Fig. 4). Oocytes expressing the transporters were voltage-clamped at -60 mV and [³H]glycine was applied for 60 s. The charge associated with transport was measured by integrating the current over the time of application and the amount of [³H]glycine taken up was measured using scintillation counting. From these measures, the charge to flux ratio was determined. The experimentally determined values for GLYT2a and GLYT1b are 2.10 ± 0.01 and 1.15 ± 0.04, respectively, which are consistent with the expected values. The charge-to-flux ratio for GLYT2a-S481G was 2.05 ± 0.10, which is similar to that of GLYT2a. This result suggests that, although the mutation has changed the substrate selectivity, the ion flux coupling ratio has not been altered. It was not possible to conduct similar experiments with the GLYT1b Y302F, G305S, G305A, and the G305S-G305A mutants because of the reduced transport current amplitudes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
One of the key predictions from the crystal structure of the bacterial Na⁺-dependent leucine transporter, LeuT_Aa, was that this protein is a good structural model for the mammalian Na⁺/Cl^--dependent neurotransmitter transporters (10). The LeuT_Aa structure is of sufficient resolution to visualize the substrate bound to the transporter, and it was possible to identify molecular contact sites between transporter and the substrate. From this information, and given the degree of amino acid sequence similarity between LeuT_Aa and mammalian neurotransmitter transporters, Yamashita et al. (10) were able to predict how the different transporters distinguish between different transmitters.

View larger version (8K): [in this window] [in a new window]   FIGURE 4. Charge-to-flux ratios for wild type GLYT1b, GLYT2a, and GLYT2a-S481G. Oocytes expressing the transporters were voltage clamped at -60 mV and 30 µM [3H]glycine was applied, and both the current and [3H]glycine uptake were measured. The total number of charges transferred across the membrane were calculated and divided by the number of glycine molecules taken up into the oocytes. Data represent mean ± S.E. (n = 6 oocytes for each transporter). See "Experimental Procedures" for more detail.

These observations raise the question as to whether it is the size of the Ser side chain or the chemical nature of the side chain. To address this point we investigated the properties of alanine substitutions at these two residues. The amplitude of transport currents were reduced for the GLYT1b G305A mutant compared with wild type, but sarcosine does generate transport currents, which indicates that the larger sarcosine can still be accommodated within the substrate binding pocket. This suggests that it is the hydroxyl group that plays an important role in determining how the amino group of the substrate interacts within the site. It is interesting to note that the affinity of glycine for the GLYT1b-G305A mutant is similar to that of wild type GLYT1b. This suggests that the mutation does not cause any significant disruption to the way in glycine interacts with the transporter. The GLYT2a S481A mutant also transported sarcosine, which suggests that this larger substrate can still be accommodated within the site with the methyl side chain, but it cannot fit into the site with the methoxy group of Ser present. Thus, it appears that the hydroxyl group of Ser influences the way in which sarcosine can fit into the substrate binding pocket. The EC₅₀ values for substrate transport by this mutant were significantly larger than for wild type GLYT2a, which suggests that substitution of the Ser residue for an Ala residue may alter the way in glycine interacts with the binding site. Thus, the Ser for Gly difference between GLYT2a and GLYT1b influences whether sarcosine is substrate or not, but in addition the difference is also likely to influence the way in which both glycine and sarcosine fit into the binding site.

Although the neighboring residues (Cys-306 in GLYT1b, Ala-482 in GLYT2a) also differ between GLYT1 and GLYT2, these differences do not appear to influence substrate selectivity. This is not surprising, because in the crystal structure of LeuT_Aa the corresponding residue, Leu-257, faces away from the substrate binding pocket. The other difference between GLYT1b and GLYT2a in close proximity to the substrate binding site (Tyr-302 in GLYT1b, Phe-478 in GLYT2a) does appear to have an impact on substrate transport. The GLYT2a-F478Y mutant was non-functional, and the GLYT1b-Y302F mutant showed reduced expression levels, reduced glycine, and sarcosine transport currents and lower EC₅₀ values for both glycine and sarcosine. In LeuT_Aa the corresponding residue is Phe-253, and the main chain carbonyl of this residue forms a hydrogen bond with the amino group of leucine. It is possible that the Tyr-Phe difference between GLYT1b and GLYT2a may alter the conformation of the unwound section of TM6 (residues 256-260 of LeuT_Aa or 305-309 in GLYT1b) or that the difference may change the way the transporter moves to either allow substrate access to the site or substrate exit from the site as part of the transport process. It is interesting to note that a mutation of human GLYT2 W482R (equivalent to W484 in the clone used in this study) has been identified as causing startle disease (23). This mutant transporter is expressed at the cell surface and binds Na⁺ ions, but does not bind glycine. Thus, this observation and the results presented in the current study suggest that TM6 plays an important role in glycine binding and transport by glycine transporters.

This study has provided an explanation for differences in substrate selectivity between glycine transporters, and we attempted to extend these observations to see if the same changes can explain differences in blocker sensitivities. The GLYT1b-G305S mutant is not functional, but the GLYT1b-G305A transporter does transport both glycine and sarcosine and did show slightly reduced sensitivity to NFPS compared with GLYT1b (31 ± 7% inhibition for GLYT1b-G305A compared with 43 ± 3% inhibition for wild type GLYT1b). The effect was marginal, and therefore, while the hydroxyl group of the Ser residue may play a minor role in influencing blocker selectivity, it is highly likely that other regions of the transporter will also play important roles in binding the blocker.

The GLYT2 mutants S481G and S481A transport sarcosine, and therefore we tested whether the ability of the transporters to bind sarcosine was related to their ability to bind NFPS. However, the GLYT2 mutants do not show any changes in NFPS sensitivity compared with wild type GLYT2. We also investigated whether differences in sensitivity to the GLYT2 inhibitor were related to this amino acid difference. However, all GLYT2 mutants retained sensitivity to the GLYT2 inhibitor, ALX1393. These results suggest that the molecular basis for selectivity of the two glycine transport blockers do not directly correspond with the same region that confers substrate selectivity. There has been one other study on the molecular basis for blocker selectivity of glycine transporters (22). In this study it was suggested that residues within TM1 of GLYT1 contributed to the formation of the NFPS binding site. There are a number of amino acid residues in TM1 that differ between GLYT1 and GLYT2 and the E40D (GLYT1 numbering) difference appears to be partially responsible for the difference in NFPS affinity. At present it is not clear as to whether this difference causes a direct or an indirect change in the structure of the blocker binding site. Glu-40 of GLYT1 is predicted to be located near the intracellular edge of TM1 (10), which is unlikely to be accessible to the bulky NFPS molecule when the transporter is in an inactive state or when both internal and external gates are closed (as depicted in the crystal structure (10)). However, it remains to be seen how the structure of the transporter changes during the transport process and whether the structure opens up sufficiently to allow NFPS access to this site. An alternate interpretation of the role of the Glu-40 residue in GLYT1 is that this residue is important for determining the conformation of a distant site of the transporter that binds NFPS. At present, there is no further understanding of how or where ALX1393 binds to GLYT2.

In the LeuT_Aa structure, two Na⁺ binding sites are identified, and on the basis of sequence similarities between LeuT_Aa, GLYT1, and GLYT2 at least one of the Na⁺ sites is likely to be conserved (10). The second Na⁺ in LeuT_Aa is not conserved in the glycine transporters and yet GLYT1 co-transports 2 Na⁺ ions and GLYT2 co-transporters 3 Na⁺, which raises the question as to where these additional Na⁺ binding sites on the glycine transporters are located. One of the interesting features of the Na⁺ recognition sites in LeuT_Aa is the role of polar, but uncharged, residues in coordinating ion binding, and given that the GLYT2 mutant converts the polar Ser residue to a Gly residue, we investigated whether the mutant altered the Na⁺ coupling ratio. However, although the mutation altered the substrate selectivity, it did not change the Na⁺ coupling ratio. Thus, we conclude that this residue difference between GLYT1 and GLYT2 does not play a role in determining differences in the Na⁺ coupling ratio.

This study has tested the hypothesis of Yamashita et al. (10) that the structure of the bacterial leucine transporter, LeuT_Aa, represents a good working model for understanding the structures of the Na⁺/Cl^--dependent glycine transporters. We have presented evidence demonstrating that the predictions from the structure of LeuT_Aa concerning substrate recognition hold true for the glycine transporters.

	FOOTNOTES

 
^* This work was supported by the Australian National Health and Medical Research Council Project Grant 307604 and Fellowship 358711 (to R. J. V.). 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.

² Current address: Victor Chang Cardiac Research Institute, University of New South Wales, Darlinghurst New South Wales 2010, Australia.

¹ To whom correspondence should be addressed. Tel.: 61-2-93516734; E-mail: robv{at}med.usyd.edu.au.

³ The abbreviations used are: NFPS, N[3-(4-fluorophenyl)-3-(4'-phenylphenoxy)propylsarcosine; TM, transmembrane.

	ACKNOWLEDGMENTS

 
We are grateful for Kong Li, Hue Tran, Xin Liu, and Suzanne Habjan for assistance in maintaining the X. laevis colony.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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