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Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2 Jane E. Carland, Michael Thomas, Shannon N. Mostyn, Nandhitha Subramanian, Megan Louise O'Mara, Renae M. Ryan, and Robert J. Vandenberg ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00407 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 13, 2017

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ACS Chemical Neuroscience

Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2

Condensed Title: Substrate interactions with GlyT2

Key Words: Neurotransmitter transport/glycine/GlyT/LeuTAa/SLC6 family/Na+ dependence

Jane E. Carland1,3, Michael Thomas2, Shannon N. Mostyn1, Nandhitha Subramanian2, Megan L. O’Mara2, Renae M. Ryan1 and Robert J. Vandenberg1 1. Discipline of Pharmacology, School of Medical Sciences, Molecular Biosciences Building, University of Sydney, Sydney, NSW, 2006, Australia 2. Research School of Chemistry, The Australian National University, Canberra, ACT, 0200, Australia 3. Current Address: St Vincents Clinical School, Theraputics Centre, Level 2 Xavier Building, St Vincents Hospital, 391 Victoria Street, Darlinghurst, NSW, 2010, Australia To whom correspondence should be addressed: Robert Vandenberg, Discipline of Pharmacology, School of Medical Sciences, Molecular Biosciences Building, University of Sydney, Sydney NSW 2006, Australia. Tel.: 61-2-9351-6734; E-mail: [email protected]

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ABSTRACT

Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homolog of the family, LeuTAa, and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuTAa, contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabelled amino acid uptake assays and electrophysiological analysis of wild type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site; and furthermore, if a secondary site exists then it is a low affinity non-selective amino acid binding site.


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INTRODUCTION Members of the SLC6 family of Na+/Cl- dependent neurotransmitter transporters play key roles in regulating neurotransmission and are the targets for a variety of therapeutic drugs for the treatment of neurological disorders. They are responsible for regulating the synaptic concentrations of monoamine (5-hydroxytryptamine, dopamine, noradrenaline) and amino acid (γ-aminobutyric acid, glycine) neurotransmitters (reviewed by 2-4). These membranebound proteins use electrochemical gradients to drive the transport of their substrates across neuronal and glial membranes, serving to terminate neurotransmission and replenish intracellular levels of neurotransmitter for future release 5. Extracellular glycine concentrations are regulated by two glycine transporters, GlyT1 and GlyT2. GlyT1 is expressed predominantly in astrocytes and is responsible for clearing glycine from synapses, whereas GlyT2 is predominantly expressed in presynaptic glycinergic terminals, as it is required for accumulating sufficient glycine for loading of synaptic vesicles 6, 7. Crystal structures of a number of members of the SLC6 family have been determined and have provided important insights into the mechanism of transport and also the specificity of inhibitor interactions 8-13. The primary substrate binding site (S1) of a variety of SLC6 transporters consists of a hydrophobic pocket that lies towards the centre of the transporters and is composed of amino acid residues drawn from the middle of transmembrane helices 3 (TM3) and 8 (TM8) and within the unwound sections of TM1 and TM6. The volume and shape of S1 is thought to determine substrate selectivity 9, 11, 12. Substrate binding to this site is dependent upon co-transported Na+ ions binding in close proximity to the substrate 12. A second substrate binding site (S2) has been proposed to exist in the bacterial leucine transporter, LeuTAa as well as the mammalian dopamine (DAT) and norepinephrine transporters (NET) 1, 14-16. The putative S2 lies above S1 within the extracellular vestibule of the transporter, and it has been proposed that substrate binding to the S2 site is required for triggering the release of substrate from the S1 site into the cytoplasm 15. However, there is conflicting evidence for the role of S2 in the mechanism of transport by LeuTAa. While S2 has been implicated in binding and flux experiments 1, 15, other structural and functional data dispute these claims 11, 17. Recently NMR has been used to demonstrate that only a single leucine molecule binds to LeuTAa 18 and extensive molecular dynamics simulations similarly suggest that there is only a single high affinity site for leucine 19. An alternate proposal is that the S2 site may be a low affinity, transient binding site that serves to direct the flow of substrates to the S1 site 9, 19, 20. 3 ACS Paragon Plus Environment


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Investigations into the existence of the S2 site have focused primarily on LeuTAa. LeuTAa is a promiscuous transporter, allowing transport of alanine, glycine, leucine, methionine and tyrosine while tryptophan is a competitive inhibitor 9. The proposed S2 site consists of a series of hydrophobic residues that appear to facilitate hydrophobic interactions between the side chain of the various amino acids and the transporter 15. In contrast, the human glycine transporter, GlyT2, is exquisitely selective for glycine. No other amino acid has been identified as a substrate or inhibitor of GlyT2. In comparison the glycine transporter GlyT1 is not as selective for glycine as GlyT2, in that GlyT1 can also transport N-methylglycine and N-ethylglycine 21, 22. Furthermore, glycine does not have a side chain and as such does not have the capacity for forming side chain hydrophobic interactions with the S2 site. Thus, GlyT2 provides a unique opportunity to explore the roles of the S1 and S2 sites in the mechanism of transport.

We have used a combination of molecular dynamics simulations of a homology model of GlyT2 and functional analysis of site directed mutants of recombinant GlyT2 to investigate the roles of the S1 and S2 sites in the transport mechanism. We provide multiple lines of evidence that suggest that glycine binding to a S2 site is not required for the transport mechanism. First, we demonstrate using molecular dynamics simulations that whilst the S1 site is selective for glycine, glycine does not form stable interactions with the S2 site. Second, analysis of the functional properties of wild type and mutant transporters confirm the central role of the S1 site in determining substrate selectivity and demonstrate that mutations to the S2 site do not prevent transport or influence substrate selectivity. Third, if glycine were to bind to both the S1 and S2 sites as part of the transport mechanism, cooperativity between the sites may be expected. However, no such cooperativity is observed under a variety of ionic conditions, suggesting that either glycine binding to the S2 site does not influence the steady state rate of glycine transport or that glycine does not bind at all to the site.

RESULTS Validation of the GlyT2 Model We have previously used a GlyT2 homology model based on the nortriptyline bound structure of dDAT (PDBid: 4M48) to identify the three Na+ binding sites and the S1 glycine site of GlyT2, and also that the binding and coordination at the Na1, Na2 and S1 sites is 4 ACS Paragon Plus Environment

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consistent with that observed in the dDAT and LeuTAa crystal structures 23 (Fig. 1a). The S2 site Hypothesis for LeuTAa was developed based on the crystal structure of the outward facing substrate occluded conformation of LeuTAa (PDBid: 2A65) and so we further validated our GlyT2 homology model to check that it forms an outward occluded structure and has the capacity to form a similar S2 site as observed in LeuTAa. We first confirmed that the GlyT2 homology models adopts the outward occluded conformation when glycine and Na+ ions are bound to the S1 site, as judged by the formation of an extracellular gate between Arg216 and Asp633 (see Fig. S1). At a more global level, the Cα RMSD calculated for GlyT2 aligned with the outward occluded conformation of LeuT (PDBid: 2A65) for TMs 1-11 is 1.85 Å, indicating no significant difference between the two structures (see Fig. S2). To investigate the similarity between the S2 sites of LeuTAa and the GlyT2 model, we first aligned the S2 site of LeuTAa with the corresponding residues in the GlyT2 model (Fig. 1b). We then plotted the distances between α-carbons of the residues that form the proposed S2 site in LeuTAa with the α-carbons of the corresponding residues in the GlyT2 model throughout a 250 ns simulation (Fig. 1c). Given the relatively close alignment of α-carbon differences (1-2.5 Å), we can conclude that the GlyT2 S2 site is very similar to the S2 site of LeuTAa and forms a reasonable starting point for simulating interactions between amino acids and the S2 site.

Molecular Dynamics Simulations of Substrate Interactions with S1 and S2 The amino acid residues that formed the initial starting position for the S1 site included Gly210, Leu211, Trp215, Tyr286, Tyr287, Leu574, Gly575 and Thr578 and the residues that formed the starting position for the S2 site included Trp215, Gln541, Gln630 and Asp633. The molecular basis for substrate interactions with the human glycine transporter GlyT2 was first investigated using branched molecular dynamics simulations of substrate interactions with the S1 and putative S2 site using a membrane-embedded homology model of GlyT2 to initiate the simulations. The simulation protocol performed in this study, and its results are summarised in Figures 2 and 3 and Table 1, and are expanded on below. In the first generation of three simulations, the substrate glycine was placed at the S1 site and the nontransportable amino acid leucine was placed at the S2 site (Fig. 1a, 3a,b,c). These systems are referred to using S1/S2 (generation) nomenclature: Gly/Leu(1) (see Fig. 2). Glycine, Na1 and Na2 remained stable in the S1 site and leucine remained stable in the S2 site in the Gly/Leu(1) system for the entire 250 ns in all three simulations. Leucine in S2 formed direct interactions with Trp215, Tyr286, Gly542, Phe547, Phe629, Gln630 and Asp633 – spending 5 ACS Paragon Plus Environment


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more than 25% of the total simulation time within 3.5 Å of these residues (Fig. 3a, 3c, Table 1).

When the S2 leucine was replaced by the substrate glycine at the start of the second generation of simulations, to give Gly/Gly(2), the glycine dissociated from the S2 site of GlyT2 and partitioned into the bulk water. When leucine was removed to give a second generation Gly/Apo(2) system, glycine in S1 remained stable for each of the three 250 ns simulations, consistent with our previous findings 24. In one of the Gly/Leu(1) simulations, the simulation was stopped half way and used to start three Gly/Gly mid(2) simulations in an attempt to identify an alternate conformation where glycine may form stable interactions with S2. However, the glycine at S2 again partitioned into the bulk water as observed for the Gly/Gly(2) simulations.

S2 Access Simulations The stable Gly/Apo(2) simulation was used to test if glycine or leucine could spontaneously bind to the S2 site. Two sets of simulations were performed (with three replicates of 250 ns each). In the first, 100 molecules of glycine were added to the bulk water and allowed to bind spontaneously. In the second, the bulk water contained 100 molecules of leucine. The distance between the centre of mass of the extracellular gate (defined as the centre of mass of Trp215, Tyr286 and Asp633) and the added glycine or leucine molecules was measured and is presented as a histogram in Figure 3d. This histogram demonstrates that both glycine and leucine enter the external vestibule and can come close to, but not quite reach the S2 site and remain 4-6 Å from the site in all three simulations. Thus, it seems probable that both leucine and glycine can approach S2 from bulk solution, but face an energetic barrier to make the final leap into the binding site.

In summary, the molecular dynamics simulations of the GlyT2 model demonstrate that the S1 site can form stable contacts with the substrate glycine, Na1 and Na2. The non-transportable amino acid leucine appears to form stable interactions with the S2 site, but the substrate glycine is unstable at this site, suggesting that substrate binding to the S2 site may not be essential for substrate transport. However, whilst the homology model is able to generate a S1 site and Na+ binding sites that are consistent with the functional characteristics of GlyT2


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(Subramanian et al., 2016) and an occluded structure that resembles the LeuTAa used to hypothesize the S2 site, the homology model was based on a 3.0Å resolution structure of dDAT that shares ~50% amino acid sequence identity with GlyT2. Thus, we cannot definitively rule out the possibility that the homology model does not faithfully generate a S2 site. To further elaborate and complement these observations we conducted a series of experiments to address the functional implications of amino acid binding to both the S1 and S2 sites using recombinant wild type and mutant GlyT2.

Characterization of Glycine Transport by GlyT2 We have utilised the Xenopus laevis oocyte expression system for analysing the functional properties of GlyT2 which provides an opportunity to study this class of transporters in a cellular environment that is a good approximation to its native environment. Application of 10 µM [3H]-labelled glycine to oocytes expressing GlyT2 for 10 minutes results in uptake of glycine that is ~24 fold above uptake by control oocytes (Fig. 4a). Co-application of either 300 µM L-leucine or 300 µM L-alanine has no effect on [3H]glycine uptake (Fig 4a), indicating that these alternate amino acids do not inhibit or stimulate glycine transport. Furthermore, incubation of oocytes expressing GlyT2 with 10 µM [14C]alanine does not result in any uptake above that of uninjected oocytes (Fig. 4b). Thus, GlyT2 is selective for glycine, which is consistent with previous studies 21, 25, 26. Glycine transport by GlyT2 is coupled to the co-transport of 3 Na+ and 1 Cl- generating a net transfer of 2 positive charges per glycine molecule transported, which allows transport to be measured by electrophysiological methods 21, 27. Application of glycine to Xenopus laevis oocytes expressing wild-type GlyT2, voltage clamped at -60 mV, produces concentration-dependent inward transport currents (GlyT2, EC50 = 13 ± 2 µM, n=11; Fig. 4c, d, Table 1), which can be exploited in more detailed analysis of the functional properties of the transporter. No other amino acids tested (300 µM leucine, isoleucine, valine, threonine, methionine, proline, phenylalanine, tyrosine or sarcosine) generate inward currents at GlyT2 (Fig. 4c, Table 2), and

neither L-alanine or L-leucine can reduce or increase the amplitude of the glycine-induced currents (Fig. 4c, d). These observations further confirm that no other amino acids can be transported, inhibit, or stimulate glycine transport by GlyT2 and that the glycine-induced currents are a direct reflection of substrate transport. Thus, although the molecular dynamics studies indicate that leucine, and possibly other non-transportable amino acids, may bind to the S2 site of GlyT2 they do not impact on the steady state rate of glycine transport. 7 ACS Paragon Plus Environment


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The S1 site of GlyT2 determines substrate selectivity The S1 site of GlyT2 is formed by Ile283 in TM3; Phe476, Ser479 and Trp482 in TM6; and Thr578 and Thr582 in TM8 (Fig. 3) 12. These residues were mutated in GlyT2 to the corresponding amino acids in either LeuTAa or the closely related GlyT1 with the aim of investigating the molecular determinants of substrate interactions with the S1 site. Introduction of the substrate binding site mutations into GlyT2 produced transporters with altered EC50s for glycine (Fig. 5b-g, Table 2). The EC50 for glycine was increased for the I283V transporter compared to wild-type GlyT2, but reduced by ~4-5-fold for S479G and T578S transporters, while transporters with the W482F mutation exhibited a substantial reduction in apparent glycine affinity (EC50 > 1 mM) (Fig. 4d-g, Table 2). Introduction of the F476Y (Vandenberg et al., 2007) and T582L mutations produced non-functional or low expression transporters, respectively. No further work was undertaken on these two mutant transporters.

A unique characteristic of GlyT2 is its exquisite selectivity for glycine (see Fig. 4 and Table 2). In addition to changes in glycine EC50s, substrate binding site mutations cause substantial changes in substrate selectivity (Fig. 5d-g, Table 2). Both GlyT1 and GlyT2 contain a tryptophan residue in the S1 site that is not found in other SLC6 transporters. It has been predicted that the large tryptophan residue may limit the range of substrates that can fit into the S1 pocket and thereby select for the smallest amino acid, glycine (12, and simulation studies above). A phenylalanine residue is found at this position in LeuTAa, as well as the Drosophila dopamine (dDAT) and human dopamine and serotonin transporters, and a leucine residue is present in this position of the γ-aminobutyric acid transporter, GAT-1 (Fig. 5a). To determine how residue properties at position 482 influence the substrate profile of GlyT2, we mutated Trp482 to Leu, Tyr, Arg and Glu. The W482F mutant allowed transport of the broadest range of substrates, with eight amino acids generating concentration-dependent inward transport currents (Fig. 5c, d). Alanine exhibited the lowest EC50 for W482F, whilst leucine and methionine were ~3-fold higher, with cysteine also showing low affinity transport. Unexpectedly, aromatic amino acids were also weak substrates of W482F, with tyrosine and phenylalanine having EC50s ~3- and 7-fold higher than alanine, respectively. Glycine and valine were also capable of acting as weak substrates of W482F. The amino acids that generated appreciable transport currents by the W482F mutant show similar predicted maximal transport currents to that of alanine (Table 2). 8 ACS Paragon Plus Environment

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Whilst the electrophysiological measures of transport suggest that the substrate selectivity profile of the W482F mutant has been substantially altered by the mutation, it is important to rule out the possibility that these alternate amino acid induced currents are due to transport and not due to substrate-induced leak currents.

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C-Alanine uptake was also measured for

oocytes expressing the W482F mutant to confirm that the amino acid-induced currents also reflect substrate transport. The W482F mutant showed a rate of uptake of 10µM 14C-alanine that was comparable to the rate of 3H-glycine uptake by WT GlyT2 (Fig. 4b) and is consistent with the electrophysiological analysis.

By mutating Trp482 to Leu and Tyr, we retained the hydrophobic characteristic of the residue, but altered the side-chain volume. Of the mutant transporters characterised in this study, W482L was the only one that did not transport glycine. Instead, five alternative amino acids were found to be weak substrates of the W482L transporter. Leucine was identified as the primary substrate, with an EC50 ~12-fold higher than glycine at wild type GlyT2. Cysteine and methionine have EC50s ~3-fold higher than leucine, while the EC50s for alanine, valine and isoleucine are ≥ 1 mM (Table 2). The W482Y mutant, although sensitive to glycine, had further reduced transport capacity, with low levels of activity observed for glycine, alanine, methionine and serine (EC50s > 3 mM). Although GAT1 contains a leucine residue at this position (Fig. 5a), γ-aminobutyric acid (GABA) or β-alanine did not generate measureable transport currents when applied to the W482L mutant. This suggests that the tryptophan residue influences side chain specificity but does not influence the specificity for compounds with differing distances between the substrates backbone carboxyl and amino groups.

The GlyT2 W482R mutation has been observed in a patient with hyperekplexia which is a disease characterised by noise- or touch-induced seizures 28, 29. Examination of W482R mutant transporters revealed that, while they are expressed on the cell surface, glycine transport has been abolished 29. In agreement with this finding, mutation of Trp482 to the charged residues Arg and Glu produced non-functional transporters in this study.

Introduction of the conservative T578S mutation into the substrate binding site also enabled a wide range of amino acids to be transported. Whilst the EC50 for glycine was lower than wildtype, other substrates (alanine, isoleucine, cysteine, methionine, serine and β-alanine) showed 9 ACS Paragon Plus Environment


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weak activity, with predicted EC50s > 1 mM (Fig. 5g). Mutations of both Ser479 and Ile283 also altered substrate selectivity, albeit to a lesser degree. In agreement with previous work, the N-methyl derivative of glycine, sarcosine, is a substrate of the S479G mutant, but is not transported by wild-type GlyT2 21. Further, N-ethylglycine was also found to be a GlyT1 substrate 22 (EC50 80 ± 9 µM, n=3) and had a low affinity for S479G (EC50 >1 mM) and no effect at GlyT2 (Fig. 5b, e, Table 2).

The isoleucine residue present at position 283 of GlyT2 is conserved in the corresponding position of GlyT1 and the serotonin transporter. Valine is present in the equivalent positions of dDAT and LeuTAa, while leucine is present in GAT-1 (Fig. 5A). In addition to glycine transport, the I283V mutant also allows low affinity transport of alanine and the larger sarcosine and β-alanine molecules (Fig. 4f). In contrast, the I283L mutant transporter retained its specificity for glycine, albeit with a significantly increased EC50 compared to wild-type. It is interesting to note that although both I283V and T578S transporters allow β-alanine to be transported, none of the mutant transporters allow the slightly longer molecule, GABA, to be transported.

All S1 site mutants of GlyT2 allow a range of amino acids to be transported (Fig. 5, Table 2). Thus, if substrate binding to S2 is required for release of substrate from S1, then these extra amino acid substrates must also interact with S2. However, although other amino acids may bind to the S2 site, they do not influence the rate of glycine transport by wild type GlyT2 (Fig. 4). Thus, these results are consistent with the conclusion from the molecular dynamics study that substrate selectivity of GlyT2 is determined entirely by the primary Na+-dependent substrate binding site.

Site Directed Mutagenesis of the Proposed S2 Site of GlyT2 We also investigated the functional implications of the GlyT2 S2 site by mutating some of the residues identified in the molecular dynamics simulations that also correspond to those proposed to form the LeuTAa S2 site 1 (see above). The impact of mutations to three tyrosine and two phenylalanine residues was investigated: Tyr286 and Tyr293 in TM3; Phe547 in EL4; and Tyr627 and Phe629 in TM10 (Fig. 6a). Given the high degree of conservation of residues lining the proposed S2 site in the SLC6 family, we introduced mutations that made


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subtle changes to steric and electrostatic characteristics. All mutants retained glycine transport, albeit with some changes in EC50 for glycine (Fig. 6b, Table 3). The EC50s for glycine were unaltered compared to wild-type for Y286F, Y293F and Y627F transporters, but decreased ~3- and 2.5-fold for the F547L and F629L transporters compared to wild-type GlyT2. The rate of 3H-glycine uptake for the F629A mutant was also measured. The rate of uptake of 3H-glycine was ~12-fold greater than uninjected control oocytes, which further confirms that the glycine induced currents for this mutant also reflect glycine transport. Of the residues proposed to form the S2 site of LeuTAa, the role of Leu400 has been controversial, with conflicting results that either implicate 1, 15, 16 or dismiss 17, 18, 30 its importance in transporter function. In studies that endorse the essential role of the S2 site in the transport mechanism, the identity of the residue at this position has been shown to be particularly important for transporter function. The L400C mutant in LeuTAa impairs transporter function, while the L400S mutation abolishes function 1. It should be noted that in binding studies by Piscitelli et al., 17 no differences in binding were observed between the mutants and wild type LeuTAa. Leu400 of LeuTAa corresponds to Phe629 in GlyT2, a residue observed to make contacts with leucine in simulations of the GlyT2 S2 site (Fig. 3). This Phe residue is conserved in GAT-1 and DAT, but is a Leu residue in GlyT1 and LeuTAa and a Val residue in SERT (Fig. 6). To further explore the importance of the predicted S2 site to GlyT2 transport function, we generated F629L, F629A and F629S mutants. All three mutants retained glycine transport with EC50s for glycine transport decreased by ~3-fold and increased by ~4- and ~10-fold compared to wild-type, respectively (Fig. 6b, Table 3). The failure of GlyT2 S2 site mutations to knock out glycine transport is consistent with the conclusion that a Phe residue within S2 is not essential for GlyT2 function. Alanine and leucine were also tested as substrates for each of the S2 site mutants and, in all cases, no transport currents were detected. Leucine and alanine do not influence the rate of glycine transport by the S2 site mutants and, the rate of 14C-alanine by F629A was not significantly greater than background uptake, which is similar to that of WT GlyT2 (Fig. 4b). All of the S2 mutants show the same high selectivity for glycine over other amino acids as observed for wild type GlyT2 and alternate amino acids are not transported or modulate glycine transport. Thus, mutations in S2 do not influence substrate selectivity or the ability of leucine or alanine to modulate glycine transport. The small changes in apparent affinity for glycine observed with some of these mutants is interesting. A possible explanation is that the mutations disrupt allosteric coupling between the two sites, or alternatively, it may reflect a disturbance in the 11 ACS Paragon Plus Environment


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structure of the S2 region caused by the mutation generates a subtle change in the S1 site which leads to altered glycine affinity at the S1 site.

Lack of co-operativity between substrate binding to S1 and S2 sites If substrate binding to S2 is required for release of substrate from S1, the binding of the second substrate may influence the transport of the first creating a substrate concentration response curve with a Hill co-efficient that deviates from 1. However, in the case of glycine transport currents, no deviation from 1 is observed (Fig. 7). This could be explained by glycine having similar affinities at S1 and S2 such that no apparent cooperativity is observed (but note that this suggestion is inconsistent with the molecular dynamics simulations above where glycine was unstable in the S2 site). In order to tease apart any cooperative interactions that may exist between the two sites, we can exploit the likely differential sensitivity of S1 and S2 to Na+ ions 31. Glycine binding to S1 is tightly coupled to Na+ binding to the Na1 and Na2 sites with the affinity of glycine dependent upon the Na+ concentration (Fig. 7) 23. In contrast, based on modelling of leucine binding to S2 of LeuTAa, and the molecular dynamics studies presented above, any glycine binding to S2 of GlyT2 is likely to be Na+ independent. Thus, if substrate binding to S2 influences the rate of glycine release from S1, it may be possible to measure differential cooperativity in glycine transport currents using different Na+ concentrations. The EC50 for Na+ is 33 ± 1 mM with a Hill coefficient of 2.5 ± 0.2 23 and so we measured glycine concentration dependent transport currents in 30 mM Na+, 15 mM Na+ and the standard ND96 buffer containing 98.5 mM Na+. The EC50s for glycine transport currents measured at 15, 30 and 98.5 mM Na+ are 157 ± 20 µM, 38 ± 3 µM and 13 ± 2 µM, respectively (Fig. 7), as may be expected for Na+-dependent transport. The relative maximal glycine currents were also reduced in 30 mM Na+ and 15 mM Na+ to 0.51 ± 0.03 and 0.27 ± 0.04, respectively, compared to 98.5 mM Na+. However, under all three conditions, the Hill coefficients for glycine do not deviate significantly from 1 (Fig. 7). The lack of change in cooperativity, despite the changes in extracellular Na+ concentration, EC50s for glycine and relative Imax values, suggests that Na+ does not influence cooperativity between the two sites and that glycine binding to the secondary site, if it binds at all, does not influence glycine transport.

DISCUSSION


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Multiple crystal structures of the prokaryotic amino acid transporter LeuTAa 9, 12, 32 have been used as a model system to better understand the mechanism of transport for the closely related mammalian neurotransmitter transporters. Using these structures, it has been proposed that substrate transport by the prokaryotic leucine transporter LeuTAa requires a binding event at a secondary allosteric site in order for substrate to be released from the primary substrate site 1, 15, 16. This concept for LeuTAa has been questioned by various groups 11, 17-19 and so we investigated whether a similar mechanism of transport exists for the related human glycine transporter, GlyT2. Whilst it is difficult to definitively prove that something does not exist, we have made four independent observations that are consistent with the suggestion that a second substrate binding site is not required for glycine transport by GlyT2. First, using molecular dynamics simulations we show that with glycine and Na+ ions bound to the S1 site, the non-transportable amino acid leucine, but not the substrate, can bind to the S2 site. This suggests that whilst some amino acids may bind to the S2 site, the lack of glycine binding to this site indicates that S2 is unlikely to play a critical role in the glycine transport mechanism (Fig 2). These observations are consistent with the recent molecular dynamics study of LeuTAa, where the affinities of leucine for LeuTAa at the two sites were estimated to be 14 nM for S1 and either 200 mM or 3.3 M for S2 19, which also suggest that the S2 site plays a minimal role in the transport mechanism at low substrate concentrations. Furthermore, Erlendsson et al., 18 used a NMR technique to demonstrate that leucine is able to bind to the S1 site of LeuTAa, but not the S2 site. However, it should be pointed out that these results are inconsistent with the report by Quick and co-workers who demonstrate using equilibrium binding assays that 3H-leucine has similar affinity at both the S1 and S2 sites 1.

Second, the only amino acid that could potentially bind to the S2 site of WT GlyT2 to influence the mechanism of transport is glycine. No other amino acid can be transported, inhibit or stimulate transport (Fig. 4). However, once conservative mutations are introduced in the S1 site, additional amino acids can be transported (Fig. 6) and this must be solely due to their interactions with the S1 site because we know that these additional amino acids do not interact with any other site on the transporter in a way that can influence function.

Third, mutations in the S2 region cause no change in selectivity and only modest changes in EC50s for glycine transport (Fig. 6). Thus, if an S2 site does exist in GlyT2, it plays no role in determining substrate selectivity. 13 ACS Paragon Plus Environment


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Fourth, for the wild type GlyT2, the Hill co-efficient for substrate transport is close to 1 (Fig. 7) and if two glycine molecules were required for transport, with the S1 site being Na+ dependent and glycine binding to S2 being Na+ independent, then changes in Hill co-efficient for transport may be observed with differing Na+ concentrations. However, under different Na+ concentrations, the Hill co-efficient for glycine remained close to 1 (0.99 ± 0.04 for 98.5 mM Na+, 0.96 ± 0.06 for 30 mM Na+ and 0.94 ± 0.11 for 15 mM Na+, Fig. 7). Thus, if there are two sites, binding of the second substrate molecule does not influence the rate of glycine transport. The most plausible, parsimonious explanation for these four independent observations is that GlyT2 does not require substrate binding to S2 for substrate to be transported from S1.

The residues in LeuTAa that correspond to the putative S2 region are highly conserved throughout the SLC6 family (Fig. 6). This is of particular interest given that the substrate selectivity profile of transporters within this family is a characteristic unique to each protein. In our simulation study, we observed that leucine can bind to the S2 site, but glycine is unstable over the course of a series of 250 ns simulations (Fig. 3), which is also difficult to reconcile with such interactions causing a specific conformational change required to modulate the S1 site to facilitate substrate release. The binding of leucine to the S2 site appears to be stabilised by hydrophobic interactions between the aliphatic side chain of leucine and the aromatic side chain of Phe629. If this observation is combined with the demonstration that leucine does not influence the rate of glycine transport it suggests that binding of an amino acid to the S2 site of GlyT2 is not important for the mechanism of transport.

These observations raise an intriguing question. The S2 site is approximately 10Å from S1 and if amino acids such as leucine can bind to the S2 site, why do they not influence glycine access to S1 site and influences the rate of glycine transport. One possible explanation for the apparent discrepancy between the various studies of the S2 site is that while this site is unable to discriminate between various substrates, it may serve to funnel all potential substrates into the S1 site. The molecular dynamics simulations, where the extracellular solution was flooded with glycine or leucine, supports the conclusion that the amino acids may enter the external vestibule and potentially interact with the S2 site. The recent molecular dynamics simulation study of LeuTAa has come to similar conclusions where substrate binding to the S2 14 ACS Paragon Plus Environment

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site is very weak (affinity constants of either 200 mM or 3.3 M) 19, which suggests that binding to this site is unlikely to be of functional significance. One interpretation of these observations is that the S2 site may serve to non-selectively capture substrates, slowing dissociation of potential substrates from the transporter, and subsequently allowing the S1 site to efficiently select substrates for transport from a more concentrated pool. So, whilst substrate binding to S2 is not required for the mechanism of transport it may serve to direct the flow of substrates to the S1 site.



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METHODS Molecular dynamics, System set-up The experimentally validated, equilibrated homology model of GlyT2 of Subramanian et al., 23

was used as a starting conformation to initiate molecular dynamics simulations. This

equilibrated GlyT2 system contained the solvated GlyT2 homology model embedded in a POPC bilayer. The bath solution of the system contained 150 mM NaCl to mimic physiological conditions. Resident Na+ ions were bound to their respective binding sites in the GlyT2 homology model and one molecule of glycine was bound in the S1 site, as described by Subramanian et al., 23. Three “generations” of molecular dynamics simulations of the GlyT2 homology model were performed to examine substrate interactions at both the S1 and S2 sites. As shown in the schematic of Figure 1, three first generation systems were simulated in which a single molecule of glycine was placed in the S1 site (as found in the crystal structures of dDAT (PDBid: 4M48) and LeuTAa (PDBid: 3TT1)) 30, 32, and S2 contained a molecule of the non-transportable amino acid leucine. Each system was energy minimised, then relaxed by placing a harmonic force constant of 1000 kJ/mol/nm2 on each alpha carbon and simulated for 25 ns. This harmonic force constant which was lowered to 100 kJ/mol/nm2 in a subsequent 25 ns simulation. All constraints were then removed and nonbiased simulations were conducted for a further 250 ns, or until the substrate dissociated from the binding site. Each system was simulated in triplicate. Each system was named according to the amino acid placed in the S1 and S2 sites and the generation of simulation. For example Gly/Leu(1) refers to a first generation (1) simulation starting with glycine in the S1 site and leucine in the S2 site. After the first generation of simulations, three further simulations were performed, each lasting 250 ns. These simulations are referred to as Systems Gly/Gly(2), Gly/Apo(2) and Gly/Gly Mid(2). The Gly/Gly(2) Mid simulation was started from the mid point of one of the first generation simulations (see Results for more explanation). The end points of the three simulations of Gly/Apo(2) were again used to initiate a third generation of branched simulations in which 100 molecules of either glycine or leucine were placed in the aqueous solution Gly/Gly flood(3) or Gly/Leu flood(3). In all cases, the systems were energy minimized and relaxed as outlined above. Non-biased simulations were performed in triplicate for 250 ns. A schematic of the branched simulation protocol is provided in Figure 1. Note: in all cases, the backbone of the substrate amino acids were zwiterionic, as appropriate at neutral pH.

Molecular dynamics, Simulation conditions 16 ACS Paragon Plus Environment

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The GROMACS version 5.1.2 MD package 33, in conjunction with the GROMOS 54A7 force field 34 was used in all MD simulations. Water was represented explicitly using the simple point charge (SPC) model 35. Each system was simulated under periodic boundary conditions in a rectangular simulation box. The temperature of the system was maintained by coupling the protein and lipids together and the solvent, ions and the ligand together to an external temperature bath at 300 K with a coupling constant of τT = 0.1 ps using a velocity rescaling thermostat. The pressure was maintained at 1 bar by weakly coupling the system to a semiisotropic pressure bath using an isothermal compressibility of 4.5x10-5 bar-1 and a coupling constant of τP = 0.5 ps. During the simulations, the length of all bonds within the protein and lipids were constrained using the LINCS algorithm 36. The SETTLE algorithm 37 was used to constrain the geometry of water molecules. In order to further extend the timescale that could be simulated, the mass of hydrogen atoms was increased to 4 a.m.u. by transferring mass from the atom to which it was attached. This allows a time step of 4 fs to be used to integrate the equation of motion without significantly affecting the thermodynamic properties of the system 38. Electrostatic interactions were calculated using particle mesh Ewald summation and nonbonded interactions were calculated with a cut-off of 1.0 nm. Both were updated each timestep.

Molecular Biology cDNAs encoding human GlyT2a and mutant GlyT2a constructs were subcloned into oocyte transcription vector (pOTV). Point mutations were introduced using standard molecular biological techniques, and all constructs were sequenced to confirm fidelity. The plasmids were linearized with SpeI and mRNA was synthesized using the T7 RNA polymerase mMESSAGE mMACHINE kit (Ambion). GlyT2a is referred to as GlyT2 in the text.

Expression of Glycine Transporters in Xenopus laevis oocytes and electrophysiology Oocytes were harvested from Xenopus laevis as described previously 39, with all procedures approved by the University of Sydney Animal Ethics Committee and in accordance with the Australian National Health and Medical Research Council guidelines for the prevention of cruelty to animals. Stage V-VI oocytes were injected with 10 ng mRNA encoding the wild type and mutant transporters and then stored at 16°C in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.5) supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 µg ml-1 gentamycin and 100 µg ml-1 tetracycline. The storage solution was changed daily. 17 ACS Paragon Plus Environment


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3-7 days after injection, recordings of transporter activity were obtained by two-electrode voltage clamp by means of a GeneClamp 500B amplifier (Axon Instruments) interfaced with a Powerlab 2/26 (ADInstruments) used in conjunction with Chart software (ADInstruments). Oocytes were voltage clamped at -60 mV and continually perfused with ND96 solution at room temperature. Known concentrations of substrates were applied to oocytes until a stable current was reached, at which time the oocyte was washed for 3 to 5 minutes, sufficient time to allow complete recovery of response to the substrate. Recordings were made for each construct from at least two batches of oocytes.

Radiolabelled uptake experiments Uptake of [3H]glycine or [14C]alanine (Perkin Elmer) was measured in oocytes expressing wild type and mutant GlyT2 and uninjected oocytes. 4-8 oocytes were incubated in ND96 buffer with 10 µM [3H]glycine or [14C]alanine at room temperature. After 10 minutes, uptake was terminated by three rapid washes in ice-cold ND96 buffer followed by lysis in 50 mM NaOH and 50% SDS. [3H]glycine or [14C]alanine was measured by scintillation counting using a Trilux beta counter (Perkin Elmer). Experiments were conducted on oocytes from at least two batches of oocytes.

Data Analysis Current (I) as a function of glycine concentration [Gly] was fitted by least-squares analysis to a derivation of the Michaelis–Menten equation, I/Imax = ([Gly])/(EC50 + [Gly]), or the Hill equation, I= ([Gly]n. Imax)/(EC50n + [Gly]n), using GraphPad Prism version 5.00 for Windows (GraphPad Software). Imax is the maximal current, EC50 is the concentration of glycine that generates half maximal current and n is the Hill coefficient for cooperativity between substrate interactions. Data for each mutant transporter was derived from oocytes collected from at least two Xenopus laevis frogs.


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[20] Topiol, S., Bang-Andersen, B., Sanchez, C., and Bogeso, K. P. (2016) Exploration of insights, opportunities and caveats provided by the X-ray structures of hSERT, Bioorg Med Chem Lett 26, 5058-5064. [21] Vandenberg, R. J., Shaddick, K., and Ju, P. (2007) Molecular basis for substrate discrimination by glycine transporters, The Journal of biological chemistry 282, 14447-14453. [22] Werdehausen, R., Kremer, D., Brandenburger, T., Schlosser, L., Jadasz, J., Kury, P., Bauer, I., Aragon, C., Eulenburg, V., and Hermanns, H. (2012) Lidocaine metabolites inhibit glycine transporter 1: a novel mechanism for the analgesic action of systemic lidocaine?, Anesthesiology 116, 147-158. [23] Subramanian, N., Scopelitti, A. J., Carland, J. E., Ryan, R. M., O'Mara, M. L., and Vandenberg, R. J. (2016) Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2, PloS one 11, e0157583. [24] Subramanian, N., Scopelitti, A.J., Carland, J.E., Ryan, R.M., O’Mara, M.L. and Vandenberg, R.J. (2016) Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2, PloS one 11, e0157583. [25] Liu, Q. R., Lopez-Corcuera, B., Mandiyan, S., Nelson, H., and Nelson, N. (1993) Cloning and expression of a spinal cord- and brain-specific glycine transporter with novel structural features, The Journal of biological chemistry 268, 22802-22808. [26] Morrow, J. A., Collie, I. T., Dunbar, D. R., Walker, G. B., Shahid, M., and Hill, D. R. (1998) Molecular cloning and functional expression of the human glycine transporter GlyT2 and chromosomal localisation of the gene in the human genome, FEBS letters 439, 334-340. [27] Roux, M. J., and Supplisson, S. (2000) Neuronal and glial glycine transporters have different stoichiometries, Neuron 25, 373-383. [28] Harvey, R. J., Carta, E., Pearce, B. R., Chung, S. K., Supplisson, S., Rees, M. I., and Harvey, K. (2008) A critical role for glycine transporters in hyperexcitability disorders, Front Mol Neurosci 1, 1. [29] Rees, M. I., Harvey, K., Pearce, B. R., Chung, S. K., Duguid, I. C., Thomas, P., Beatty, S., Graham, G. E., Armstrong, L., Shiang, R., Abbott, K. J., Zuberi, S. M., Stephenson, J. B., Owen, M. J., Tijssen, M. A., van den Maagdenberg, A. M., Smart, T. G., Supplisson, S., and Harvey, R. J. (2006) Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease, Nat Genet 38, 801-806. [30] Penmatsa, A., Wang, K. H., and Gouaux, E. (2013) X-ray structure of dopamine transporter elucidates antidepressant mechanism, Nature 503, 85-90. [31] Reyes, N., and Tavoulari, S. (2011) To be, or not to be two sites: that is the question about LeuT substrate binding, J Gen Physiol 138, 467-471. [32] Krishnamurthy, H., and Gouaux, E. (2012) X-ray structures of LeuT in substrate-free outwardopen and apo inward-open states, Nature 481, 469-474. [33] Abraham MJ, M. T., Schulz R, Páll S Smith JC, Hess B, Lindahl E (2015) GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers, SoftwareX 1, 19-25. [34] Schmid, N., Eichenberger, A. P., Choutko, A., Riniker, S., Winger, M., Mark, A. E., and van Gunsteren, W. F. (2011) Definition and testing of the GROMOS force-field versions 54A7 and 54B7, European biophysics journal : EBJ 40, 843-856. [35] Hermans J, B. H., van Gunsteren WF, Postma JPM (1984) A Consistent Empirical Potential for Water-Protein Interactions, Biopolymers 23, 1513-1518. [36] Hess B, B. H., Berendsen HJC, Fraaije JGEM. (1997) LINCS: A Linear Constraint Solver for Molecular Simulations., J Comput Chem 18, 1463-1472. [37] Miyamoto S, K. P. (1992) SETTLE: An Analytical Version of the Shake and Rattle Algorithm for Rigid Water Models, J Comput Chem 13, 952-962. [38] Feenstra KA, H. B., Berendsen HJC (1999) Improving Efficiency of Large Time-Scale Molecular Dynamics Simulations of Hydrogen-Rich Systems, J Comput Chem 20, 786-798. 20 ACS Paragon Plus Environment

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[39] Wiles, A. L., Pearlman, R. J., Rosvall, M., Aubrey, K. R., and Vandenberg, R. J. (2006) NArachidonyl-glycine inhibits the glycine transporter, GLYT2a, Journal of neurochemistry 99, 781-786.

ACKNOWLEDGEMENTS: We thank Cheryl Handford for expert technical assistance and maintenance of the Xenopus laevis facility at the University of Sydney. This work was supported by a National Health and Medical Research Council Project Grant (APP1082570) and the Merit Allocation Scheme on the NCI National Facility at the ANU. MLO is supported by an ARC DECRA (DE120101550). SM is supported by an Australian Postgraduate Award. The authors declare no competing financial interests. AUTHOR CONTRIBUTIONS: JEC, MT, SM and RV conducted and planned experiments, analysed data, helped write the manuscript. MLO, NS and RR planned experiments, analysed data, helped write the manuscript. COMPETING FINACIAL INTERERST All authors declare that they have any conflicting financial interests SUPPORTING INFORMATION The Second Site Hypothesis was developed using the outward occluded structure of LeuTAa (PDBid: 2A65), whereas our model of GlyT2 was generated from the structure of the outward open structure of dDAT bound to nortriptyline (PDBid: 4M48). In order to address the question as to whether the GlyT2 model is an appropriate model for testing the S2 site hypothesis, we have investigated whether the GlyT2 model forms similar structures to both the outward occluded and outward open structures of LeuTAa.



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Figure Legends Figure 1 Validation of the GlyT2 model. a The membrane-embedded homology model used in this study was derived from the crystal structure of Drosophila dopamine transporter 23, 30.

Protein is in grey glass, substrates are solid spheres, Na1 and Na2 are yellow spheres. Glycine is bound in the S1 site and leucine is bound in the proposed S2 site. POPC head groups are shown as large tan spheres and the lipid tails are shown as pink spheres b. Overlay structures of the side chains of a select number of residues that form the proposed S2 site in LeuTAa with the Gly/Apo model of GlyT2 after a 250 ns simulation. Homologous residues between LeuT and the GlyT2 model have the same coloured side chains. Leu29, Tyr 107, Leu400, Asp401 and Asp404 are from LeuT. Trp215, Tyr286, Phe629, Gln630 and Asp633 are from the GlyT2 model. c. Histogram of α-carbon distances between LeuT crystal structure (2A65.pdb) and GlyT2 Gly/Apo simulations of S2 residues highlighted in part a. The first amino acid in the legend refers to the LeuT structure and the second refers to the corresponding residue in the GlyT2 model. One of the 250 ns simulations was aligned to the LeuT structure and the difference in α carbon distances between the two corresponding residues were measured. These distances are presented as a histogram with a bin size of 0.25 Å. Figure 2 Simulation protocol. Schematic diagram of the branched molecular dynamics simulations protocol for investigating substrate interactions with the S1 and putative S2 sites using the validated membrane-embedded homology model of GlyT2. Each simulation was named according to the amino acid in S1 and S2 sites and the generation of simulation. For example Gly/Leu(1) refers to a first generation (1) simulation starting with glycine in the S1 site and leucine in the S2 site. The three end points of the Gly/Leu(1) simulation were used as the start of the subsequent Gly/Gly(2) and Gly/Apo(2) simulations. The Gly/Leu(1) simulation was also stopped mid-way and used in three Gly/Gly mid(2) simulations. Red lines represent structures where the added amino acid was unstable, green lines represent structures where the amino acid remained bound for the entire simulation, blue lines represent a flooding simulation where glycine or leucine are added to the bulk water phase and allowed to freely diffuse into the external vestibule of the transporter. Figure 3 Simulations of substrate interactions with homology models of wild type GlyT2. a. Homology model of GlyT2. Protein is in grey glass, substrates are solid spheres, Na1 and Na2 are yellow spheres. Glycine is bound in the S1 site and leucine is


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bound in the proposed S2 site. b Access of Gly (red line) and Leu (blue line) from the flooding simulation (see text for details). The extracellular gate consisting of Trp215, Tyr286 and Asp633 was used as the reference point from which distances were measured. The black line represents the location of the putative S2 site measured using the Gly/Leu(1) simulation and is 4Å from the extracellular gate. c The spatial relationship between S1 and S2. Sodium ions and the amino acids glycine and leucine are presented as spheres (Na+ is yellow, carbon is cyan, nitrogen is blue, oxygen is red, hydrogen is white) while the residues forming the S1 and S2 sites are represented sticks (hydrophobic residues are white, polar are green, positively charged are red. d Close up of S1 binding site. Residues shown interact with the substrate (Gly) for more than 25% of simulation time (except brown). Colours are as for c with the addition of brown sticks for residues that are in close proximity to the sites, and were investigated by the mutagenesis in this study, but did not directly interact with Gly in S1 during simulations. e Close up of S2 binding site. Residues shown interact with the substrate (Leu) for more than 25% of simulation time. Figure 4 Characterization of substrate selectivity of GlyT2. a. 10 µM [3H]glycine uptake (black bars) by oocytes expressing GlyT2 in the presense and absence of 300 µM Glycine, 300 µM L-alanine and 300 µM L-leucine (n=8 for each). Rate of uptake for control (uninjected oocytes, n=8) were subtracted from oocytes expressing GlyT2 (n=8). b 10 µM [14C]alanine uptake (grey bars) by oocytes expressing wild type, W482F and F629A GlyT2 (n=8 for each). Rates of [3H]-glycine/[14C]-alanine for control oocytes were substrated from oocytes expressing each on the WT and mutant transporters. c A representative current trace from an oocyte expressing GlyT2 upon appplication of 30 µM glycine in the presence of increasing concentrations of L-alanine. d. Concentration-dependent glycine transport currents in the presence and absense of 300 µM L-alanine.

Figure 5 The S1 binding site determines substrate selectivity of GlyT2. a. Amino acid sequence alignment of S1 site residues in TM3, TM6 and TM8 of human GlyT2, human GlyT1, human GABA transporter type 1 (GAT1), Drosophila DAT (dDAT) and LeuTAa. The GlyT2 sequence and where other transporters have identical residues is shown with black background with white text. Residues mutated in GlyT2 and the corresponding residues in the other transporters are highlighted in yellow. The sequence alignment was performed using Clustal Omega 1.2.1. b. Glycine concentration responses for wild-type GlyT2. c. Representative current traces for substrate induced inward currents for W482F. d-g. Substrate 23 ACS Paragon Plus Environment


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concentration reponses for GlyT2 mutants W482F (d), S479G (e), I283V (f) and T578S (g). Closed red circles for glycine in each graph. Additional substrates are: alanine (▲), leucine (■), valine (▼), tyrosine (o), methionine (□), cysteine (∆), phenylalanine (◊), isoleucine (×), serine(*), β-alanine (●) and sarcosine ( ). Data are the mean ± S.E.M and are summarized in Table 2.

Figure 6 Mutations of residues that line the S2 binding site do not disrupt function of GlyT2. a. Amino acid sequence alignment of proposed S2 site residues in TM1b, TM3, EL4 and TM10 of human GlyT2, human GlyT1, human GABA transporter type 1 (GAT1), Drosophila DAT (dDAT), SERT and LeuTAa. The GlyT2 sequence, and where other transporters have identical residues, is shown with black background with white text. Residues mutated in GlyT2 and the corresponding residues in the other transporters are highlighted in yellow. The sequence alignment was performed using Clustal Omega 1.2.1 b. Glycine concentration responses for wild-type (●) and GlyT2 mutants Y219F (■), Y286F (▲), I290V (▼), Y293F (o), F547L (∆), Y627F (◊) and F629L (●). Data are the mean ± S.E.M.and are summarized in Table 2.

Figure 7 The Hill co-efficient for glycine transport is unaffected by extracellular Na+ concentration. a. Glycine concentration responses were measured in the presence of standard ND96 (containing 96 mM Na+) (●). Data are raw current measurements from 11 cells from 3 batches of oocytes. The variability in current measurement reflects differences in expression levels between cells. b Glycine concentration responses measured in the presence of 96 mM Na+ (●), 30 mM Na+ (■) and 15 mM Na+ (▲). The data is normalised to the maximal current generated in 96 mM Na+ for each cell. c. The data from B was normalised to the maximal current generated for each Na+ concentration to allow comparison of the slopes of the curves under the three conditions. Data were fit to the Hill equation (see methods) and Hill co-efficients are 0.99 ± 0.04, 0.96 ± 0.06 and 0.94 ± 0.11 in 98.5 mM Na+, 30 mM Na+ and 15 mM Na+, respectively. Data are the mean ± S.E.M. (n=11 for 96 mM Na+, n=8 for 30 mM Na+ and n=3 for 15 mM Na+).


Page 25 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TABLE 1. Residues of the S1 and S2 sites that bind to Glycine and Leucine in the molecular dynamic simulations S1a

S2

Residue

% Bound b

Residue

% Bound

Trp215

97

Tyr207

44

Ala208

64

Tyr286

75

Val209

55

Gly542

35

Gly210

60

Phe547

39

Leu211

89

Phe629

67

Gly212

90

Gln630

93

Tyr287

97

Asp633

67

Phe249

54

Ser477

33

Ser479

91

Thr578

60

Water

533c

Water

416

a.

Results for S1 are averaged over the three Gly/Apo (2) simulations. Results for S2 are averaged over the three Gly/Leu (1) simulations.

b.

Percentage bound is the fraction of time each residue spends within 3.5 Å of the substrate. Only values greater than 25% are presented

c.

Results for water indicated the average number of water molecules bound to each substrate in the binding site. The value of 533 indicates that there are 5.33 water molecules surrounding Glycine bound to S1.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 26 of 36

TABLE 2. EC50s and Relative Imax values for Amino Acid Transport by GlyT2 S1 Site Mutants Substrate

Gly

Ala

Leu

WT

W482F

W482L

W482Y

I283V

I283L

T578S

T582V

S479G

EC50 (µM)

EC50 (µM)

EC50 (µM)

EC50 (µM)

EC50 (µM)

EC50 (µM)

EC50 (µM)

EC50 (µM)

EC50 (µM)

I/Imax

I/Imax

I/Imax

I/Imax

I/Imax

I/Imax

I/Imax

I/Imax

I/Imax

>1 mM (3)

7.9 ± 0.8 (4)

149 ± 15 (4)

53 ± 3 (4)

5.5 ± 0.6 (5)

72 ± 5 (9)

81±4

48±6

172±27

46±8

13±3

61±15

>1 mM (3)

214 ± 25 (3)

NR

>1 mM (3)

>1 mM (3)

NR

NR

NR

13± 2 (11)

a

>1 mM (8)

b

117±14d

92±11

NR

138 ± 14 (4)

NR

c

NR

916 ± 48 (4)

1.13 ± 0.03

104±29(4)

d

328 ± 64 (4)

159 ± 22 (5)

1.09 ± 0.05

0.67 ± 0.06

0.84 ± 0.07 >1 mM (3)

NR

Val

NR

>1 mM (4)

>1 mM (4)

NR

NR

>1 mM (3)

Ile

NR

NR

>1 mM (3)

NR

NR

>1 mM (3)

Tyr

NR

348 ± 47 (3)

-

NR

NR

NR

NR

813 ± 68 (4)

550 ± 84 (3)

NR

NR

>1 mM (4)

NR

1.23 ± 0.06

0.98 ± 0.03 >1 mM (3)

NR

1.5 ± 0.2 Cys

NR

Met

NR

348 ± 32 (4)

496 ± 77 (5)

>1 mM (3)

NR

1.3 ± 0.01

0.79 ± 0.05

Ser

NR

NR

>1 mM (3)

NR

NR

>1 mM (3)

NR

Phe

NR

771 ± 112 (3)

NR

NR

NR

NR

NR

NR

NR

NR

NR

NR

NR

>1 mM (4)

NR

NR

29 ± 1 (3)

0.84 ± 0.08 Asn

NR

NR

Sarcosine

NR

NR

NR

1.06 ± 0.03 N-Ethylglycine

NR

NR

β-Alanine

NR

NR

GABA

NR

NR NR

NR

NR

NR

NR

NR

>1 mM (3) NR

>1 mM (3)

>1 mM (3)

a. Number of oocytes tested, n, is given in brackets. 26

ACS Paragon Plus Environment

Page 27 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


b. Amino acid concentrations greater than 3mM generate currents in uninjected oocytes and so concentration responses curves were limited to concentrations up to 3mM. For substrates where transport currents were generated, but saturation was not achieved at concentrations up to 3mM, the EC50s are listed as being greater than 1mM. In these cases, no I/Imax value is given. c. NR refers to amino acids that were tested but did not generate a response. d. The maximal currents generated by glycine for WT and each mutant is given in nA. There is significant cell to cell and also batch to batch variation in expression levels leading to different current amplitudes. For the W482L mutant, glycine did not generate a significant current and as such the current generated by L-alanine is given. For all other amino acids, the transport currents were normalised to the maximal currents generated by glycine for each transporter, except for W482L, which are normalised to the maximal currents generated by L-alanine.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 36

TABLE 3 EC50 and Imax values for Glycinea Transport by GlyT2 S2 site mutants

Transporter

EC50 (µM)

Imax (nA)b

WT

13.1 ± 1.6 (11) c

117 ± 14 (11)

Y219F

53.9 ± 5.1 (5)

46 ±6 (5)

Y286F

20.9 ± 1.4 (6)

34 ± 4(6)

I290V

8.9 ± 0.8 (5)

29 ± 5 (5)

Y293F

11.8 ± 0.5 (5)

39 ± 9 (5)

F547L

3.9 ± 0.7 (4)

14 ± 3 (4)

Y627F

15.6 ± 2.4 (5)

109 ±23 (5)

F629L

5.5 ± 0.2 (5)

20 ± 2 (5)

F629A

50.8 ± 4.3 (6)

74 ± 13 (6)

F629S

131 ± 32 (4)

67 ± 23 (4)

a. The amino acids L-alanine and L-leucine were also tested on each transporter, but did not generate responses. b. Measurements were made from at least two batches of oocytes for each mutant and considerable variation in maximal current amplitude is often observed and as such no significance is attributed to variations in Imax values. c. Number of oocytes tested, n, is given in brackets.

28


Page 29 of 36

a

b

c

Phe629

500 Gln630

Leu29

400

Leu400

Trp215 Asp401

Tyr286

Asp633

Tyr107 Asp404

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


300 200

LeuT-GlyT2 Tyr107-Tyr286 Leu29-Trp215 Leu400-Phe629 Asp401-Gln630 Asp404-Asp633

100 0

0


1 2 3 Distance (Angstroms)

4


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

Simulation Time Generation

(total: 5.125μs)

Gly/Leu(1)

1

250 ns

Gly/Gly(2)

Gly/Apo(2)

Gly/Gly(2) 250 ns

2

Gly/Gly flood

Gly/Leu flood

Gly/Apo(3) 250 ns

3


Page 31 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

b

c

d S2

S1


e


b pmol 3H-gly or 14C-ala/oocyte/min

a 4 pmol 3H-gly/oocyte/min

3 2 1

2 1 0

u

a

W482F

00

Le

Al 00 -G

ly

+3

+3 ly

-G

3

d 1 .0

1000 ala 100 300 30 10

glycine + 300 µM alanine

0 .8 I/Imax

30 gly

4

WT

3H

c

3H

-G

ly

+3

00

3H

G

-G

ly

ly

0

3H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

0 .6 glycine

0 .4 0 .2 0 .0

20 nA 1 min

0 .1

1

10 1 00 [Glycine] (µM )


F629A

a GlyT2 GlyT1 GAT1 dDAT SERT LeuT

TM3 276 M L I I S V L 171 M M V V S T Y 129 A A V L S F W 113 V V L I A F Y 165 I C I I A F Y 97 G L W I P L V

I I L V I V

A G N D A A

I I I F S I

Y Y Y Y Y Y

Y Y Y Y Y Y

474 354 292 317 333 311

I I I V I I

F Y F F F F

S S S S S T

L L Y L L L

TM6

S G G G G S

A C L P P L

A A G G G G

W W L F F F

G G G G G G

G G S V V A

L L L L L I

574 454 392 417 434 351

L L L L L A

G G G G G G

L L I L L L

D G D D D T

Ala

Asn

Phe

40 nA 1

10 100 1000 [Substrate] (µM)

0.0 0.1

10 100 1000 [Substrate] (µM)

E E E E E Q

T T G A G P

0.5 1

1.0 0.5 0.0 0.1

1

10

100 1000

[Substrate] (µM)

T578S I/Imax

I/Imax 1

I L V S L M

1.0

I283V

S479G

T L T G G I

g

f

0.5

A C C G A A

0.0 0.1

2 min

e 1.0

F F F F F I

W482F

0.5 0.0 0.1

TM8

M Q Q S T S

d

Gly

1.0

T T S S S S

W482F

Wild Type I/Imax

F F F F F F

c

b

I/Imax

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


I/Imax

Page 33 of 36

10 100 1000 [Substrate] (µM)


1.0 0.5 0.0 0.1

1

10

100 1000

[Substrate] (µM)


a GlyT2 212 GlyT1 107 GAT1 65 dDAT 48 SERT 100 LeuT 26

G G G A G G

N N N N N N

V V V V V F

W W W W W L

R R R R R R

GlyT2 GlyT1 GAT1 dDAT SERT LeuT

G G G G G A

P P P P P F

G G G G S N

I L L L L L

A A A V L G

542 422 360 385 402 354

TM1b F F F F F F

P P F P P P

EL4b F F F F F F

V V L V I I

Y Y Y Y Y V

L L L L I Q

A C C C C A

F Y G Y Y A

Q R K K Q E

289 184 142 126 178 110

V V V V T Y

I V I I I I

I I I I M E

C C S A A S

Y I W W W W

V A A V T T

Y Y Y Y Y L

P P P P A P

E E E A E A

A A A A A I

627 508 445 469 487 400

Y Y Y Y Y -

M W V F V -

F L F F V L

Q L K H K D

L L L L L E

b 1.0 I/Imax

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

0.5

0.0

0.1

1

10

100

1000

[Glycine] (µM)

Figure 6


TM3 T A A S A T

L F I L L L

TM10 V M F L L M

D D D D E D

F Y Y R Y G

Y Y Y F Y F

L F L F L A

F F Y F I I

A S N A S K

T N Y R E F

Y Y Y Y Y W

A A S A A A

A A A A T G

S S S G G T

Page 35 of 36

I(nA)

a

80 60 40 20 0

I/Imax(96mM Na+)

b

c I/Imax

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0

0.5

0.0 1.0

0.5

0.0 0.1

1

10 100 glycine (µ M)

1000



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

G

Page 36 of 36

G G S2

OR

G S1

G S1

G


G

Molecular Determinants for Substrate Interactions ... - ACS Publications

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