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Our previous work suggested that collapsing the Na+ gradient and membrane potential converts the dopamine (DA) transporter (DAT) to an inward-facing conformation with a different substrate binding profile. Here, DAT expressing human embryonic kidney 293 cells were permeabilized with digitonin, disrupting ion/voltage gradients and allowing passage of DAT substrates. The potency of p-tyramine and other non-catechols (d-amphetamine, β-phenethylamine, MPP+) in inhibiting cocaine analog binding to DAT in digitonin-treated cells was markedly weakened to a level similar to that observed in cell-free membranes. In contrast, the potency of DA and another catechol, norepinephrine, was not significantly changed by the same treatment, whereas epinephrine showed only a modest reduction. These findings suggest that catechol substrates interact symmetrically with both sides of DAT and non-catechol substrates, favoring binding to outward-facing transporter. In the cocaine analog binding assay, the mutant W84L displayed enhanced intrinsic binding affinity for substrates in interacting with both outward- and inward-facing states; D313N showed wild-type-like symmetric binding; but D267L and E428Q showed an apparent improvement in the permeation pathway from the external face towards the substrate site. Thus, the structure of both substrate and transporter play a role in the sidedness and mode of interaction between them.
The dopamine (DA) transporter (DAT) is a member of the neurotransmitter sodium symporter (NSS) family that includes transporters for norepinephrine, serotonin (SERT), GABA, glycine, proline, or taurine (Busch and Saier 2002). It is located on the plasma membrane of dopaminergic neurons and transports DA across the membrane. By taking up DA into neurons from the extracellular space, DAT plays a critical role in modulating dopaminergic neurotransmission (Giros et al. 1996; Rice and Cragg 2008). On the other hand, by mediating reverse transport of intracellular DA, DAT can also serve as a pathway for DA release at dopaminergic dendrites (Falkenburger et al. 2001). Direct interaction with DAT is crucial for the effects of psychostimulants with drug abuse liability such as d-amphetamine and of neurotoxins gaining access to DA neurons through the DAT such as MPP+ [active metabolites of MPTP] (Amara and Sonders 1998; Gainetdinov et al. 2002; Bannon 2005).
d-Amphetamine, phenethylamine psychostimulants and MPP+ are substrates for DAT as DA itself (Meiergerd and Schenk 1994; Chen and Justice 2000; Rothman et al. 2001). Substrate interaction with monoamine transporters is thought to be defined by the alternate access model (Androutsellis-Theotokis and Rudnick 2002). Thus, the transporter protein is assumed to alternate between at least two different conformational states which differ in the accessibility of the binding site for substrates. For inward transport, external substrates are proposed to initially bind at the outward-facing state where the binding site for substrates is exposed only to the external medium. For outward transport, internal substrates bind to the inward-facing state where the site is only exposed to the cytoplasmic fluid (Levi and Raiteri 1993; Chen and Justice 1998). This is also supported by detailed simulation of substrate transport by the bacterial neurotransmitter sodium symporter (NSS) homolog leucine transporter (LeuT) (Shi et al. 2008).
Gramicidin as a monovalent cation ionophore increases intracellular Na+ and decreases K+ to extracellular levels causing membrane depolarization (Chen and Reith 2004). Our previous studies demonstrated that gramicidin treatment of cells promoted accumulation of DAT in the inward-facing state (Chen and Reith 2004; Zhen et al. 2005). With DA being relatively impermeable and DAT activity inhibited by gramicidin, internal DA levels remained very low. Gramicidin reduced the affinity of DA for binding to intact cells. We speculated this was because DA had reduced access to outward-facing binding sites (measured as average affinity for multiple states interconverting rapidly within the time frame of the binding assay) (Figs. 1a and b). Furthermore, in membrane preparations with complete collapse of membrane Na+ gradient and potential, most DATs are inward-facing but DA can readily act at the side normally facing internally, and in consonance the potency of DA in inhibiting cocaine analog, 2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), binding was normal (Fig. 1c). Thus, DA appears to have no preference for inward- or outward-facing recognition sites. In contrast, the Ki value of non-catechol substrates such as β-phenethylamine, p-tyramine, and MPP+ was increased (i.e., reduced affinity) for cells in the presence of gramicidin as well as in membrane preparations. We speculated that these substrates cannot readily bind to DAT when it is facing inwardly.
Figure 1. Cartoon of access model for DA at wild type DAT. (a) In intact cells under physiological condition, most DATs reside in outward-facing state where extracellular DA binding sites are accessible. K+ prevents internally accumulated DA to bind to the few inward-facing transporters; for simplicity internal DA (or other substrate) has been omitted from the cartoon here and in Figs. 6 and 7 (Cell Control). (b) Gramicidin (GRAM) treatment causes the collapse of membrane Na+ gradient (high [Na+]o and low [Na+]i), leading to accumulation of the transporter in inward-facing state. Under this condition, the majority of binding sites is unavailable to extracellular DA. Intracellular DA is sparse. (c) In cell-free membranes, the complete disruption of the membrane Na+ gradient causes DAT redistribution similar to that accruing in GRAM-treated cells. Ambient DA can approach its binding site either externally or internally. (d) Digitionin (DIG) treatment causes a similar change of transporter conformation as in GRAM-treated cells or cell-free membranes, but (in contrast to the situation with GRAM) DA can access from both sides similar to the case for cell-free membranes; DIG holds the cellular milieu similar to that occurring in GRAM-treated cells. Note that all panels illustrate only the initial conformational state for ligand binding. Cartoons are drawn according to those presented for Tyt1 (Quick et al. 2006), another Na+-dependent bacterial transporter for tyrosine operating with the same gated pore mechanism for transport as the DAT.
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In this work, we tested our previous speculations regarding interaction of substrates with the intracellular face of the transporter with digitonin, which in addition to creating pores for ions, also allows small molecules such as DA and other substrates to cross the membrane (Dunn and Holz 1983; Wilson and Kirshner 1983; Frye and Holz 1985). With digitonin, therefore, we can test the speculation that the binding of the catechol DA is symmetric regarding binding to the external and internal face of DAT (Fig. 1d) as opposed to the asymmetry or sidedness for the interaction of non-catechol substrates. Norepinephrine and epinephrine were tested as other catechol substrates in addition to DA, whereas d-amphetamine, β-phenethylamine, p-tyramine, and MPP+ were studied as non-catechol substrates. Mutant DATs with altered DA recognition were used to study the preferential impact of external versus internal residues in DAT on externally versus internally facing substrate binding sites.
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- Materials and methods
Knowledge regarding the mode of interaction between ligands and DAT is important for understanding the mechanisms underlying the addictive and neurotoxic effects of cocaine and various substrate-like drugs including psychostimulants. So far, most studies have focused on how ligands bind to DAT from the extracellular side. To our knowledge, this is the first report to show different substrates have diverse ability to interact with the transporter from inside the cell with DAT facing inwardly. We present here experimental evidence supporting different models for how catechol and non-catechol substrates interact with DAT in the plasma membrane either from the outside or inside.
Our previous results with the ionophore gramicidin suggested that the catechol DA can interact with DAT from either side whereas non-catechol substrates only bind to outward-facing DAT (Zhen et al. 2005). The limitation of gramicidin as a tool is that the access pathway from the cytosol is unavailable for substrates including DA applied outside the cell (Fig. 1b). In cell-free membranes, both the ion and voltage gradient is lacking, as with gramicidin, but substrate can access from either side (Fig. 1c). However, the complete loss of the cytosolic milieu might impact the binding property of the transporter. Therefore in the present study, cells were permeabilized with digitonin (for cartoons see Figs. 1d, 6 and 7) to disrupt the TM gradient of Na+, K+, and other ions as well as the voltage gradient, and to enable substrate to access both sides of DAT as in membrane preparations with the important difference that the main cellular cytosolic architecture is intact.
Figure 6. Cartoon of binding model for DA at WT and mutant DATs. Conformational distributions between outward and inward states are drawn qualitatively based on the Zn2+ experiments. WT: DATs in control cells are mostly outward-facing; in digitonin (DIG)-treated cells and membranes (either with or without DIG treatment), DAT is mostly inward-facing. DA binds DAT with similar potency in both situations. W84L: Conformational preference is the same as that for WT, except for a somewhat reduced conversion to inward-facing transporters by DIG. DA is able to reach both external and internal binding sites as WT. However, the intrinsic binding affinity is enhanced at both sides (indicated by a better fit of the red diamond). D313N: Conformational preference and DA binding pattern are similar as for WT except for a somewhat reduced conversion to inward-facing transporters by DIG. D267L: In control cells, less transporter is facing outward compared with that in WT, but DA can access its binding site on DAT with greater ease (indicated by blue arrow) resulting in a higher apparent affinity compared with that at WT. In DIG-treated cells and in membranes, DAT is mostly inward-facing and the DA–DAT interaction is similar to that for WT; by analogy with the comparable case in Fig. 7, the enhanced access to outward-facing DAT at the right hand has been removed (see Fig. 7 legend). E428Q: In cells, DATs are mostly outward facing as for WT; in membranes, the reverse is true as is the case for WT. DA has free access to external and internal binding sites. However, outward-facing DAT displays higher apparent affinity for DA compared with WT because of easier access of DA to its binding site (blue arrow); by analogy with the comparable case in Fig. 7, the enhanced access to outward-facing DAT at the right hand has been removed (see Fig. 7 legend). For each cell type, the number in the left parentheses is the DA Ki in control cells; the first number in the right parentheses is the DA Ki in cells treated with DIG, the second number is the mean of DA Ki values from membranes treated with or without DIG. Changes in DA recognition or access in the mutants are drawn only when the data indicated statistically significant differences with WT.
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Figure 7. Cartoon of binding model for p-tyramine (p-TYR) at WT and mutant DATs. DAT conformational distribution in control cells, digitonin (DIG)-permeabilized cells, and in membranes is the same as in Fig. 5. p-TYR only binds to outward-facing transporters. For control cells, W84L is prone to have a higher affinity for DA compared with WT (indicated by better fit of violet diamond). W267L and E428Q have a higher apparent affinity by easier access (indicated by blue arrow). For DIG-permeabilized cells and for membranes, p-TYR binds to WT and the mutants similarly, except in the case of W84L that displays a higher affinity (in analogy with control cells and with DA in Fig. 6 in the comparable case). Depolarization combined with disruption of the transmembrane ion gradient (panels on the right) is speculated to remove the enhanced access for W267L and E428Q. For each cell type, the number in the left parentheses is the p-TYR Ki in control cells; the first number in the right parentheses is the p-TYR Ki in cell treated with DIG, the second number is the mean of p-TYR Ki values from membranes treated with or without DIG.
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For WT as studied with [3H]CFT as the radioligand in digitonin-permeabilized cells and in broken membranes where DAT was predominantly in the inward-facing state, DA bound with a similar Ki value as that observed in control cells where DAT was predominantly outward-facing, indicating a symmetric binding pattern (Table 1, for cartoon see Fig. 6, WT). This is different from the pattern of the non-catechol substrate p-tyramine (Table 2, Fig 7, WT). p-Tyramine only binds DAT that is outward facing, as evidenced by an increased Ki for digitonin-permeabilized cells and for membranes, where DAT is predominantly inward facing. Mutant W84L control cells showed stronger DA binding compared with WT (Table 1). This is very likely because of mutation-enhanced intrinsic binding affinity per se, rather than an improvement in the permeation pathway for substrate from the extracellular face towards the substrate binding site, which according to the crystallized bacterial homolog transporter LeuT, lies midway inside the membrane in a region of unwound helix 1 and 6 (Yamashita et al. 2005). When DAT turned to be inward-facing as in digitonin-treated cells and in membranes, DA binding was similarly improved (Fig. 6, W84L). It is easier to conceptualize a single mutation-enhanced binding affinity at the substrate site than a dual improvement in both the pathway from the extracellular as well as the pathway from the intracellular face towards the substrate site midway in the membrane. Consistent with this, p-tyamine showed enhanced binding in digitonin-permeabilized cells and in membranes with W84L, also with such a tendency in control cells (Table 2, see better fit of the violet diamond in the W84L cartoon in Fig 7, as well as better fit of the red diamond for DA in Fig. 6). For D313N, the DA binding profile was similar to that for WT: external and internal binding was symmetric (Table 1, Fig. 6). Compared with WT, the mutants W267L and E428Q in control cells where DAT was outward facing, showed improved binding (Table 1). Our interpretation is that this is because of an improvement in the permeation pathway from the external face towards the substrate site midway inside the membrane (indicated by blue arrow in bottom left two cartoons in Fig. 6), rather than to enhanced intrinsic binding affinity. In consonance, in digitonin-permeabilized cells and in membranes where DAT becomes inward-facing and the permeation pathway is now directed from the inner face of the membrane towards the substrate site, DA bound DAT just as in WT (Table 1 and Fig. 6) which would not have been the case if a higher intrinsic binding affinity at the substrate site had been the underlying mechanism for enhanced affinity. A second possible interpretation is that the intrinsic binding affinity is enhanced in these two mutants but because the internal access pathway is blocked DA binding becomes asymmetric (preference for outward-facing state) just as the non-catechol substrate p-tyramine. However, the ratio of Ki in membranes over that in control intact cells was much higher for p-tyramine (15.1 for W267L, 22.57 for E428Q) than for DA (9.93 for W267L, 5.70 for E428Q) (calculated from data in Figs. 6 and 7). The p-tyramine data indicate that without binding to inward-facing DAT, the Ki for DA in membranes should increase (compared with control intact cells) to a much higher degree than what is actually seen in these two mutants, because the conversion from outward- to inward-facing DATs (by depolarization and collapse of ion gradients) is independent of the interacting ligand. A third possibility is that the mutations have a differential impact on the intrinsic binding affinities in outward- and inward-facing transporters. For reasons of simplicity we prefer the first interpretation but acknowledge that current experimental data are lacking to rule out the latter possibility.
Most DAT substrates are phenethylamine derivatives with differences in substitutions for the hydrogen atom in the backbone of phenethylamine. Work with striatal slices where structural variants of DA were tested either as inhibitors of DA uptake or as substrates themselves, emphasized the catechol feature as mediating substrate recognition (Meiergerd and Schenk 1994), but subsequent work with cells expressing hDAT has been equivocal (Wang et al. 2003; Appell et al. 2004). In addition, the classical view of the catechol hydroxyls interacting with serine residues in TM7 of DAT (Kitayama et al. 1992) has been questioned (Wang et al. 2003; Xhaard et al. 2008). Our previous results from [3H]CFT competition studies suggest that DA and the substrates with a modified catechol moiety differ in their binding interactions with DAT as revealed under conditions where the intracellular part of DAT was facing higher [Na+]in (Chen and Reith 2004; Zhen et al. 2005). Here in [3H]CFT binding assays on digitonin-permeabilized cells where substrates can go inside the cells freely, catechol substrates (DA and norepinephrine) showed binding activity similar as in intact cells (symmetric binding). On the contrary, the non-catechol substrates p-tyramine, d-amphetamine, β-phenethylamine, and MPP+ had a much lower affinity for DAT with digitonin, consonant with their lack of interaction at the inward face of the transporter (asymmetric binding) under the conditions used (Fig. 3). Thus, even the lack of just one of the two phenolic hydroxyl groups has a dramatic effect on binding of substrates to the inward-facing transporter, and this will impact their ability to undergo reversed transport. This would provide an additional explanation as to how amphetamine, while it is itself accumulated intracellularly, causes efflux of DA (catechol) rather than of amphetamine (non-catechol) through DAT, with DA efflux known to be severely impacted by the absence of DAT in preparations from DAT knockout mice (Jones et al. 1998). Be that as it may, at high enough intracellular concentrations, even non-catechol substrates will be able to leave the cell through DAT in the reversed mode. It can also be thought that for asymmetrically acting substrates lipophilicity will be a crucial additional property, enabling passage through the lipid phase of the plasma membrane, diminishing a role for DAT as the passage conduit. Another example of the relevance of the catechol group in determining ligand interaction with proteins important in monoaminergic neurotransmission relates to the β2-adrenergic receptor (Swaminath et al. 2005) where the aromatic ring of the non-catechol salbutamol (m-methylhydroxyl instead of hydroxyl) occupies a binding space that does not overlap with that for catecholamines. Our results do not suggest much impact on binding affinity for outward facing DAT in intact cells upon removing one hydroxyl (p-tyramine) or two hydroxyls (β-phenethylamine) from the catechol DA (Fig. 3, see cell control bars), but the catechol feature appears essential for the interaction with inward-facing DAT as deduced from the digitonin experiments (Fig. 3, see cell digitonin and membrane bars). This could be thought to be related to the proposed coordination of catechol hydroxyls by Na+ (Xhaard et al. 2008) if one postulates that this chelation process plays different roles depending on DAT conformation. There was a modest decrease in the potency of epinephrine for inward-facing DAT in digitonin-permeabilized cells or in membranes (Fig. 3), which we interpret as resulting from the extra methyl group on the aliphatic chain portion of the molecule. Although the catechol feature is crucial for symmetric binding, additional structural features appear to impact substrate recognition such that the same domain when switching from the outward- to the inward-facing state looses some of its binding strength.
All the residues mutated in this work are highly conserved in the superfamily of Na+/Cl−-dependent transporters (Chen et al. 2001). The mutations are located at the external (W84L and D313N) or internal (W267L and E428Q) side of the transporter, they tend to constitutively hold transporter in the outward (W84L and D313N) or inward (W267L) facing state, and the residue mutated is either a tryptophan (W84L and W267L), which is prominent in providing cation–π interactions, or an acidic amino acid (E428Q and D313N), which provides a negative charge. None of these mutations cause affinities for DA in membranes or digitonin-treated cells lower than those observed in WT as measured via [3H]CFT binding (Table 1), and none of the speculated mechanisms (Figs. 6 and 7) require an impediment or enhancement for DA in the ease of accessing the substrate binding site from the internal side through the internal permeation pathway in order to describe the data (Table 1). It will be important to assess what residues in DAT line this permeation pathway, as well as the external pathway spanning the external side and the substrate site in the center of the plasma membrane. According to a recent study on the SERT (Forrest et al. 2008), the external and internal permeation pathway is made up of TMs 1b, 3, 6a, 10 and 1a, 5, 6b, 8, respectively, which contain the residues studied here (84 and 313 in TM 1b and 6a, respectively, and 267 and 428, in TM 5 and 8, respectively). The same study also indicates which residues line the internal pathway based on modeling and substituted accessibility of cysteine to methanethiosulfonate: W282 in SERT, equivalent to W267 in DAT, does not face the internal permeation pathway, but E444 corresponding to E428 in DAT does. Such information is not available for residues lining the external pathway. Residues 84, 313, 267, or 428 in DAT are clearly not part of the substrate binding site (Yamashita et al. 2005). W84 is next to R85, which is likely to form the extracellular gate by ion-pairing with D476; this ion-pairing helps holding the transporter in the inward-facing form, and therefore mutation of the neighboring W84 can be thought to disrupt the 85–476 ionic bond leading to destabilization of the inward-facing conformation as observed in our experiments. E428 is modeled to interact with Y335, which is part of an intracellular interaction network with bonds between residues assisting in holding the DAT in an outward-facing conformation (Kniazeff et al. 2008). Nevertheless, mutation of E428 to Q did not promote an inward form in our Zn2+ experiments (Fig. 4), but it is possibly an effect could have been uncovered if lower Na+ concentrations had been tested. Three out of the four mutations studied here displayed enhanced apparent affinity for DA binding from the external side (Fig. 6), and given their locations in relation to the primary substrate site as described above, these effects appear to be indirect.
Treatment of cells with digitonin is known to increase the membrane permeability of cells to ions and proteins (Dunn and Holz 1983; Wilson and Kirshner 1983; Frye and Holz 1985). Thus, proteins as large as phenylethanolamine-N-methyltransferase (38 kDa) and lactic dehydrogenase (134 kDa) pass through membranes with digitonin, and therefore substrates less than 200 Da as used in the present study can easily permeate. CFT will also pass through easily but it always does (regardless of the presence of digitonin) because of its lipophilicity as does its parent compound, cocaine. Digitonin permeabilization might cause perturbations other than the intended membrane leakiness for ions and phenethylamine-related compounds and MPP+, potentially adding complexity to the interpretation of the result. Acute regulation of the DAT includes a receptor- or protein kinase-mediated internalization of the DAT (Melikian and Buckley 1999; Sorkina et al. 2006; Bolan et al. 2007; Zapata et al. 2007; Boudanova et al. 2008; Mortensen et al. 2008). In experiment involving digitonin, one could consider that changes in intracellular ion concentration and membrane potential cause reduced availability of the transporter at the cell surface impacting DAT measurements. If that is the case, one should observe a low Bmax in cells after their treatment with digitonin, which is contrary to the present results. In addition, our previous work with gramicidin-treated cells showed that after similar ionic and membrane potential changes, DAT mostly remained on cell surface and its CFT binding characteristics (Kd and Bmax) underwent only minor changes (Chen and Reith 2004). It should be noted that in broken membranes, digitionin had no effect on the binding of various substrates in different cell types (Tables 1 and 2), indicating a lack of direct effect on substrate–DAT interactions by the low concentration (15 μM) and short time (15 min) of digitonin exposure used in this study. Digitonin-permeabilized cells have also been used successfully to analyze the intracellular topology of the SERT (Androutsellis-Theotokis and Rudnick 2002). Most importantly, the ability of DAT and SERT to bind the cocaine analogs CFT and (−)-2β-carbomethoxy-3β-(4-iodophenyl)-tropane (β-CIT) is preserved in digitonin-solubilized striatal membranes or digitonin-permeabilized cells (Gracz and Madras 1995; Androutsellis-Theotokis and Rudnick 2002). There are also evidences that digitonin renders cell permeable but still preserves membraneous protein function and physiological activity. Thus, plasma membranes of adrenal medullary chromaffin cells can be made leaky by digitonin, allowing free entry of extracellular Ca2+ and ATP, but importantly these cells are still able to undergo Ca2+-induced exocytosis (Frye and Holz 1985).
As p-tyramine appears to bind only to outward-facing DAT, and CFT has been speculated to prefer the outward-facing conformation as other cocaine-like compounds (Reith et al. 1992; Chen and Reith 2004; Chen et al. 2004b; Loland et al. 2008), it is instructive to compare the change in their binding affinities for DAT-expressing intact cells upon shifting the equilibrium from outward- towards inward-facing transporters. In [3H]CFT binding assays, digitonin reduced the affinity of p-tyramine for WT and W84L three- to fourfold (Table 2), and for W267L 15-fold if the membrane affinity is taken to represent the digitonin-intact cell value as is generally true for substrates (Tables 1 and 2, Fig. 3). In contrast, the affinity of CFT was virtually unchanged by digitonin in WT, W84L, and W267L (Fig. 2). This suggests that CFT binding is much less susceptible to the conformational state of DAT than substrate binding. This is consonant with the modest effect of gramicidin on [3H]CFT binding (1.3- to 1.5-fold reduction) observed in our previous studies (Chen and Reith 2004; Zhen et al. 2005) under comparable conditions of membrane depolarization and ion gradient collapse. It could be considered that closure of the DA permeation pathway on the extracellular side with DAT facing inwardly does not close off access to cocaine-like compounds, even though the cocaine site has been modeled to overlap with the substrate site by Beuming et al. (2008). In this context, the effects of DAT mutations themselves are of course complex in those that can affect both the conformational equilibrium of DAT and the intrinsic binding affinity at the substrate or cocaine binding site (or access through entry pathway to binding pocket). Thus, the W84L and W267L mutants both showed enhanced affinity for p-tyramine compared with WT (Table 2), but only W84L displayed higher affinity for CFT (Fig. 2). With W84L tending towards an outward-facing and W267 towards an inward-facing conformation; clearly, there are mutation-dependent effects beyond conformational impacts. The DAT mutant Y335A studied by Loland et al. (2008) tends to be inward facing, and the potency of CFT in inhibiting [3H]DA uptake into this mutant was reduced as much as 100-fold compared with WT, again emphasizing mutational effects on recognition in addition to conformation.
The present Zn2+ effects deserve a comment in comparison with available literature from other labs and with our own previous results. In the present work, Zn2+ generally affected [3H]CFT binding through changes in Bmax (Fig. 4). This is consonant with observations by Norregaard et al. (1998) and can be rationalized by considering that Zn2+ fixates a portion of the transporters in a conformation with high affinity for [3H]CFT, thereby reducing the contribution of low-affinity [3H]CFT binding sites that can escape detection by rapid dissociation during the filtration process for capturing binding. However, there is also the oscillation between various conformations that normally gives a time-averaged measure over the window of the binding assay; and Zn2+ could be thought to increase this average affinity consonant with our previous observations (Wu et al. 1997; Chen et al. 2004a). A recent study (Pifl et al. 2008) shows that Zn2+ regulation of DAT is additionally susceptible to the influence of membrane potential and ion (K+, Cl−) concentration. Such complexities may contribute to observations for [3H]CFT binding of either Bmax and/or Kd changes with Zn2+. In this context, there were also some minor differences between the present comparisons between WT and D313N or E428Q cells in binding affinity for DA or CFT, respectively, and those reported by us previously (Chen et al. 2001; Chen and Reith 2004). Generally, the protocols followed in the present experiments were the same as in the previous studies, but small methodological differences cannot be ruled out. Overall, however, conclusions are not impacted by these differences.
The elegant study by Shi et al. (2008) postulates a secondary substrate site, a vestibular site situated between the primary substrate site in the center of the plasma membrane (in the region of the unwound portions of helices 1 and 6) and the cytoplasmic end of TM domains 1, 3, and 10 capped by the tip of extracellular loop 4. This vestibular site is also the binding site for tricyclic antidepressants in the bacterial homolog transporter, the LeuT (Singh et al. 2007; Zhou et al. 2007). If the secondary site also exists in hDAT, the question is how it would affect the interpretation of the present results. This site would bind DA with or without Na+ from the external side in cells in analogy with LeuT (Shi et al. 2008) or in membranes from the side of the DAT normally facing external medium. In the context of the present results, one needs to know the relationship between the secondary (and primary) substrate site to the binding pocket for CFT, as [3H]CFT is the radioligand used here for assessing substrate affinities. If the computational model of Beuming et al. (2008) and a recent photoaffinity labeling study favoring the same model (Parnas et al. 2008) are correct, DA and [3H]CFT interact at the primary binding site in the center of the membrane. It could be thought that DA binding to the secondary, more extracellularly located site reduces the affinity of CFT for the primary site as it also weakens the binding of DA at that site (Shi et al. 2008). What argues against this idea is the lack of complexities observed in inhibition of [3H]CFT binding by DA which rather appears to obey general kinetics of competitive inhibition in both rat brain tissue and hDAT expressing cells (Amejdki-Chab et al. 1992; Reith et al. 1992; Li and Reith 1999), as well as the lack of effect of DA on the dissociation rate of [3H]mazindol binding (Zimanyi et al. 1989) which is known to overlap with CFT binding (Reith and Selmeci 1992; Tidjane et al. 2001). So far, the data support a simple interaction of CFT and DA at the primary substrate site, with the caveat that the above kinetic evidence does not prove that CFT physically binds to the primary DA site itself (just that binding of DA and CFT is mutually exclusive). Based on these considerations, we suggest that the secondary substrate site, if it exists in hDAT, plays no role in the present experiments.
Asymmetry for DAT properties, as reported here for non-catechol interactions, is not a novel phenomenon for DAT. Thus, outward and inward transport of DA are regulated differentially as revealed by hDAT constructs with alanine substituted for S528 (Chen and Justice 2000) or with substitutions of five N-terminal serines (Khoshbouei et al. 2004). It is possible that in addition to differences in recognition and translocation constants, different external and internal environments in contact with the DAT play a role. The present experiments with digitonin are an attempt to equalize two parts of these environments: the ionic composition and the membrane potential. Our previous study showed the importance of simultaneous influx of Na+ and membrane depolarization in turning the DAT inward (Chen and Reith 2004). Such an increase in intracellular Na+ under depolarizing conditions impedes binding and uptake of extracellular DA while facilitating outward transport of intracellular DA. In brain, such events could be thought to be triggered by depolarizing Na+ influx through voltage-gated Na+ channels resulting from pre- or post-synaptic excitation. In future studies it will be of interest to define the role of Na+ influx via Na+ channels in DAT activity. Prolonged opening of Na+ channels has been implicated in inherited diseases of neuronal hyperexcitability, glutamate excitotoxicity, and brain ischemia (Catterall 2000; Sugawara et al. 2001; Hammarstrom and Gage 2002).