Getting the Message Across: A Recent Transporter Structure Shows the Way
Authors: L. Keith Henry, Louis J. DeFelice, Randy D. Blakely
Journal: Neuron (2006)

Efforts to define the mechanisms governing neurotransmitter uptake and drug action have moved into high gear with the publication of a high-resolution structure of a leucine transporter from Aquifex aeolicus, a bacterial member of the SLC6 transporter family. Solved with the substrate leucine bound, the new structure corroborates extensive biochemical and mutagenesis studies performed with related mammalian neurotransmitter transporters and provides exciting suggestions as to how coupling arises between ions and substrates to permit efficient neurotransmitter clearance. Efforts to define the mechanisms governing neurotransmitter uptake and drug action have moved into high gear with the publication of a high-resolution structure of a leucine transporter from Aquifex aeolicus, a bacterial member of the SLC6 transporter family. Solved with the substrate leucine bound, the new structure corroborates extensive biochemical and mutagenesis studies performed with related mammalian neurotransmitter transporters and provides exciting suggestions as to how coupling arises between ions and substrates to permit efficient neurotransmitter clearance. Thousands of riders gain access to city subways every day by plunking tokens into clanking turnstiles that limit transit access (for the most part) to paying customers. At the synapse, membrane transporters are the turnstiles. The would-be travelers are neurotransmitters like GABA, dopamine, norepinephrine, and serotonin (but also other solutes such as neurotransmitter precursors). The coins of this realm are sodium, chloride, and potassium ions, profitably engaged in a process the transporter cognoscenti call "coupling," or the transformation of ion gradients into the energy needed to pump neurotransmitters across membranes, often against their own concentration gradients. Ion-coupled solute transfer across membranes is as essential for the less complex forms of life as it is for neurons. Indeed, such transporter genes make up significant fractions of microorganismal genomes, allowing essential nutrients to be gobbled from the environment, permitting cells to buffer themselves against osmotic stress via transfer of organic osmolytes, and permitting the elimination of toxins arising from hostile organisms or metabolic trash. In mammalian cells, ion-coupled transporters perform these same actions, quite often doing so in parallel with so-called "facilitated transport" systems, transporters that rely solely on solute gradients for transport across the membrane (similar in many ways to channels), and "pumps," membrane proteins that hydolyze ATP to achieve solute flux. Although we have known about ion-coupled neurotransmitter transport for decades (Iversen and Kravitz, 1966Iversen L.L. Kravitz E.A. Mol. Pharmacol. 1966; 2: 360-362PubMed Google Scholar), there is still only a vague understanding of how coupling is achieved at a molecular level informed by authentic transporter protein structures. The fact is that one need not understand the particulars of a turnstile's mechanical innards to appreciate what they do. Analogously, what channels do and how they work anticipated much of the fine structural data—and so it has been with neurotransmitter transporters. It is clear that their chief activity is to clear neurotransmitter after release, thereby shaping the magnitude and duration of chemical signaling, limiting neurotransmitter spillover to neighboring synapses, and recycling neurotransmitter for subsequent rerelease (Iversen, 1971Iversen L.L. Br. J. Pharmacol. 1971; 41: 571-591Crossref PubMed Scopus (653) Google Scholar). Need we know more? Like turnstiles, sometimes neurotransmitter transporters grind to a halt (from mutations), get blocked (e.g., cocaine, antidepressants), or, with a little forced ratcheting, allow passengers to slip through without a token (uncoupled substrate movements). Some drugs, particularly the amphetamines, can send transporters spinning madly in reverse or allow many travelers to go through with a single token (Kahlig et al., 2005Kahlig K.M. Binda F. Khoshbouei H. Blakely R.D. McMahon D.G. Javitch J.A. Galli A. Proc. Natl. Acad. Sci. USA. 2005; 102: 3495-3500Crossref PubMed Scopus (216) Google Scholar). Transporters also sometimes leave the plasma membrane (trafficking) or just refuse to work even though prompted to do so (inactivation), processes that can be impacted by the neurotransmitter itself (Ramamoorthy and Blakely, 1999Ramamoorthy S. Blakely R.D. Science. 1999; 285: 763-766Crossref PubMed Scopus (311) Google Scholar). Finally, the basic process of ion coupling is central to how these transporters get the job done, and perhaps it is here that we understand the least. The number or "stoichiometry" of ions required for coupling varies among transporters and can therefore result in some transporters being either "electrogenic" or "electroneutral," assignations given on the basis of cumulative charge required for a single transport "event." Whether variance in ion-to-solute ratios is indicative of different transporters having distinct molecular features for ion binding embedded in their structure is unknown, but there are even more perplexing issues. In addition to the normal substrate/ion-coupled transport exhibited by many members of the NSS family, substrate-independent "leak" currents occur (see Figure 1) as if transporters occasionally decide to imitate ion channels. Even more perplexing are channel-like flux states identified for solutes themselves (Blakely et al., 2005Blakely R.D. Defelice L.J. Galli A. Physiology (Bethesda). 2005; 20: 225-231Crossref PubMed Scopus (76) Google Scholar), blurring distinctions so seemingly well-defined by the use of distinct names like "transporter" and "channel." It is possible that all three modes illustrated in Figure 1 exist at the same time in the membrane or are interconverted in response to intra- and extracellular input. Recently, transporter-interacting proteins have been found to modify flux stoichiometry, seemingly tampering with the fundamental mechanisms that support standard transporter definitions (Quick, 2003Quick M.W. Neuron. 2003; 40: 537-549Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Thus, although the fine structure of ion-coupled transporters is not required to comprehend their function, to make sense of these functional nuances one has to gain access to a transporter's inner workings. Gouaux and coworkers have recently done just that by achieving the first high-resolution (1.65 Å) X-ray structure of a transporter in the SLC6 (or Na:Solute Symporter, NSS) family, the leucine transporter (LeuTAa) from Aquifex aeolicus (Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar). Prokaryotic homologs of mammalian membrane proteins have proved to be the path of least resistance to obtaining high-resolution structures, e.g., K channels (Doyle et al., 1998Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5732) Google Scholar), Cl/H exchangers (Accardi and Miller, 2004Accardi A. Miller C. Nature. 2004; 427: 803-807Crossref PubMed Scopus (517) Google Scholar), and the proton-coupled, lactose transporter (Abramson et al., 2003Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1221) Google Scholar). The advantages of prokaryotic proteins derive from their general lack of posttranslational processing (e.g., N-glycosylation) and the ease of production to a scale needed for crystallization. The strategy to seek insights of neuronal transporters from prokaryotic homologs now seems an obvious path needing little defense; one need only review the accelerated pace of ion channel research that has followed the structural elucidation of prokaryotic homologs of voltage-gated potassium channels (MacKinnon, 2003MacKinnon R. FEBS Lett. 2003; 555: 62-65Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) to understand the driving forces behind the LeuTAa work. One concern, however, is that the LeuTAa sequence is only ∼25% identical with mammalian neurotransmitter transporters, such as transporters for the brain's major inhibitory neurotransmitter GABA (GAT-1–3), the cocaine-sensitive dopamine transporter (DAT), or the antidepressant-sensitive norepinephrine and serotonin transporters (NET and SERT, respectively). Though only distantly related to mammalian Na/Cl-coupled neurotransmitter transporters, SLC6-conserved residues and LeuTAa topology make a strong case that LeuTAa is clearly in the bloodline. As predicted by initial hydropathy models, the binding site for substrates is surrounded by twelve transmembrane domains (TMs) connected to cytoplasmic N and C termini (Figure 2). However, not every feature known to mammalian SLC6 devotees is well represented in the LeuTAa structure. For example, the extracellular loop between TMs 3 and 4 is a noticeably smaller domain in LeuTAa, and the loop that is present lacks sites for N-glycosylation as well as a critical disulfide linkage. Although these missing features do not diminish the relevance of the core structure of LeuTAa to the SLC6 family, it is important to note that regulation of ion/substrate ratios and transport function may be mediated through features absent in the LeuTAa structure. Evident in the LeuTAa structure is a previously unrecognized 2-fold symmetry relating TMs 1–5 to TMs 6–10, with these two domain sets interdigitated and reflected in the plane of the membrane. In this regard, LeuTAa joins a growing number of bacterial transporters like NhaA, EmrE, Amt-1, and others whose crystal structures indicate use of antiparallel architecture, giving rise to a pseudo or true 2-fold axis of symmetry (Pornillos and Chang, 2006Pornillos O. Chang G. FEBS Lett. 2006; 580: 358-362Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Therefore, whereas it is exciting that the LeuTAa structure has broad resemblances to bona fide neurotransmitter transporters, more exciting are the fine details that give us clues as to how these nanomachines actually work, how neurotransmitters and drugs are recognized, and how coupling is achieved. With further study, the common organizational theme of antiparallel symmetry may provide insight into not only the necessary structural components used to support neurotransmitter transport, but also clues to mechanisms used by other ion-driven transporters. As expected, LeuTAa has an absolute requirement for Na (as in all SLC6 members) though not Cl (as in some other SLC6 members). Indeed, leucine and Na ions were trapped in the solved structure. Actually, two Na ions were found in the structure, cradled by TMs 1, 3, 6, and 8 and in proximity to substrate, though whether both Na ions are involved in supporting leucine transport is unknown. TMs 1 and 6 are two of the most conserved domains throughout the SLC6 family, consistent with an evolutionarily constrained contribution to the transport process. Interestingly, these domains are not simple helical structures spanning the bilayer but are interrupted halfway through the membrane, just adjacent to the sites for leucine and Na binding, by extended, nonhelical structures. TMs 3 and 8 make important, but far more discrete, contributions to the substrate binding area due to a striking tilt of the two helices, leaning ∼50° off the vertical plane and intersecting TMs 1 and 6 to bring only a few residues in proximity to substrates. In turn, TMs 3 and 8 are nestled between V-shaped intersections of TMs 9 and 10 and TMs 4 and 5, respectively. These features of interhelical packing are quite distinct from first generation "circle the wagons" models (Edvardsen and Dahl, 1994Edvardsen O. Dahl S.G. Brain Res. Mol. Brain Res. 1994; 27: 265-274Crossref PubMed Scopus (47) Google Scholar), which organized TMs next to one another, circling a central pore, and they are also very different from the solved structures of the bacterial glutamate transporter (Yernool et al., 2004Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (666) Google Scholar) and LacY, the prokaryotic transporter for lactose (Abramson et al., 2003Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1221) Google Scholar). One feature in common, however, between glutamate and leucine transporter structures is the postulated role of two gates at the cytoplasmic and periplasmic sides of each protomer in the structure. The two-gate hypothesis for Na-substrate coupling is a version of the much older alternating access model of coupled transport and has been proposed for SERT (Lester et al., 1994Lester H.A. Mager S. Quick M.W. Corey J.L. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 219-249Crossref PubMed Scopus (127) Google Scholar) and for NA/K ATPase (Artigas and Gadsby, 2003Artigas P. Gadsby D.C. Proc. Natl. Acad. Sci. USA. 2003; 100: 501-505Crossref PubMed Scopus (131) Google Scholar). A more conventional alternating access model with large conformational changes has been proposed for the LacY mechanism (Abramson et al., 2003Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1221) Google Scholar). In ion channels, the interface of the protomers typically forms the conduction pathway. The LeuTAa structure indicates that a single gene product is responsible for substrate transport, though monomer complexes may pack together in a higher-order structure through perimeter helices. There is evidence for monomer-supported substrate flux from a fluorescent study of substrate binding to NET (Schwartz et al., 2003Schwartz J.W. Blakely R.D. DeFelice L.J. J. Biol. Chem. 2003; 278: 9768-9777Crossref PubMed Scopus (88) Google Scholar). Nevertheless, there are strong structural and functional analogies between transporters and channels that are yet to be fully explored (Accardi and Miller, 2004Accardi A. Miller C. Nature. 2004; 427: 803-807Crossref PubMed Scopus (517) Google Scholar, DeFelice, 2004DeFelice L.J. Trends Neurosci. 2004; 27: 352-359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Evolution appears to have solved the problem of moving solutes across membranes by multiple strategies. For transporters, we are just getting started in this game, as many transporter gene families pepper the genomes of all organisms and most have yet to be resolved structurally. Importantly, biochemical evidence from homologous mammalian transporters elevates our confidence in the LeuTAa crystal structure. One of the most useful and widely used methods of probing amino acid environments in membrane proteins is the substituted cysteine accessibility method (SCAM). Prior to the current LeuTAa crystal structure, SCAM analysis of human SERT (hSERT) predicted that TM 1 formed part of the permeation pathway for 5-HT and suggested that the pathway becomes occluded at residues in the helix cytoplasmic to Tyr95 (Henry et al., 2003Henry L.K. Adkins E.M. Han Q. Blakely R.D. J. Biol. Chem. 2003; 278: 37052-37063Crossref PubMed Scopus (86) Google Scholar). In agreement with the LeuTAa crystal structure, hSERT Gly94, Tyr95, Asp98, Gly100, and Asn101 (residues homologous to participants of leucine binding in LeuTAa) were predicted to lie near the substrate binding pocket (Henry et al., 2003Henry L.K. Adkins E.M. Han Q. Blakely R.D. J. Biol. Chem. 2003; 278: 37052-37063Crossref PubMed Scopus (86) Google Scholar). SCAM analysis of the GABA transporter GAT-1 TM1 provided similar findings and suggested that TM1 is a conserved element supporting substrate recognition in the SLC6 family (Zhou et al., 2004Zhou Y. Bennett E.R. Kanner B.I. J. Biol. Chem. 2004; 279: 13800-13808Crossref PubMed Scopus (41) Google Scholar). As confirmed in the LeuTAa structure, GAT-1 TM1 was also predicted to be unwound near the center of the membrane. But how is differential substrate recognition supported? What makes LeuTAa substrate recognition distinct from that of GAT-1 or SERT, permitting the wide variety of substrates transported in the SLC-6 family? Certainly one can envision remodeling of the LeuTAa hydrophobic substrate binding site during evolution to accommodate various neurotransmitters. As mutation studies stride forth with the LeuTAa blueprint, we may be able to explain both the specificity and the promiscuity of transporters for their substrates. Returning to the turnstile analogy, if we truly understand how substrate recognition is achieved, we should be able to make the turnstile accept a penny in place of a token. With this in mind, we initiated a cursory modeling procedure to map the hSERT residues onto the LeuTAa structure by overlay of the hSERT sequence directly onto the coordinates of the LeuTAa PDB ID 2A65 per the alignment reported by Yamashita et al. As a coarse method to account for the few amino acid substitutions in the binding site, the resulting PDB file was analyzed (with help from Drs. Jens Meiler and Eric Dawson, Vanderbilt Center for Structural Biology) using SCWRL3 (Canutescu et al., 2003Canutescu A.A. Shelenkov A.A. Dunbrack Jr., R.L. Protein Sci. 2003; 12: 2001-2014Crossref PubMed Scopus (873) Google Scholar) (a simple energy function based on a backbone-dependent rotamer library and a linear repulsive steric energy) to alter only the χ1 and χ1+2 angles of the side chain to minimize steric clashes while leaving the backbone rigid to assign the most probable rotomer angles to SERT side chains. Visualization of a SERT-like remodeled leucine binding pocket allows for initial comparisons between the hSERT and LeuTAa binding pockets. The model combined with previous biochemical and pharmacological analyses of substrate interaction with TMs 1 and 3 of hSERT (Adkins et al., 2001Adkins E.M. Barker E.L. Blakely R.D. Mol. Pharmacol. 2001; 59: 514-523Crossref PubMed Scopus (77) Google Scholar, Barker et al., 1998Barker E.L. Perlman M.A. Adkins E.M. Houlihan W.J. Pristupa Z.B. Niznik H.B. Blakely R.D. J. Biol. Chem. 1998; 273: 19459-19468Crossref PubMed Scopus (137) Google Scholar, Henry et al., 2003Henry L.K. Adkins E.M. Han Q. Blakely R.D. J. Biol. Chem. 2003; 278: 37052-37063Crossref PubMed Scopus (86) Google Scholar, Henry et al., 2006Henry L.K. Field J.R. Adkins E.M. Parnas M.L. Vaughan R.A. Zou M.F. Newman A.H. Blakely R.D. J. Biol. Chem. 2006; 281: 2012-2023Crossref PubMed Scopus (147) Google Scholar) enables us to propose a rough illustration of the binding pocket and the residues that appear to play a major role in substrate recognition (Figure 3). Although this presentation is not meant to be a full computational analysis of the 5-HT binding pocket in SERT, this first glimpse suggests the features that may be evident in the 5-HT binding site where a larger binding space may be afforded by the presence of smaller side chains nestling the substrate binding pocket in SERT and its close relatives. Residues Ile359 and Ser256 in the hydrophobic binding pocket of LeuTAa become glycines in SERT and DAT, and these smaller side chains may permit binding of the bulkier 5-HT and DA structures. Two other domains ascribed to substrate/antagonist recognition, TMs 11 and 12 (Hahn et al., 2005Hahn M.K. Mazei-Robison M.S. Blakely R.D. Mol. Pharmacol. 2005; 68: 457-466Crossref PubMed Scopus (72) Google Scholar), appear to be on the outs with respect to the main party of intertwined helices, consistent with evidence of prokaryotic relatives lacking these domains altogether and the low degree of sequence conservation in these two domains. Data describing functional contributions to ligand binding in mammalian TMs 11 and 12 may thus reflect indirect, allosteric perturbations of transport. Another interesting residue in this region is a highly conserved TM 1 Gly residue that shifts to an Asp in the monoamine transporters (Barker et al., 1999Barker E.L. Moore K.R. Rakhshan F. Blakely R.D. J. Neurosci. 1999; 19: 4705-4717Crossref PubMed Google Scholar). The LeuTAa structure indicates that the β-carboxy group of Asp may compensate for the absence of a carboxyl group on 5-HT and collaboratively organize Na ion binding as a critical feature of coupled transport (Barker et al., 1999Barker E.L. Moore K.R. Rakhshan F. Blakely R.D. J. Neurosci. 1999; 19: 4705-4717Crossref PubMed Google Scholar). Moreover, the structural model of LeuTAa predicts that leucine binding is stabilized by two distinct hands within the binding pocket. One is this charged network that includes Na itself, which interacts with the charged carboxyl and amino moieties in the dehydrated leucine. The other hand is a hydrophobic binding surface formed by the side chains of Val104, Tyr108, Phe253, Ser256, Phe259, Ser355, and Ile359, encompassing residues from TMs 3, 6, and 8 that appear to nestle the aliphatic methyl groups of the leucine side chain. A common interaction site for substrate and Na ions in TM 1 also reveals to us that both Na and organic substrate pass through a common pore, an idea supported by electrophysiological experiments on Drosophila SERT (dSERT) (Petersen and DeFelice, 1999Petersen C.I. DeFelice L.J. Nat. Neurosci. 1999; 2: 605-610Crossref PubMed Scopus (60) Google Scholar). Na interaction within TM 1 was also suggested by mutagenesis studies that link ion sensitivity to specific TM 1 residues of both SERT and GAT-1. For example, a conservative mutation in rSERT TM1, viz., Asp98Gln (LeuTAa Gly24), results in diminished Na coupling (Barker et al., 1999Barker E.L. Moore K.R. Rakhshan F. Blakely R.D. J. Neurosci. 1999; 19: 4705-4717Crossref PubMed Google Scholar), similar to homologous mutations found by the Kanner group in GAT-1, Gly63Ser/Cys (Gly 24). How other aspects of substrate structure are discriminated will soon, no doubt, come under attack by modeling and site-directed mutagenesis. SCAM analysis of hSERT TM 1 further suggested that the conserved three amino acids Arg-Phe-Pro, located at the top of the helix, are important for folding and translocation to the plasma membrane (Henry et al., 2003Henry L.K. Adkins E.M. Han Q. Blakely R.D. J. Biol. Chem. 2003; 278: 37052-37063Crossref PubMed Scopus (86) Google Scholar). Yamashita and colleagues (Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar) propose that Arg40 in this domain forms a gate that ion-pairs with Asp404 of TM 10. As mutation of Arg104 in hSERT leads to loss of transporter protein surface expression, it seems possible that this ionic interaction has a 2-fold function by first providing contacts necessary for correct folding and later is employed in the folded structure to support the proposed "hinged-gate" for substrate access to the core binding residues. Although the two proposed gating residues could form a salt bridge, Asp404 is not conserved in Drosophila, mosquito, or hornworm SERTs, which instead have Asn in the homologous position in TM 10. Since the charged pair in LeuTAa appears to interact via interspanning water molecules, the oxygen of the Asn side chain could compensate for the loss of the charged Glu. Experiments within LeuTAa and homologs to interconvert these charge pairs may reveal more about their functional roles. What does the structure tell us about where transporter antagonists bind? In a SCAM analysis of hSERT TM3, residues homologous to LeuTAa Val104 (Ile172) and Tyr108 (Tyr176) were predicted to lie proximal to substrate and inhibitor binding sites (Chen et al., 1997Chen J.-G. Sachpatzidis A. Rudnick G. J. Biol. Chem. 1997; 45: 28321-28327Crossref Scopus (166) Google Scholar). Recently, we substituted hSERT Ile172 with Met (the homologous residue in dSERT) and observed a large decrease in antidepressant potency (as much as 1000×) but remarkably no effect on substrate binding or transport (Henry et al., 2006Henry L.K. Field J.R. Adkins E.M. Parnas M.L. Vaughan R.A. Zou M.F. Newman A.H. Blakely R.D. J. Biol. Chem. 2006; 281: 2012-2023Crossref PubMed Scopus (147) Google Scholar). Furthermore, a TM 1 (Tyr95Phe) mutation combined with the Ile172Met substitution resulted in a 10,000-fold loss of antagonist potency. This greater than additive effect on inhibitor binding in combination with formation of high-affinity metal binding sites through engineered cysteines in TMs 1 and 3 of GAT-1 (Zomot et al., 2005Zomot E. Zhou Y. Kanner B.I. J. Biol. Chem. 2005; 280: 25512-25516Crossref PubMed Scopus (25) Google Scholar) and hSERT (Henry et al., 2006Henry L.K. Field J.R. Adkins E.M. Parnas M.L. Vaughan R.A. Zou M.F. Newman A.H. Blakely R.D. J. Biol. Chem. 2006; 281: 2012-2023Crossref PubMed Scopus (147) Google Scholar) strongly suggests cooperation of these two domains in high-affinity antagonist recognition. In a preliminary model of hSERT based on the LeuTAa structure, we can see that indeed Y95 and I172M sit across from one another in the substrate binding pocket (Figure 4). Substitution of Met for the Ile172 homologous residue in NET and DAT preserves binding of substrates but results in complete loss of transport (Henry et al., 2006Henry L.K. Field J.R. Adkins E.M. Parnas M.L. Vaughan R.A. Zou M.F. Newman A.H. Blakely R.D. J. Biol. Chem. 2006; 281: 2012-2023Crossref PubMed Scopus (147) Google Scholar) and other hDAT substitutions at the residue analogous to hSERT Ile172 reduced transport of the neurotoxin MPP+ (Lee et al., 2000Lee S.H. Chang M.Y. Lee K.H. Park B.S. Lee Y.S. Chin H.R. Lee Y.S. Mol. Pharmacol. 2000; 57: 883-889PubMed Google Scholar). Together, these findings indicate that some, but not all, antagonists clearly make extensive contacts within the substrate binding pocket and that mutations in the region can collectively impact binding of substrates and antagonists as well as modulate substrate translocation. Below the bound substrate, an intracellular gate is formed in LeuTAa by TMs 1, 6, and 8, which converge to form a more elaborate and extensive barrier when compared to regions visualized as supporting the external gate. The intracellular gate also contains a universally conserved charge pair that forms a true salt bridge and may act to preferentially stabilize an outward facing transporter whose pore is occluded at the intracellular face. Gouaux and colleagues suggest that unwound regions within the TMs may provide flexibility and underlie the mechanism for opening and closing of these gates. The V-shape of TMs 4–5 and 9–10 nestle TM 3 and 8 like pincers. If binding substrate and Na causes TM 1 and 6 as well as TM 3 and 8 to move, it is conceivable that the "pincers" could effect conformational change and coordinate opening and closing of the gates for the permeation pathway (Sen et al., 2005Sen N. Shi L. Beuming T. Weinstein H. Javitch J.A. Neuropharmacology. 2005; 49: 780-790Crossref PubMed Scopus (37) Google Scholar). Imperfect coordination has been suggested as a general means to account for channels in transporters (Gadsby, 2004Gadsby D.C. Nature. 2004; 427: 795-797Crossref PubMed Scopus (44) Google Scholar). Further biochemical modeling and dynamic studies are needed before we truly understand how substrate docking is translated into the swinging of these "gates." Besides offering insight into substrate and ion binding, the packing of LeuTAa in the crystal lattice hints at possible aspects of transporter oligomerization, which features prominently in the function of channels and transporters (DeFelice, 2004DeFelice L.J. Trends Neurosci. 2004; 27: 352-359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, Sitte et al., 2004Sitte H.H. Farhan H. Javitch J.A. Mol. Interv. 2004; 4: 38-47Crossref PubMed Scopus (109) Google Scholar). Although LeuTAa arranges in dimers in the crystal, it is unclear whether the exact dimer presented occurs naturally (packing in an ordered fashion is necessary for crystallization, but in many cases such packing is simply what packs well under the chosen conditions, not the true multimeric structure) or even whether LeuTAa forms higher-order assemblages as proposed for its eukaryotic counterparts (Sitte et al., 2004Sitte H.H. Farhan H. Javitch J.A. Mol. Interv. 2004; 4: 38-47Crossref PubMed Scopus (109) Google Scholar). The dimer interface of LeuTAa is formed by EL2, TMs 9 and 12 such that TMs 9 and 12 from each protomer form a four-helix bundle. Recent data suggest that TMs 11 and 12 of the eukaryotic NSS family members are involved in oligomerization and that the transporter exists as a tetramer (Sitte et al., 2004Sitte H.H. Farhan H. Javitch J.A. Mol. Interv. 2004; 4: 38-47Crossref PubMed Scopus (109) Google Scholar). Other data suggest that DAT TM 6 is involved in dimerization, but in LeuTAa TM6 is buried in the core of the transporter and unlikely to be involved in dimerization (Sitte et al., 2004Sitte H.H. Farhan H. Javitch J.A. Mol. Interv. 2004; 4: 38-47Crossref PubMed Scopus (109) Google Scholar). However, in hDAT, residue C306 in the loop between TMs 6 and 7 appears accessible in the LeuTAa model for intermolecular interactions and could be involved in dimerization. Finally, a role for TM2 in dimerization of mammalian transporters has been proposed, based on conserved intramembrane leucine heptad repeats. This motif is intact in GAT-1 but only partially conserved in DAT and SERT, and the crystal structure of LeuTAa reveals that the portion of TM 2, which contains the leucines in the incomplete motif, is buried and unlikely to act as a dimer interface. Understanding oligomerization domains and their impact on intermolecular communication within the homomeric complex could help us understand emerging data of drug allosterism (Chen et al., 2005Chen F. Larsen M.B. Neubauer H.A. Sanchez C. Plenge P. Wiborg O. J. Neurochem. 2005; 92: 21-28Crossref PubMed Scopus (86) Google Scholar) and possibly also the growing number of functional coding variants linked to neurological/psychiatric disorders (Sutcliffe et al., 2005Sutcliffe J.S. Delahanty R.J. Prasad H.C. McCauley J.L. Han Q. Jiang L. Li C. Folstein S.E. Blakely R.D. Am. J. Hum. Genet. 2005; 77: 265-279Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). One area of SLC6 transporter research that the structure of LeuTAa cannot address well is regulation mediated by cytoplasmic domains (Sung et al., 2005Sung U. Jennings J.L. Link A.J. Blakely R.D. Biochem. Biophys. Res. Commun. 2005; 333: 671-678Crossref PubMed Scopus (32) Google Scholar). The cytoplasmic N and C termini in LeuTAa are quite short compared to their mammalian counterparts. However, the small N terminus in LeuTAa does insinuate at the base of TMs 1 and 6, which organize part of the leucine binding site, thus suggesting that the N-tail may affect the flow of ions and/or organic substrates. Intriguing in this regard is the mode of action of syntaxin-1A, a plasma membrane SNARE protein whose cytoplasmic domain binds the N terminus of multiple SLC6 family members and in SERT has been shown to alter the relative flux stoichiometry of Na and 5-HT (Quick, 2003Quick M.W. Neuron. 2003; 40: 537-549Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Since the N terminus leads directly to TM1, perhaps syntaxin modulates aspects of the TM 1 and TM 6 interface to enhance the fidelity of Na coupling. Deriving mechanism from a transporter crystal structure is like reviewing a movie with only a single frame. The solved LeuTAa structure contains bound substrates with both presumed gates (extracellular and intracellular) closed. This was taken by Gouaux and coworkers as support of the alternating access transport model, where substrates enter through an extracellular aqueous pore to a centrally located binding site. The extracellular gate closes, preventing back flow, then the intracellular gate opens allowing the substrates to enter the cytoplasm. The transporter returns empty to an outward conformation allowing the next transport cycle to begin. However, realistically, how much in the way of mechanism may we ask of a single structure? It is unreasonable to expect the cotransport machinery proposed by Gouaux and colleagues to explain all of the existing transport data, and many questions remain, though certainly much has been learned. Is it possible for example that binding either the co-ion or substrate alone could lead to gate closure and translocation? Is the ratcheting turnstile ever partially open at both ends? Transport from such states might be termed uncoupled and could perhaps explain the evidence that suprastoichiometric ion currents are known to pass through many SLC6 transporters, sometimes in the absence of organic substrates (DeFelice and Blakely, 1996DeFelice L.J. Blakely R.D. Biophys. J. 1996; 70: 579-580Abstract Full Text PDF PubMed Scopus (71) Google Scholar, Lester et al., 1996Lester H.A. Cao Y. Mager S. Neuron. 1996; 17: 807-810Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Clarifying this point should help explain the propensity of some of the transporters in this family to masquerade as ion channels. Or should we even care about the distinctions? There is evidence of single amino acid mutations changing a channel into a transporter and vice versa. A prototypical example of the channel/transporter conundrum is the divalent metal transporter DMT1. A point mutation in this transporter associated with anemia converts DMT1 into a Ca2+ channel (Xu et al., 2004Xu H. Jin J. DeFelice L.J. Andrews N.C. Clapham D.E. PLoS Biol. 2004; 2: E50https://doi.org/10.1371/journal.pbio.0020050Crossref PubMed Scopus (52) Google Scholar). In line with this phenomenon, CLC-ec1, which was thought to be a Cl− channel, is actually a counter-transporter (exchanger) that can be converted into a Cl− channel by a point mutation near the Cl− binding site (Accardi and Miller, 2004Accardi A. Miller C. Nature. 2004; 427: 803-807Crossref PubMed Scopus (517) Google Scholar). The switch of transporters to channels and the ability of expression density and interacting proteins to modulate flux stoichiometry in SLC6 family members warn us against a premature acceptance of the of LeuTAa snapshot as a complete statement of the permeation mechanism. Certainly, the LeuTAa crystal structure is the gateway to future travels, but, of course, we have only just gotten to the subway station, and complete mechanistic insight will require a few more tokens, and maybe a train transfer or two.