Neurotransmitter Transporters: Structure Meets Function
2013; Elsevier BV; Volume: 21; Issue: 5 Linguagem: Inglês
10.1016/j.str.2013.03.002
ISSN1878-4186
AutoresPaul J. Focke, Xiaoyu Wang, H. Peter Larsson,
Tópico(s)Ion channel regulation and function
ResumoAt synapses, sodium-coupled transporters remove released neurotransmitters, thereby recycling them and maintaining a low extracellular concentration of the neurotransmitter. The molecular mechanism underlying sodium-coupled neurotransmitter uptake is not completely understood. Several structures of homologs of human neurotransmitter transporters have been solved with X-ray crystallography. These crystal structures have spurred a plethora of computational and experimental work to elucidate the molecular mechanism underlying sodium-coupled transport. Here, we compare the structures of GltPh, a glutamate transporter homolog, and LeuT, a homolog of neurotransmitter transporters for the biogenic amines and inhibitory molecules GABA and glycine. We relate these structures to data obtained from experiments and computational simulations, to draw conclusions about the mechanism of uptake by sodium-coupled neurotransmitter transporters. Here, we propose how sodium and substrate binding is coupled and how binding of sodium and substrate opens and closes the gates in these transporters, thereby leading to an efficient coupled transport. At synapses, sodium-coupled transporters remove released neurotransmitters, thereby recycling them and maintaining a low extracellular concentration of the neurotransmitter. The molecular mechanism underlying sodium-coupled neurotransmitter uptake is not completely understood. Several structures of homologs of human neurotransmitter transporters have been solved with X-ray crystallography. These crystal structures have spurred a plethora of computational and experimental work to elucidate the molecular mechanism underlying sodium-coupled transport. Here, we compare the structures of GltPh, a glutamate transporter homolog, and LeuT, a homolog of neurotransmitter transporters for the biogenic amines and inhibitory molecules GABA and glycine. We relate these structures to data obtained from experiments and computational simulations, to draw conclusions about the mechanism of uptake by sodium-coupled neurotransmitter transporters. Here, we propose how sodium and substrate binding is coupled and how binding of sodium and substrate opens and closes the gates in these transporters, thereby leading to an efficient coupled transport. Review of sodium coupling and conformational change in neurotransmitter transporter Side-by-side structural comparison of transporter homologs GltPh and LeuT Relating crystal structures of GltPh and LeuT to functional data and simulations Structural and functional implications of sodium coupling in these transporters Communication between cells in the nervous system is mainly chemical, through presynaptic release of neurotransmitters, diffusion across the synapse, and activation of receptors in the postsynaptic cell (Figure 1A). The released molecules, for example, glutamate, GABA, serotonin, or dopamine, are subsequently removed from the extracellular space and transported back into the neuron or surrounding glial cells by neurotransmitter transporters. Their removal allows for subsequent release to exert full effect, as well as to localize signaling action to a synapse (Figure 1B). Removal also prevents the prolonged presence of high concentrations of neurotransmitter, which can be detrimental in other ways. For example, high concentrations of extracellular glutamate are neurotoxic; basal extracellular glutamate concentration must be kept low (Danbolt, 2001Danbolt N.C. Glutamate uptake.Prog. Neurobiol. 2001; 65: 1-105Crossref PubMed Scopus (3764) Google Scholar; Grewer and Rauen, 2005Grewer C. Rauen T. Electrogenic glutamate transporters in the CNS: molecular mechanism, pre-steady-state kinetics, and their impact on synaptic signaling.J. Membr. Biol. 2005; 203: 1-20Crossref PubMed Scopus (131) Google Scholar). Extracellular glutamate is removed from the synapse by transporters called excitatory amino acid transporters (EAATs), which are expressed in neurons and glia. EAATs belong to solute carrier family 1 (SLC1; Figure 1C). Serotonin, noradrenaline, dopamine, GABA, and glycine are removed by neurotransmitter sodium symporters (NSSs), belonging to solute carrier family 6 (SLC6; Figure 1D). Due to their crucial role of keeping basal concentrations of neurotransmitters low, malfunction or improper regulation of these transporters contributes to neurological and neuropsychiatric disorders (Gether et al., 2006Gether U. Andersen P.H. Larsson O.M. Schousboe A. Neurotransmitter transporters: molecular function of important drug targets.Trends Pharmacol. Sci. 2006; 27: 375-383Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). For example, during ischemia in the brain caused by stroke, EAATs can malfunction and release glutamate, thereby elevating glutamate levels in the extracellular space to neurotoxic levels and causing massive neuronal death (Grewer and Rauen, 2005Grewer C. Rauen T. Electrogenic glutamate transporters in the CNS: molecular mechanism, pre-steady-state kinetics, and their impact on synaptic signaling.J. Membr. Biol. 2005; 203: 1-20Crossref PubMed Scopus (131) Google Scholar; Rossi et al., 2000Rossi D.J. Oshima T. Attwell D. Glutamate release in severe brain ischaemia is mainly by reversed uptake.Nature. 2000; 403: 316-321Crossref PubMed Scopus (1201) Google Scholar). In addition, many drugs target the transporters, including drugs of abuse, such as cocaine and amphetamine, as well as drugs to treat depression, anxiety, obesity, and epilepsy (Kristensen et al., 2011Kristensen A.S. Andersen J. Jørgensen T.N. Sørensen L. Eriksen J. Loland C.J. Strømgaard K. Gether U. SLC6 neurotransmitter transporters: structure, function, and regulation.Pharmacol. Rev. 2011; 63: 585-640Crossref PubMed Scopus (559) Google Scholar). A number of structures of the transporters GltPh and LeuT, homologous to the mammalian EAATs and NSSs, respectively, have been elucidated in various states by X-ray crystallography (Boudker et al., 2007Boudker O. Ryan R.M. Yernool D. Shimamoto K. Gouaux E. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter.Nature. 2007; 445: 387-393Crossref PubMed Scopus (394) Google Scholar; Krishnamurthy and Gouaux, 2012Krishnamurthy H. Gouaux E. X-ray structures of LeuT in substrate-free outward-open and apo inward-open states.Nature. 2012; 481: 469-474Crossref PubMed Scopus (388) Google Scholar; Piscitelli and Gouaux, 2012Piscitelli C.L. Gouaux E. Insights into transport mechanism from LeuT engineered to transport tryptophan.EMBO J. 2012; 31: 228-235Crossref PubMed Scopus (39) Google Scholar; Quick et al., 2009Quick M. Winther A.M. Shi L. Nissen P. Weinstein H. Javitch J.A. Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation.Proc. Natl. Acad. Sci. USA. 2009; 106: 5563-5568Crossref PubMed Scopus (145) Google Scholar; Reyes et al., 2009Reyes N. Ginter C. Boudker O. Transport mechanism of a bacterial homologue of glutamate transporters.Nature. 2009; 462: 880-885Crossref PubMed Scopus (337) Google Scholar; Singh et al., 2007Singh S.K. Yamashita A. Gouaux E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.Nature. 2007; 448: 952-956Crossref PubMed Scopus (338) Google Scholar, Singh et al., 2008Singh S.K. Piscitelli C.L. Yamashita A. Gouaux E. A competitive inhibitor traps LeuT in an open-to-out conformation.Science. 2008; 322: 1655-1661Crossref PubMed Scopus (346) Google Scholar; Verdon and Boudker, 2012Verdon G. Boudker O. Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog.Nat. Struct. Mol. Biol. 2012; 19: 355-357Crossref PubMed Scopus (127) Google Scholar; Wang et al., 2012Wang H. Elferich J. Gouaux E. Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context.Nat. Struct. Mol. Biol. 2012; 19: 212-219Crossref PubMed Scopus (88) Google Scholar; Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl—dependent neurotransmitter transporters.Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar; Yernool et al., 2004Yernool D. Boudker O. Jin Y. Gouaux E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii.Nature. 2004; 431: 811-818Crossref PubMed Scopus (666) Google Scholar; Zhou et al., 2007Zhou Z. Zhen J. Karpowich N.K. Goetz R.M. Law C.J. Reith M.E. Wang D.N. LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake.Science. 2007; 317: 1390-1393Crossref PubMed Scopus (282) Google Scholar, Zhou et al., 2009Zhou Z. Zhen J. Karpowich N.K. Law C.J. Reith M.E. Wang D.N. Antidepressant specificity of serotonin transporter suggested by three LeuT-SSRI structures.Nat. Struct. Mol. Biol. 2009; 16: 652-657Crossref PubMed Scopus (205) Google Scholar). In-depth analysis of these structures and other proteins with similar structures has been conducted in several excellent reviews (Abramson and Wright, 2009Abramson J. Wright E.M. Structure and function of Na(+)-symporters with inverted repeats.Curr. Opin. Struct. Biol. 2009; 19: 425-432Crossref PubMed Scopus (162) Google Scholar; Boudker and Verdon, 2010Boudker O. Verdon G. Structural perspectives on secondary active transporters.Trends Pharmacol. Sci. 2010; 31: 418-426Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar; Forrest et al., 2011Forrest L.R. Krämer R. Ziegler C. The structural basis of secondary active transport mechanisms.Biochim. Biophys. Acta. 2011; 1807: 167-188Crossref PubMed Scopus (320) Google Scholar; Krishnamurthy et al., 2009Krishnamurthy H. Piscitelli C.L. Gouaux E. Unlocking the molecular secrets of sodium-coupled transporters.Nature. 2009; 459: 347-355Crossref PubMed Scopus (265) Google Scholar). Here, we compare side by side the structures of GltPh and LeuT and relate these structures to data obtained from experiments and computational simulations, to draw conclusions about the mechanism of uptake by neurotransmitter transporters. We focus on how sodium and substrate binding is coupled and how binding of sodium and substrate affects the gates in these transporters, thereby leading to an efficient coupled transport. Neurotransmitter transporters are mainly powered by the Na+ gradient across the plasma membrane. The NSS-type transporters cotransport one to three Na+ (depending on the specific transporter), most cotransport one Cl−, and some countertransport one K+ or H+ per substrate molecule transported (reviewed in Kristensen et al., 2011Kristensen A.S. Andersen J. Jørgensen T.N. Sørensen L. Eriksen J. Loland C.J. Strømgaard K. Gether U. SLC6 neurotransmitter transporters: structure, function, and regulation.Pharmacol. Rev. 2011; 63: 585-640Crossref PubMed Scopus (559) Google Scholar) (Figure 2A). The EAAT-type transporters cotransport three Na+ and one H+, and countertransport one K+, for each glutamate molecule (Figure 2A) (Billups et al., 1998Billups B. Rossi D. Oshima T. Warr O. Takahashi M. Sarantis M. Szatkowski M. Attwell D. Physiological and pathological operation of glutamate transporters.Prog. Brain Res. 1998; 116: 45-57Crossref PubMed Google Scholar; Zerangue and Kavanaugh, 1996Zerangue N. Kavanaugh M.P. Flux coupling in a neuronal glutamate transporter.Nature. 1996; 383: 634-637Crossref PubMed Scopus (706) Google Scholar). The coupling stoichiometries for the bacterial/archaeal homologs of NSS (LeuT) and EAATs (GltPh) are as follows: LeuT cotransports two Na+ (Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl—dependent neurotransmitter transporters.Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar) and GltPh cotransports three Na+ (Groeneveld and Slotboom, 2010Groeneveld M. Slotboom D.J. Na(+):aspartate coupling stoichiometry in the glutamate transporter homologue Glt(Ph).Biochemistry. 2010; 49: 3511-3513Crossref PubMed Scopus (87) Google Scholar) per transported substrate molecule (Figure 2A). How is coupled transport thought to be accomplished by neurotransmitter transporters? The models made for most secondary active transporters involve "alternating access": a binding site for both substrate and transported ions is alternately accessible either to the external or the internal solution, but never to both solutions at the same time (Mitchell, 1957Mitchell P. A general theory of membrane transport from studies of bacteria.Nature. 1957; 180: 134-136Crossref PubMed Scopus (142) Google Scholar). Two versions have been proposed for alternating access: the rocker switch (Figure 2B) (Jardetzky, 1966Jardetzky O. Simple allosteric model for membrane pumps.Nature. 1966; 211: 969-970Crossref PubMed Scopus (876) Google Scholar; Vidaver, 1966Vidaver G.A. Inhibition of parallel flux and augmentation of counter flux shown by transport models not involving a mobile carrier.J. Theor. Biol. 1966; 10: 301-306Crossref PubMed Scopus (84) Google Scholar) and the two-gated pore (Figure 2C) (Patlak, 1957Patlak C.S. Contributions to the theory of active transport. II. The gate type non-carrier mechanism and generalizations concerning tracer flow, efficiency, and measurement of energy expenditure.Bull. Math. Biophys. 1957; 19: 209-235Crossref Scopus (98) Google Scholar). In the rocker switch, the transporter is composed of two domains able to undergo a rigid-body rocking motion relative to one another so that external access to the binding site is closed and the internal access to the binding site is simultaneously opened, or vice versa (Figure 2B). In the two-gated pore (Figure 2C), a pore across the membrane is terminated by a gate at each end. Only one gate is open at any time; both can be closed, but both gates cannot be open simultaneously. Binding of substrate and the ions from the exposed side of the membrane closes the gate on that side. The state with both gates closed around the trapped substrate is referred to as the occluded state. From the occluded state, the gate on the opposite side of the membrane can open and allow the substrate and the ions to diffuse out of the pore, thereby completing the coupled transport (Figure 2C). Recent models based on crystal structures of GltPh and LeuT combine aspects of both the rocker-switch and the two-gate pore models. Alternating access can produce either cotransport of ions and substrate (both transported in same direction across membrane, as described above) or countertransport of ions and substrate (ions and substrate transported in opposite directions across membrane), depending on the postulated rules of switching between the outward-facing to inward-facing conformations. A cotransporter switches from one conformation to the other only when both substrate and coupled ions are bound to the transporter or when neither is bound (Figure 2D). In cotransport, the transporter with either only substrate bound or only the coupled ions bound does not switch between outward and inward conformations, lest it generate a leak flow of substrate or the coupled ions. This leak flow would alter the measured stoichiometry of substrate and coupled ions and degrade the energy coupling for coupled uptake. EAATs have been shown to have an uncoupled Cl− leak current (Wadiche et al., 1995Wadiche J.I. Amara S.G. Kavanaugh M.P. Ion fluxes associated with excitatory amino acid transport.Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (454) Google Scholar), but because this current is not coupled to glutamate uptake it does not affect the stoichiometry of coupled uptake. However, some NSSs display Na+ leak currents (Lester et al., 1996Lester H.A. Cao Y. Mager S. Listening to neurotransmitter transporters.Neuron. 1996; 17: 807-810Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and these would be predicted to degrade the energy for coupled uptake in NSS uptake. A transporter (such as in Figure 2C) functions as a countertransporter if it can switch between the outward- and inward-facing conformations only when either substrate or ions are bound to the transporter, but not when both substrate and ions are bound or when neither is bound to the transporter (as for glutamate and K+ exchange in Figure 2E). More complicated models for co- and countertransport are possible, but, for example, the EAATs seem to use these simple rules to cotransport Na+/H+/glutamate and countertransport K+ using two gates that control access to a binding pocket (Figure 2E). The transport models can accomplish substrate uptake into the cell by clockwise stepping through the states of the cycle, or substrate release by counterclockwise stepping through the states (Figures 2D and 2E). In which direction does the transport normally occur? The requirement for net secondary active transport is that the free energy drop in transporting the coupled ions down their electrochemical gradient exceeds the free energy required for transporting the substrate against its electrochemical gradient. An EAAT transporter operating under physiological ionic conditions and voltage is thought to be able to establish a transmembrane glutamate concentration ratio of 106 (10 mM inside/10 nM outside the cell) (Zerangue and Kavanaugh, 1996Zerangue N. Kavanaugh M.P. Flux coupling in a neuronal glutamate transporter.Nature. 1996; 383: 634-637Crossref PubMed Scopus (706) Google Scholar). The direction of transport or maximal gradient achieved by the transporter is only determined by the stoichiometry of the substrate and transported ions and the thermodynamic gradient for transport, and not the molecular details of transport or the affinity for the different molecules in the different states (but these factors could determine the kinetics of transport). So what is the structural basis for how neurotransmitter transporters implement alternating access and accomplish a coupled co- or countertransport necessary for an efficient uptake of neurotransmitters against steep concentration gradients of neurotransmitters? In the next section, we review which states in the transport cycle have been identified for GltPh and LeuT. Atomic resolution 3D structural information on sodium-coupled neurotransmitter transporters started to arrive in 2004 and 2005 with reports of the structures of GltPh, an archaeal EAAT homolog from Pyrococcus horikoshii (Yernool et al., 2004Yernool D. Boudker O. Jin Y. Gouaux E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii.Nature. 2004; 431: 811-818Crossref PubMed Scopus (666) Google Scholar), and LeuT, a bacterial NSS homolog from Aquifex aeolicus (Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl—dependent neurotransmitter transporters.Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar). Structures of these transporters have now been determined in various conformations, beginning to reveal the structural basis of substrate and ion binding, mechanisms of inhibition, and mechanisms of transport. The first published structures revealed substantial differences in the three-dimensional fold of GltPh (Figure 3A) and LeuT (Figure 3B), yet presented a common theme of 2-fold internal structural symmetry and discontinuous membrane helices (Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl—dependent neurotransmitter transporters.Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar; Yernool et al., 2004Yernool D. Boudker O. Jin Y. Gouaux E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii.Nature. 2004; 431: 811-818Crossref PubMed Scopus (666) Google Scholar). GltPh assembles as a bowl-shaped trimer with a large solvent-filled basin open to the extracellular solution (Figure 3A) (Yernool et al., 2004Yernool D. Boudker O. Jin Y. Gouaux E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii.Nature. 2004; 431: 811-818Crossref PubMed Scopus (666) Google Scholar). Each protomer in GltPh is made up of two sets of inverted repeats (AA−1BB−1: Figure 3C). The first inverted repeat forms a scaffold domain consisting of six transmembrane segments (TM1–6) folded into a cylinder that houses the second repeat, a core domain (Figure 3D) consisting of two reentrant helical hairpin loops (HP1 and HP2) and two transmembrane helices (TM7 and TM8) (Figure 3C). In contrast, LeuT is a monomer made up of 12 transmembrane helices and with a fold resembling a shallow "shot glass" (Figures 3E and 3F) (Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl—dependent neurotransmitter transporters.Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar). In LeuT, the first ten transmembrane helices constitute an internal structural repeat relating the first five helices to the second five by a pseudo-2-fold axis parallel to the membrane plane (ABA−1B−1: Figure 3E). TMs 1, 2, 6, and 7 form a centrally located core domain, while TMs 3–5 and 8–10 form a surrounding scaffold domain (Figure 3F). For both GltPh and LeuT, the inverted repeat structural elements were not a priori predicted from sequence analysis, but only apparent in the crystal structures. The inverted repeats have proved integral to understanding the basic transport mechanisms of GltPh and LeuT and are a common theme for many transporters with different architectures (Abramson and Wright, 2009Abramson J. Wright E.M. Structure and function of Na(+)-symporters with inverted repeats.Curr. Opin. Struct. Biol. 2009; 19: 425-432Crossref PubMed Scopus (162) Google Scholar; Boudker and Verdon, 2010Boudker O. Verdon G. Structural perspectives on secondary active transporters.Trends Pharmacol. Sci. 2010; 31: 418-426Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar; Forrest et al., 2011Forrest L.R. Krämer R. Ziegler C. The structural basis of secondary active transport mechanisms.Biochim. Biophys. Acta. 2011; 1807: 167-188Crossref PubMed Scopus (320) Google Scholar; Forrest and Rudnick, 2009Forrest L.R. Rudnick G. The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters.Physiology (Bethesda). 2009; 24: 377-386Crossref PubMed Scopus (215) Google Scholar; Krishnamurthy et al., 2009Krishnamurthy H. Piscitelli C.L. Gouaux E. Unlocking the molecular secrets of sodium-coupled transporters.Nature. 2009; 459: 347-355Crossref PubMed Scopus (265) Google Scholar). Furthermore, the LeuT fold itself has been found in a number of seemingly unrelated transporters, establishing the 2-fold-related "5+5" transmembrane repeat as an integral aspect of numerous transporters (Abramson and Wright, 2009Abramson J. Wright E.M. Structure and function of Na(+)-symporters with inverted repeats.Curr. Opin. Struct. Biol. 2009; 19: 425-432Crossref PubMed Scopus (162) Google Scholar; Boudker and Verdon, 2010Boudker O. Verdon G. Structural perspectives on secondary active transporters.Trends Pharmacol. Sci. 2010; 31: 418-426Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar; Forrest et al., 2011Forrest L.R. Krämer R. Ziegler C. The structural basis of secondary active transport mechanisms.Biochim. Biophys. Acta. 2011; 1807: 167-188Crossref PubMed Scopus (320) Google Scholar; Forrest and Rudnick, 2009Forrest L.R. Rudnick G. The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters.Physiology (Bethesda). 2009; 24: 377-386Crossref PubMed Scopus (215) Google Scholar; Krishnamurthy et al., 2009Krishnamurthy H. Piscitelli C.L. Gouaux E. Unlocking the molecular secrets of sodium-coupled transporters.Nature. 2009; 459: 347-355Crossref PubMed Scopus (265) Google Scholar), while the GltPh-like folds are quite rare by comparison (Johnson et al., 2012Johnson Z.L. Cheong C.G. Lee S.Y. Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å.Nature. 2012; 483: 489-493Crossref PubMed Scopus (90) Google Scholar). In the first published structures of GltPh and LeuT, the substrates were occluded from solution on both sides of the membrane (Figure 4) (Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl—dependent neurotransmitter transporters.Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar; Yernool et al., 2004Yernool D. Boudker O. Jin Y. Gouaux E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii.Nature. 2004; 431: 811-818Crossref PubMed Scopus (666) Google Scholar). In GltPh, the substrate was occluded by HP2 on the extracellular side (Figures 4A and 5B ), suggesting HP2 forms the extracellular gate (Yernool et al., 2004Yernool D. Boudker O. Jin Y. Gouaux E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii.Nature. 2004; 431: 811-818Crossref PubMed Scopus (666) Google Scholar). In LeuT, Tyr 108 and Phe 253 sequestered the substrate and ion binding sites from the extracellular solution. These residues, together with residues Arg 30 and Asp 404 and the extracellular loop 4 (EL4), were proposed to form the extracellular gate in LeuT (Figures 4B and 5B) (Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl—dependent neurotransmitter transporters.Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar). Additionally, in both structures the proposed extracellular gates were observed to be "thin" sections of protein, while access to the substrate and ion-binding sites from the intracellular side of the membrane was obstructed by 15–20 Å of "thick" sections of protein (Figure 4) (Krishnamurthy et al., 2009Krishnamurthy H. Piscitelli C.L. Gouaux E. Unlocking the molecular secrets of sodium-coupled transporters.Nature. 2009; 459: 347-355Crossref PubMed Scopus (265) Google Scholar; Yamashita et al., 2005Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl—dependent neurotransmitter transporters.Nature. 2005; 437: 215-223Crossref PubMed Scopus (1344) Google Scholar; Yernool et al., 2004Yernool D. Boudker O. Jin Y. Gouaux E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii.Nature. 2004; 431: 811-818Crossref PubMed Scopus (666) Google Scholar). These crystal structures were thus proposed to represent outward-facing occluded states of the transporters with both gates closed.Figure 5Crystal Structures of Multiple States in GltPh and LeuTShow full caption(A) D,L-TBOA locks GltPh in an outward-facing state by preventing closure of HP2. Tryptophan locks LeuT in an outward-facing state by increasing the distance between aromatic and charged extracellular gating residues. TMs 1a, 6b, and EL4 are outwardly rotated in the presence of tryptophan, widening the extracellular cavity.(B) In the outward occluded state of GltPh, substrate is trapped between HP1 and HP2. In the outward occluded state of LeuT, substrate is blocked from the extracellular solution by the extracellular gate comprised of aromatic and charged amino acids, and EL4.(C) In the inward-facing occluded state of GltPh, the core domain is moved toward the cytosol, with substrate remaining trapped between HP1 and HP2.(D) The inward-open state of LeuT is the result of an inward tilt of TMs 1b and 6a, inwardly directed movement of EL4, and outward movement of TM1a. No crystal structures have been solved for the inward-occluded state of LeuT or the inward-open state of GltPh (indicated by ?).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) D,L-TBOA locks GltPh in an outward-facing state by preventing closure of HP2. Tryptophan locks LeuT in an outward-facing state by increasing the distance between aromatic and charged extracellular gating residues. TMs 1a, 6b, and EL4 are outwardly rotated in the presence of tryptophan, widening the extracellular cavity. (B) In the outward occluded state of GltPh, substrate is trapped between HP1 and HP2. In the outward occluded state of LeuT, substrate is blocked from the extracellular solution by the extracellular gate comprised of aromatic and charged amino acids, and EL4. (C) In the inward-facing occluded state of GltPh, the core domain is moved toward the cytosol, with substrate remaining trapped between HP1 and HP2. (D) The inward-open state of LeuT is the result of an inward tilt of TMs 1b and 6a, inwardly directed movement of EL4, and outward movement of TM1a. No crystal structures have been solved for the inward-occluded state of LeuT or the inward-open state of GltPh (indicated by ?). Direct evidence supporting the proposed nature of the extracellular gates in GltPh and LeuT has been provided by crystal structures solved in complex with various inhibitors. The crystal structure of GltPh in complex with the nontransportable, competitive inhibitor D,L-threo-β-benzyloxyaspartate (TBOA) revealed a structure in which the tip of HP2 was displaced by about 10 Å (Figure 5A) from its position in an aspartate-bound structure (Figure 5B) (Boudker et al., 2007Boudker O. Ryan R.M. Yernool D. Shimamoto K. Gouaux E. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter.Nature. 2007; 445: 387-393Crossref PubMed Scopus (394) Google Scholar). Displacement of HP2 was found to be due to a steric hindrance of HP2 induced by the bulky benzyl group of TBOA (Figure 5A), with the aspartate moiety of TBOA residing in the substrate-binding pocket (Boudker et al.,
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