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Unlocking the molecular secrets of sodium-coupled transporters
Abstract
Transmembrane sodium-ion gradients provide energy that can be harnessed by 'secondary transporters' to drive the translocation of solute molecules into a cell. Decades of study have shown that such sodium-coupled transporters are involved in many physiological processes, making them targets for the treatment of numerous diseases. Within the past year, crystal structures of several sodium-coupled transporters from different families have been reported, showing a remarkable structural conservation between functionally unrelated transporters. These atomic-resolution structures are revealing the mechanism of the sodium-coupled transport of solutes across cellular membranes.
... This architecture is now considered to represent a superfamily, termed as LeuT-fold transporters 5,6,11,13 , inasmuch as LeuT is the first structurally solved member 15 . Structural alignment of LeuT-fold transporters unveils a similar substrate-binding site 6,11 , and a conserved cation-binding site 6,11,25,35 known as the Na2 site in LeuT 15 . This Na2 site has been proven critical for binding or ...
... symport of the coupled substrate in LeuT-fold transporters accross various families 6,11 , listed here but not limited to, LeuT 36 and DAT 37,38 of the neurotransmitter:sodium symport (NSS) family, BetP 39 and CaiT 40,41 of the betaine/carnitine/choline (BCCT) family, vSGLT 22 of the solute:sodium symporter (SSS) family, ApcT 25 of the amino acid-polyamine-organocation (APC) family, and the focus of this study, Mhp1 23 of the nucleobase:cation symporter-1 (NCS1) family (Fig. 1). ...
... The structural commonalities among LeuT-fold transporters, i.e., same 5-helix inverted repeats, similar substrate-binding sites, and conserved Na + -binding sites, strongly suggest a similar mechanism of transport 5,6,11 . However, some of the recent studies have proposed completely different ion-coupling mechanisms for different transporter families within the LeuT-fold superfamily 36,44,45,47,50,54,[58][59][60] . ...
Secondary active transporters use electrochemical gradient of ions to fuel the “uphill” translocation of the substrate following the alternating-access model. The coupling of ions to conformational dynamics of the protein remains one of the least characterized aspects of the transporter function. We employ extended molecular dynamics (MD) simulations to examine the Na+-binding effects on the structure and dynamics of a LeuT-fold, Na+-coupled secondary transporter (Mhp1) in its major conformational states, i.e., the outward-facing (OF) and inward-facing (IF) states, as well as on the OF ↔ IF state transition. Microsecond-long, unbiased MD simulations illustrate that Na+ stabilizes an OF conformation favorable for substrate association, by binding to a highly conserved site at the interface between the two helical bundles and restraining their relative position and motion. Furthermore, a special-protocol biased simulation for state transition suggests that Na+ binding hinders the OF ↔ IF transition. These synergistic Na+-binding effects allosterically couple the ion and substrate binding sites and modify the kinetics of state transition, collectively increasing the lifetime of an OF conformation with high substrate affinity, thereby facilitating substrate recruitment from a low-concentration environment. Based on the similarity between our findings for Mhp1 and experimental reports on LeuT, we propose that this model may represent a general Na+-coupling mechanism among LeuT-fold transporters.
... As will be argued in Section 5., such occluded states are a necessary consequence of a molecular architecture in which the alternating access conformations are formed by gate domains. The hallmark of the alternating access mechanism are relatively large conformational changes in the protein conformation and these appear to exist in many secondary transporters for which the alternating access mechanism remains the standard structural framework in which to understand transporter function (Boudker and Verdon 2010;Law, Maloney, and Wang 2008;Forrest and Rudnick 2009;Gouaux 2009;Krishnamurthy, Piscitelli, and Gouaux 2009;Abramson and Wright 2009;Boudker and Verdon 2010;H. Ronald Kaback et al. 2011;Forrest, Krämer, and Ziegler 2011;Schweikhard and Ziegler 2012;Henzler-Wildman 2012;Yan 2013;Shi 2013;Slotboom 2014;Diallinas 2014;Li et al. 2015;Drew and Boudker 2016;Bai, Moraes, and Reithmeier 2017;Kazmier, Claxton, and Mchaourab 2017;Henderson, Fendler, and Poolman 2019). ...
... Läuger (1980) envisaged ion channels as pores with a single free energy barrier, which can be identified with the gate of the channel that controls ion flow in response to external stimuli (Hille 2001). He could model transporters as pores with two coupled barriers (Läuger 1980); motivated by the suggestive original "gated pore model" (Klingenberg 1979) (which was more precisely renamed the "single binding center gated model" (SBGP) (Klingenberg 2007)) one may also call these two barriers gates (Figure 6) (Abramson and Wright 2009;Krishnamurthy, Piscitelli, and Gouaux 2009;Forrest, Krämer, and Ziegler 2011). Such a gate should be thought of as a switch or bi-stable element that can exist in two states that are generally called "open" and "closed" although it might also carry the meaning "outward facing" or "inward facing". ...
... X-ray crystallographic structures of Mhp1 in outward facing and inward facing conformations together with computer simulations revealed the structural basis for the alternating access mechanism in this secondary transporter (Weyand et al. 2008;Shimamura et al. 2010;Simmons et al. 2014). The transporter can be understood as a gated pore with two thin and one thick gate (Krishnamurthy, Piscitelli, and Gouaux 2009), i.e., as a pore with three gates: The thick gate regulates the passage through the center of the membrane by means of the large conformational change that switches the transporter from its outward facing to its inward facing conformation. In Mhp1 it consists of the hash motif (formed by helices TM3, TM4 and their inverted-repeat counterparts TM8 and TM9; see Figures 7A, C and 4B) that can rotate by about 30 • on an axis parallel to TM3 relative to the four-helix "bundle" (TM1, TM2 and TM6, TM7) (Shimamura et al. 2010). ...
Transport of ions and small molecules across the cell membrane against electrochemical gradients is catalyzed by integral membrane proteins that use a source of free energy to drive the energetically uphill flux of the transported substrate. Secondary active transporters couple the spontaneous influx of a "driving" ion such as Na+ or H+ to the flux of the substrate. The thermodynamics of such cyclical non-equilibrium systems are well understood and recent work has focused on the molecular mechanism of secondary active transport. The fact that these transporters change their conformation between an inward-facing and outward-facing conformation in a cyclical fashion, called the alternating access model, is broadly recognized as the molecular framework in which to describe transporter function. However, only with the advent of high resolution crystal structures and detailed computer simulations has it become possible to recognize common molecular-level principles between disparate transporter families. Inverted repeat symmetry in secondary active transporters has shed light on how protein structures can encode a bi-stable two-state system. More detailed analysis (based on experimental structural data and detailed molecular dynamics simulations) indicates that transporters can be understood as gated pores with at least two coupled gates. These gates are not just a convenient cartoon element to illustrate a putative mechanism but map to distinct parts of the transporter protein. Enumerating all distinct gate states naturally includes occluded states in the alternating access picture and also suggests what kind of protein conformations might be observable. By connecting the possible conformational states and ion/substrate bound states in a kinetic model, a unified picture emerges in which symporter, antiporter, and uniporter function are extremes in a continuum of functionality.
... According to current hypothesis on the transport mechanism by the alternate access model (Jardetzky 1966;Zhou et al. 2008;Forrest and Rudnick 2009;Krishnamurthy et al. 2009;Shimamura et al. 2010;Radestock and Forrest 2011), a critical feature of substrate transport is the equilibrium established between the outward-and the inward-facing orientations, allowing for an efficient transport turnover (Figure 4.11). ...
... For MelB, this is substantiated by the similar accessibility of the substrates to the substrate-binding sites from both sides of the vesicles (Figure 4.8), even if the frequency of opening to the periplasmic space might be much lower than opening to the cytoplasmic space . Under normal conditions, the binding of the substrates from the periplasmic side is followed by the PART I 118 reorientation of the outward-facing to the inward-facing conformation (Forrest and Rudnick 2009;Krishnamurthy et al. 2009). The binding process itself might also contribute to speed up the reorientation process by decreasing its free energy barrier, as it occurs in enzymatic reactions. ...
... Based on previous studies of the topology of MelB, Ala155 should be located at the middle of the helix 5. In general, the main structural component of the transmembrane part of the membrane transport proteins is α-helix, except for the discontinue structures, which always play key role in the substrates binding or/ and substrate-dependent conformational changes (Krishnamurthy et al. 2009). Therefore, it may be reasonable to suppose that Ala155 is located at or close to discontinue structures. ...
As membrane transporters play a very important role to maintain the normal
physiology of the organism as well as in drug safety and efficacy, it is very important
to study the structure and function of these types of membrane proteins. Melibiose
permease (MelB), a membrane transporter, couples the uphill transport of the sugar to
the downhill electrochemical ion gradient. MelB is of great interest because it can use
different sugars (either α- or β-galactosides) and cations (Na+
, Li+
, and H+
), whereas
other symporterslike lactose permease, a memberof the major facilitator superfamily
(MFS) uses only H+
(Saier, M. H., MicrobiolMolBiol Rev 2000). This implies
thatMelB should present some unique substrate recognition
transportcharacteristics.The in-depth study of the structure and function of MelB may
provide us with key advancements in the understandingof the cotransport mechanism
of membrane transporters.
The current biochemical, biophysical, and structuraldata for MelB fail to give us a
clear sceneof the substrates recognition mechanism, and explain the structural
reorganization that occurs and that ultimately forces the conformational changes
needed for transport through the membrane. Although an enormous amount of data on
MelBhas been published, until now many questions remain unanswered:
- Which residues areinvolved in cations and sugar binding?
- Why does the binding of Na+
or Li+
to MelB greatly enhance its affinity to sugar?
- What is the mechanism by which the sugaris carried through the membrane?
- Why can MelB use Na+
, Li+
, H+
as a couplingcation to uptake sugar?
-How can MelB bind α- and β-galactosides?
To answer these questions, more biochemical andbiophysical evidences, as well as a
high resolution structure are required. Fourier transform infrared difference
(IRdiff)spectroscopy, as well as fluorescene spectroscopy combined with site-directed
mutagenesishas been shown tobe a powerful technique to detect conformational
changes induced by cation and sugar bindingto MelB.
In this thesis, I plan to use these techniques to explore the structure and function of
melibiose prermease. Firstly, I will focus on the R149C mutant, which cannot bind
sugar and cannot transport (Abdel-Dayem M, J BiolChem 2003). In this work, I am
going to reveal how R149C affects sugar binding and translocation. Then I will
continue to study the role of helix V by cysteine scanning mutagenesis. As previous
studies indicate, helix V may be involved in the sugar binding (Basquin 2001) and in a
3D model, helix V was shown to be close to theimportant helix II(Yousef and Guan
PNAS 2009). Finally, I will tryto crystallyze the MelB transporter and apply X-ray
diffraction to obtain its 3D structure.
... In recent years, crystallization-based structural analyses revealed important insights into the three-dimensional structures of various transporters including sodium-dependent systems. The analyses led to the discovery of common structural folds (for example, MFS fold, LeuT fold) [11,12]. Thereby, common core structures are shared even by transporters that do not have a significant sequence similarity or fulfill different functions (for example, sodium or proton-coupled solute uptake or solute antiport). ...
... The resulting inwardly oriented apo state of the transporters changes to the outwardopen conformation to allow for a new transport cycle. Reciprocal opening and closing of inwardly and outwardly oriented cavities may involve movement of thin gates (vSGLT: Y263; PmSiaT: I67, F78; AaLeuT: Y108, F253), rearrangements of interactions between TMDs as well as between TMDs and inner and outer loops (for example the loop connecting TMDs 7 and 8[11,64,84]. out: outwardopen conformation, occ: occluded conformation, in: inward-open conformation. ...
The solute/sodium symporter family (SSS family; TC 2.A.21; SLC5) consists of integral membrane proteins that use an existing sodium gradient to drive the uphill transport of various solutes, such as sugars, amino acids, vitamins, or ions across the membrane. This large family has representatives in all three kingdoms of life. The human sodium/iodide symporter (NIS) and the sodium/glucose transporter (SGLT1) are involved in diseases such as iodide transport defect or glucose -galactose malabsorption. Moreover, the bacterial sodium/proline symporter PutP and the so-dium/sialic acid symporter SiaT play important roles in bacteria-host interactions. This review focuses on the physiological significance and structural and functional features of prokaryotic members of the SSS family. Special emphasis will be given to the roles and properties of proteins containing an SSS family domain fused to domains typically found in bacterial sensor kinases.
This article is dedicated to the memory of Ron Kaback, an exceptional scientist and great mentor.
... Surprisingly, transporters of distant evolutionary families and of distinct function and energetic requirement might share a common architectural fold, one among the four currently identified, namely the LeuT, the major facilitator superfamily (MFS), the GltPh and the NhaA fold [1][2][3]. Notably, however, while different folds are associated with distinct mechanisms of transport, known as the rocking-bundle (LeuT), rocker-switch (MFS) or elevator-like sliding ( LeuT-like transporters), the function of all transporters is based on a common mechanism which drives the alteration from an outward-to an inward-facing topology [4][5][6][7][8][9]. This common mechanism is elicited by substrate binding, translocation and release. ...
... (b) Interactions of the cytosolic N-tail (deep blue) with internal loops in dDAT (PDB entry 4m48). (c) Mhp1 dynamic topology showing the rocking movement of the 'hash' domain(TMS 3,4,8,9) from the outward (gold) to the inward (blue) conformation. Notice also the displacement of TMS5 (PDB entries 2jln, 2x79). ...
Cell nutrition, detoxification, signalling, homeostasis and response to drugs, processes related to cell growth, differentiation and survival are all mediated by plasma membrane (PM) proteins called transporters. Despite their distinct fine structures, mechanism of function, energetic requirements, kinetics and substrate specificities, all transporters are characterized by a main hydrophobic body embedded in the PM as a series of tightly packed, often intertwined, α-helices that traverse the lipid bilayer in a zigzag mode, connected with intracellular or extracellular loops and hydrophilic N- and C-termini. Whereas longstanding genetic, biochemical and biophysical evidence suggests that specific transmembrane segments, and also their connecting loops, are responsible for substrate recognition and transport dynamics, emerging evidence also reveals the functional importance of transporter N- and C-termini, in respect to transport catalysis, substrate specificity, subcellular expression, stability and signalling. This review highlights selected prototypic examples of transporters in which their termini play important roles in their functioning.
... At the end of the cavity is a conserved substrate-binding site shared by all characterized APC transporters (50). The structure revealed an empty space without any prominent density ( Fig. 3c and Fig. S4b), indicating the apo state of the transporter. ...
... A possible deviation from the general model is that CD98hc-ED is on top of LAT1, and contacts EL4a. In most APC members, EL4 has been implicated in the latch function (50), where it opens and closes during the transport cycle. Therefore, during the conformational changes, EL4a of LAT1 may rearrange its interaction with CD98hc, leading to a widening of the substrate pathway in an outward-open state (Fig. 7a). ...
The L-type amino acid transporter 1 (LAT1) transports large neutral amino acids and drugs across the plasma membrane and is crucial for nutrient uptake, brain drug delivery and tumor growth. LAT1 is a unique solute carrier that forms a disulfide-linked heterodimer with the cell-surface glycoprotein CD98 heavy chain (CD98hc), but the mechanisms of its molecular assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.4 Å resolution, revealing the hitherto unprecedented architecture of a solute carrier-glycoprotein heterocomplex. LAT1 features a canonical LeuT-fold while exhibiting an unusual loop structure on transmembrane helix 6, creating an extended cavity to accommodate bulky hydrophobic amino acids and drugs. CD98hc engages with LAT1 through multiple interactions, not only in the extracellular and transmembrane domains but also in the interdomain linker. The heterodimer interface features multiple sterol molecules, corroborating previous biochemical data on the role of cholesterols in heterodimer stabilization. We also visualized the binding modes of two anti-CD98 antibodies and show that they recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities despite the glycan heterogeneity. Furthermore, we mapped disease-causing mutations onto the structure and homology models, which rationalized some of the phenotypes of SLC3- and SLC7-related congenital disorders. Together, these results shed light on the principles of the structural assembly between a glycoprotein and a solute carrier, and provide a template for improving preclinical drugs and therapeutic antibodies targeting LAT1 and CD98.
... Its functional role of co-transporting substrates across the membrane is associated with highly coordinated structural transitions between its corresponding conformational states, which occur generally in an active manner. SGLT1 belongs to the membrane protein family of sodium solute symporters, sharing a common structural core motif called 'LeuT-fold' and playing a crucial role in the physiology of the intestines, brain, kidney, thyroid, and skin, representing an important target for therapeutic intervention in the treatment of diabetes, diarrhea, depression, obesity, etc. [3,4]. The two major conformations that the SGLT1 cotransporter occupies during its working cycle are the inward-facing (IF) and outward-facing (OF) states [5]. ...
The human sodium–glucose cotransporter protein (SGLT1) is an important representative of the sodium solute symporters belonging to the secondary active transporters that are critical to the homeostasis of sugar, sodium, and water in the cell. The underlying transport mechanism of SGLT1 is based on switching between inward- and outward-facing conformations, known as the alternating access model, which is crucial for substrate transport, and has also been postulated for water permeation. However, the nature of water transport remains unclear and is disputed along the passive and active transport, with the latter postulating the presence of the pumping effect. To better examine the water transport in SGLT1, we performed a series of equilibrium all-atom molecular dynamics simulations, totaling over 6 μs of sample representative conformational states of SGLT1 and its complexes, with the natural substrates, ions, and inhibitors. In addition to elucidating the basic physical factors influencing water permeation, such as channel openings and energetics, we focus on dynamic flexibility and its relationship with domain motion. Our results clearly demonstrate a dependence of instantaneous water flux on the channel opening and local water diffusion in the channel, strongly supporting the existence of a passive water transport in SGLT1. In addition, a strong correlation found between the local water diffusion and protein domain motion, resembling the “rocking-bundle” motion, reveals its facilitating role in the water transport.
... The preparation wizard was used for the preparation of protein (Schrodinger, LLC, New York). The downloaded crystal structure (PBD ID: 3DH4) was processed [21] and converted into single entity by removal of water molecule. Furthermore, optimization of hydrogen bonds, removal of water molecule, and minimization were performed using OPLS_2005 force field. ...
Sodium glucose cotransporter 2 (SGLT2) inhibitors work by controlling the blood glucose levels, through limiting reabsorption of glucose from the blood therefore, and promoting glucose excretion in the urine. As it is reason for the 90% of reabsorption of glucose through insulin-independent mechanism. The present study described the screening of potential SGLT2 inhibitors using docking studies. In silico studies were carried out with help of the Schrödinger software using PDB ID:3DH4. Inhibitors were docked which resulted that phlorizin is one of the most potent compound having highest docking score −12.118 kcal/mol showing binding interaction with the Asn64, Ser66, Ala63, Ser91, Tyr263, Glu88, and Gln 428 (PBD ID: 3DH4) amino acids. Various ADME properties were studied and numerous properties were also analyzed. The forecast model can also be used for the further development of the potential compounds against SGLT2.
... Indeed, the mutation F252A completely suppresses the transport activity, whereas the conservative mutation F252W, resembling the corresponding residue in AdiC, causes only a partial loss of activity [28]. Another bacterial leucine transporter (LeuT) [47,48] that shares the same fold of LAT1, has a phenylalanine residue, namely F253, corresponding to F252 of LAT1. Similarly, the CAT subfamily member Geobacillus Kaustophilus amino acid-polyamine-organocation transporter (GkapcT) presents the corresponding F231 as involved in the transport mechanism [49]. ...
The large Amino Acid Transporter 1 (LAT1) is an interesting target in drug discovery since this transporter is overexpressed in several human cancers. Furthermore, due to its location in the blood-brain barrier (BBB), LAT1 is interesting for delivering pro-drugs to the brain. In this work, we focused on defining the transport cycle of LAT1 using an in silico approach. So far, studies of the interaction of LAT1 with substrates and inhibitors have not considered that the transporter must undergo at least four different conformations to complete the transport cycle. We built outward-open and inward-occluded conformations of LAT1 using an optimized homology modelling procedure. We used these 3D models and the cryo-EM structures in outward-occluded and inward-open conformations to define the substrate/protein interaction during the transport cycle. We found that the binding scores for the substrate depend on the conformation, with the occluded states as the crucial steps affecting the substrate affinity. Finally, we analyzed the interaction of JPH203, a high-affinity inhibitor of LAT1. The results indicate that conformational states must be considered for in silico analyses and early-stage drug discovery. The two built models, together with the available cryo-EM 3D structures, provide important information on the LAT1 transport cycle, which could be used to speed up the identification of potential inhibitors through in silico screening.
... By sequence and structural alignment with NKCC1, which also transports Na + ions 16 , we identified the conserved Na + -binding site in the NCC. This 'Na2' site has been found in many APC transporters 38,39 . In the cryo-EM map of NCC cryo2 , we observed a density at the centre of Na2 (Extended Data Fig. 5d), potentially Na + . ...
The sodium–chloride cotransporter (NCC) is critical for kidney physiology¹. The NCC has a major role in salt reabsorption in the distal convoluted tubule of the nephron2,3, and mutations in the NCC cause the salt-wasting disease Gitelman syndrome⁴. As a key player in salt handling, the NCC regulates blood pressure and is the target of thiazide diuretics, which have been widely prescribed as first-line medications to treat hypertension for more than 60 years5–7. Here we determined the structures of human NCC alone and in complex with a commonly used thiazide diuretic using cryo-electron microscopy. These structures, together with functional studies, reveal major conformational states of the NCC and an intriguing regulatory mechanism. They also illuminate how thiazide diuretics specifically interact with the NCC and inhibit its transport function. Our results provide critical insights for understanding the Na–Cl cotransport mechanism of the NCC, and they establish a framework for future drug design and for interpreting disease-related mutations.
... Na + may more loosely bind to NKCC1 than K + and Cl − do, as only weak density can be observed in the putative Na + site of NKCC1 maps [72,75,88]. Notwithstanding this caveat, the Na + site can be inferred by analogy from the conserved Na + coordination geometry of so-called Na + site 2 observed in other Na + -coupled APC transporters [89][90][91]. In DrNKCC1, Na + is coordinated by the backbone carbonyl oxygen atoms of L219 and W222 on TM1, A535 on TM8, and the sidechain oxygens of S538 and S539 on the TM8 helix [72]. ...
Loop and thiazide diuretics have been cornerstones of clinical management of hypertension and fluid overload conditions for more than five decades. The hunt for their molecular targets led to the discovery of cation-chloride cotransporters (CCCs) that catalyze electroneutral movement of Cl− together with Na+ and/or K+. CCCs consist of two 1 Na+-1 K+-2 Cl− (NKCC1-2), one 1 Na+-1 Cl− (NCC), and four 1 K+-1 Cl− (KCC1-4) transporters in human. CCCs are fundamental in trans-epithelia ion secretion and absorption, homeostasis of intracellular Cl− concentration and cell volume, and regulation of neuronal excitability. Malfunction of NKCC2 and NCC leads to abnormal salt and water retention in the kidney and, consequently, imbalance in electrolytes and blood pressure. Mutations in KCC2 and KCC3 are associated with brain disorders due to impairments in regulation of excitability and possibly cell volume of neurons. A recent surge of structures of CCCs have defined their dimeric architecture, their ion binding sites, their conformational changes associated with ion translocation, and the mechanisms of action of loop diuretics and small molecule inhibitors. These breakthroughs now set the stage to expand CCC pharmacology beyond loop and thiazide diuretics, developing the next generation of diuretics with improved potency and specificity. Beyond drugging renal-specific CCCs, brain-penetrable therapeutics are sorely needed to target CCCs in the nervous system for the treatment of neurological disorders and psychiatric conditions.
... At present, the nucleoside transport mechanism of CNTs has been extensively studied (Krishnamurthy et al., 2009;Jardetzky, 1966;Yamashita et al., 2005;Hirschi et al., 2017;Faham et al., 2008;Weyand et al., 2008). Based on a series of structural and computational biology studies on CNT, a multistep elevator-like nucleoside transport mechanism has been widely recognized (Hirschi et al., 2017;Duan et al., 2021). ...
The human equilibrative nucleoside transporter 1 (hENT1) is an effective controller of adenosine signaling by regulating its extracellular and intracellular concentration, and has become a solid drug target of clinical used adenosine reuptake inhibitors (AdoRIs). Currently, the mechanisms of adenosine transport and inhibition for hENT1 remain unclear, which greatly limits the in-depth understanding of its inner workings as well as the development of novel inhibitors. In this work, the dynamic details of hENT1 underlie adenosine transport and the inhibition mechanism of the non-nucleoside AdoRIs dilazep both were investigated by comparative long-time unbiased molecular dynamics simulations. The calculation results show that the conformational transitions of hENT1 from the outward open to metastable occluded state are mainly driven by TM1, TM2, TM7 and TM9. One of the trimethoxyphenyl rings in dilazep serves as the adenosyl moiety of the endogenous adenosine substrate to competitively occupy the orthosteric site of hENT1. Due to extensive and various VDW interactions with N30, M33, M84, P308 and F334, the other trimethoxyphenyl ring is stuck in the opportunistic site near the extracellular side preventing the complete occlusion of thin gate simultaneously. Obviously, dilazep shows significant inhibitory activity by disrupting the local induce-fit action in substrate binding cavity and blocking the transport cycle of whole protein. This study not only reveals the nucleoside transport mechanism by hENT1 at atomic level, but also provides structural guidance for the subsequent design of novel non-nucleoside AdoRIs with enhanced pharmacologic properties.
... The cryo-EM map also resolved a Ca 2+ ion and six N-linked glycans for each rBAT molecule, as discussed later in detail. b 0,+ AT shares structural and sequence homology with the APC superfamily and shows a typical LeuT-fold (Fig. 2c), consisting of 12 transmembrane helices (TM1-12) that contain 5 + 5 TM inverted repeats 19 . Other structural features involve two helices in the extracellular loop 4 (EL4a and EL4b), one helix in intracellular loop 1 (IL1) and the cytoplasmic C-terminal helix (CH) running parallel to the membrane ( Supplementary Fig. 5). ...
Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter b0,+AT, which constitute system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+ mediates higher-order assembly of system b0,+. Ca2+ stabilizes the interface between two rBAT molecules, leading to super-dimerization of b0,+AT–rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca2+ site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b0,+ biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases. Cystinuria is caused by mutations in heterodimeric amino acid transporter known as system b0,+. Here, authors discover that Ca2+ stabilizes the interface between two system b0,+ regulatory subunits rBAT, leading to super-dimerization of the b0,+AT–rBAT heterodimer, facilitating system b0,+ maturation.
... LeuT Aa functions as a sodium/leucine transporter in Aquifex aeolicus and is a member of the neurotransmitter sodium symporter (NSS or SLC6) family (Yamashita et al., 2005;Krishnamurthy et al., 2009;Navratna and Gouaux, 2019). In humans, NSS proteins are responsible for the reuptake of neurotransmitters involved in synaptic transmission (Joseph et al., 2019;Möller et al., 2019). ...
The majority of alpha helical membrane proteins fold co-translationally during their synthesis on the ribosome. In contrast, most mechanistic folding studies address refolding of full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation of membrane proteins is emerging as a useful tool to address folding during translation by a ribosome. We summarise the benefits of this approach and show how it can be successfully extended to a membrane protein with a complex topology. The bacterial leucine transporter, LeuT can be synthesised and inserted into lipid membranes using a variety of in vitro transcription translation systems. Unlike major facilitator superfamily transporters, where changes in lipids can optimise the amount of correctly inserted protein, LeuT insertion yields are much less dependent on the lipid composition. The presence of a bacterial translocon either in native membrane extracts or in reconstituted membranes also has little influence on the yield of LeuT incorporated into the lipid membrane, except at high reconstitution concentrations. LeuT is considered a paradigm for neurotransmitter transporters and possesses a knotted structure that is characteristic of this transporter family. This work provides a method in which to probe the formation of a protein as the polypeptide chain is being synthesised on a ribosome and inserting into lipids. We show that in comparison with the simpler major facilitator transporter structures, LeuT inserts less efficiently into membranes when synthesised cell-free, suggesting that more of the protein aggregates, likely as a result of the challenging formation of the knotted topology in the membrane.
... The Na + binding site is highly conserved throughout sodium-dependent APC transporters ( Fig. S5A-C) (Faham et al., 2008;Khafizov et al, 2012;Krishnamurthy et al, 2009;Malinauskaite et al., 2014;Penmatsa et al., 2013;Perez et al, 2012;Weyand et al, 2008;Yamashita et al., 2005). The crucial role of the Na2 site seems also to be retained in many sodium-independent transporters adopting the LeuT-fold ( Fig. S5D-F). ...
The sodium-potassium-chloride transporter NKCC1 (SLC12A2) performs Na+-dependent Cl- and K+ ion uptake across plasma membranes. NKCC1 is important for regulating e.g. cell volume, hearing, blood pressure, and chloride gradients defining GABAergic and glycinergic signaling in brain. Here, we present a 2.6 Å resolution cryo-electron microscopy (cryo-EM) structure of human NKCC1 in the substrate-loaded (Na+, K+, 2 Cl-) and inward-facing conformation adopting an occluded state that has also been observed for the SLC6 type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provide a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- ion binding at the Cl2 site seems to undertake a structural role similar to a conserved glutamate of SLC6 transporters and may allow for chloride-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations we describe the Na+ binding site and a putative Na+ release pathway along transmembrane helix 5. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.
... The structure and function of SLC6 neurotransmitter transporters has been extensively reviewed elsewhere (Forrest and Rudnick, 2009;Kristensen et al., 2011;Navratna and Gouaux, 2019) and thus will only be briefly described here. NSS consist of twelve transmembrane α-helices (TMs) that are pseudo-symmetrically arranged with respect to the membrane and are connected via a series of intracellular and extracellular loops (Yamashita et al., 2005;Krishnamurthy et al., 2009;Shi, 2013). The transport process is proposed to occur via an alternating access mechanism in which substrate and co-transported ions are exposed to either the extracellular or cytoplasmic side (Forrest et al., 2008). ...
Neurotransmitter sodium symporters (NSS) are a subfamily of SLC6 transporters responsible for regulating neurotransmitter signalling. They are a major target for psychoactive substances including antidepressants and drugs of abuse, prompting substantial research into their modulation and structure-function dynamics. Recently, a series of allosteric transport inhibitors have been identified, which may reduce side effect profiles, compared to orthosteric inhibitors. Allosteric inhibitors are also likely to provide different clearance kinetics compared to competitive inhibitors and potentially better clinical outcomes. Crystal structures and homology models have identified several allosteric modulatory sites on NSS including the vestibule allosteric site (VAS), lipid allosteric site (LAS) and cholesterol binding site (CHOL1). Whilst the architecture of eukaryotic NSS is generally well conserved there are differences in regions that form the VAS, LAS, and CHOL1. Here, we describe ligand-protein interactions that stabilize binding in each allosteric site and explore how differences between transporters could be exploited to generate NSS specific compounds with an emphasis on GlyT2 modulation.
... The complex structure of CCCs allows formation of discrete ion-binding sites nested within the TM domains, although ion selectivity and stoichiometry can differ among NCC, NKCCs, and KCCs [17]. Ion translocation occurs via the alternating access model, which involves switching between an inward-open (IO) ↔ outward-open (OO) conformational state that exposes the ion-binding sites to either side of the membrane (Box 1) [18,19]. ...
Cation-coupled chloride cotransporters (CCCs) modulate the transport of sodium and/or potassium cations coupled with chloride anions across the cell membrane. CCCs thus help regulate intracellular ionic concentration and consequent cell volume homeostasis. This has been largely exploited in the past to develop diuretic drugs that act on CCCs expressed in the kidney. However, a growing wealth of evidence has demonstrated that CCCs are also critically involved in a great variety of other pathologies, motivating most recent drug discovery programs targeting CCCs. Here, we examine the structure–function relationship of CCCs. By linking recent high-resolution cryogenic electron microscopy (cryo-EM) data with older biochemical/functional studies on CCCs, we discuss the mechanistic insights and opportunities to design selective CCC modulators to treat diverse pathologies.
... The cryo-EM map also resolved a Ca 2+ ion and six Nlinked glycans for each rBAT molecule, as discussed later in detail. b 0,+ AT shares structural and sequence homology with the APC superfamily and shows a typical LeuT-fold (Fig. 2c), consisting of 12 transmembrane helices (TM1-12) that contain 5 + 5 TM inverted repeats (18). Other structural features involve two helices in the extracellular loop 4 (EL4a and EL4b), one helix in intracellular loop 1 (IL1) and the cytoplasmic C-terminal helix (CH) running parallel to the membrane (Fig. S5). ...
Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, which causes recurrent kidney stones and occasionally severe kidney failure. Mutations of the two responsible proteins, rBAT and b0,+AT, which comprise system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Although recent structural insights into human b0,+AT-rBAT suggested a model for transport-inactivating mutations, the mechanisms by which type I mutations trigger trafficking deficiencies are not well understood. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+-mediated higher-order assembly of system b0,+ is the key to its trafficking on the cell surface. We show that Ca2+ stabilizes the interface between two rBAT molecules to mediate super-dimerization, and this in turn facilitates the N-glycan maturation of system b0,+. A common cystinuria mutant T216M and mutations that disrupt the Ca2+ site in rBAT cause the loss of higher-order assemblies, resulting in protein trafficking deficiency. Mutations at the super-dimer interface reproduce the mis-trafficking phenotype, demonstrating that super-dimerization is essential for cellular function. Cell-based transport assays confirmed the importance of the Ca2+ site and super-dimerization, and additionally suggested which residues are involved in cationic amino acid recognition. Taken together, our results provide the molecular basis of type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and trafficking diseases in general.
... This is known as the alternative access model ( Figure 8B). 40 In contrast, cavitand 1, with an approximate size of 14 Å by 10 Å , is not able to access simultaneously both sides of biological membranes ($30 Å ). This remark suggests that cavitand 1 might diffuse within the membrane (e.g., liposomes or the plasma membrane of HeLa cells), and eventually, upon reaching the membrane surface, binds the substrate (e.g., L-Pro). ...
The transport of anions and cations through liposomal membranes facilitated by synthetic carriers has been widely described. In contrast, analogous studies describing the facilitated transport of amino acids (aa) are scarce. We describe the use of calix[4]pyrrole receptors as synthetic carriers for the facilitated diffusion of aa across membranes of liposomes and living cells. We demonstrate that a calix[4]pyrrole cavitand is highly effective and selective for the facilitated diffusion of L-proline (L-Pro). We propose a mobile carrier diffusion mechanism to explain the observed aa facilitated diffusion process. The transport process involves the formation of a 1:1 complex between the carrier and the aa (e.g., L-Pro⊂1). We also describe the unprecedented application of a synthetic carrier to contribute to the uptake of L-Pro in human cells in addition to that mediated by the natural transporters. The reported results augur well for the potential use of aa synthetic carriers in therapeutic applications.
... All NSS transporters possess 12 transmembrane helices with both the amino-and carboxy-termini (N-term and C-term, respectively) modeled in the cytoplasm. Historically, much of the structural information about the NSS family had been inferred from the known structure of LeuT (6)(7)(8) though there is only 20-25% amino acid homology between LeuT and members of NSS family (9,10). Despite this limited sequence homology, LeuT possesses a high structural homology in its agonist binding site when compared to NSS and has high affinity and is inhibited by SSRIs (8,11). ...
Altered serotonin (5-HT) levels contribute to disease states such as depression and anxiety. Synaptic serotonin concentration is partially regulated by the serotonin transporter (SERT), making this transporter an important therapeutic target. This study seeks to examine the lipid accessible domains of hSERT to provide critical information regarding the apo-state of this transporter in a lipid environment. Recombinant hSERT was inducibly expressed in a human cell line. Solubilized SERT was purified by affinity chromatography using a FLAG Tag and reconstituted into mixed lipid vesicles containing our photoactivatable lipid probe. The lipid-accessible domains of the reconstituted transporter in membranes in its apo-state were probed via photocrosslinking to azi-cholesterol followed by quadrupole time of flight liquid chromatography-mass spectrometry (QTOF-LC-MS). MS studies identified crosslinks in three transmembrane loops consistent with the known topology of SERT. Surprisingly, the amino- and carboxy-terminal domains were similarly crosslinked by cholesterol, suggesting that these regions may also be intimately associated with the lipid bilayer. The data presented herein assist in further refining our understanding of the topography of the apo-state of hSERT via analysis of lipid accessibility.
... This is known as the alternative access model ( Figure 8B). 40 In contrast, cavitand 1, with an approximate size of 14 Å by 10 Å , is not able to access simultaneously both sides of biological membranes ($30 Å ). This remark suggests that cavitand 1 might diffuse within the membrane (e.g., liposomes or the plasma membrane of HeLa cells), and eventually, upon reaching the membrane surface, binds the substrate (e.g., L-Pro). ...
The transport of anions and cations through liposomal membranes facilitated by synthetic carriers has been widely described. In contrast, analogous studies describing the facilitated transport of amino acids (aa) are scarce. We describe the use of calix[4]pyrrole receptors as synthetic carriers for the facilitated diffusion of aa across membranes of liposomes and living cells. We demonstrate that a calix[4]pyrrole cavitand is highly effective and selective for the facilitated diffusion of L-proline (L-Pro). We propose a mobile carrier diffusion mechanism to explain the observed aa facilitated diffusion process. The transport process involves the formation of a 1:1 complex between the carrier and the aa (e.g., L-Pro⊂1). We also describe the unprecedented application of a synthetic carrier to contribute to the uptake of L-Pro in human cells in addition to that mediated by the natural transporters. The reported results augur well for the potential use of aa synthetic carriers in therapeutic applications.
... The extracellular side is tightly sealed by main chain packing of TM helices 1b, 3, 6a, and 10, while on the cytoplasmic side we observe a wide solvent-filled tunnel, lined by polar residues (D117, Y118, E233, N237, T317, S320, Q324) (Fig. 4a, b). A thin gate 29 , formed by the side chains of D36, T121, T230 and Y377, separates this tunnel from a smaller cavity located next to the discontinuous region of TM helices 1 and 6, which we hypothesize to be a potassium ion binding site (Fig. 4a, b). ...
Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K+/H+ symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins. KUP transporters facilitate potassium uptake by the co-transport of protons and are key players in potassium homeostasis. Here authors identify the potassium importer KimA from Bacillus subtilis as a new member of the KUP transporter family and show the cryo-EM structure of KimA in an inward-occluded, trans-inhibited conformation.
... Na + is a commonly cotransported ion in "secondary transporters," which utilize the electrochemical gradient to translocate a second ion or a molecule against its gradient across membranes (Krishnamurthy, Piscitelli, & Gouaux, 2009). For this reason, the enrichment in Na + transport documented here is difficult to allocate to any process in the algal cell at this stage. ...
Abstract Coral–dinoflagellate symbiosis underpins the evolutionary success of corals reefs. Successful exchange of molecules between the cnidarian host and the Symbiodiniaceae algae enables the mutualistic partnership. The algae translocate photosynthate to their host in exchange for nutrients and shelter. The photosynthate must traverse multiple membranes, most likely facilitated by transporters. Here, we compared gene expression profiles of cultured, free‐living Breviolum minutum with those of the homologous symbionts freshly isolated from the sea anemone Exaiptasia diaphana, a widely used model for coral hosts. Additionally, we assessed expression levels of a list of candidate host transporters of interest in anemones with and without symbionts. Our transcriptome analyses highlight the distinctive nature of the two algal life stages, with many gene expression level changes correlating to the different morphologies, cell cycles, and metabolisms adopted in hospite versus free‐living. Morphogenesis‐related genes that likely underpin the metamorphosis process observed when symbionts enter a host cell were up‐regulated. Conversely, many down‐regulated genes appear to be indicative of the protective and confined nature of the symbiosome. Our results emphasize the significance of transmembrane transport to the symbiosis, and in particular of ammonium and sugar transport. Further, we pinpoint and characterize candidate transporters—predicted to be localized variously to the algal plasma membrane, the host plasma membrane, and the symbiosome membrane—that likely serve pivotal roles in the interchange of material during symbiosis. Our study provides new insights that expand our understanding of the molecular exchanges that underpin the cnidarian–algal symbiotic relationship.
... Additionally, the EL4 of LAT1 swings out during the transition, leading to the expansion of the substrate pathway in the outward-open state. This observation is coherent with the findings of Lee and co-workers, that EL4 might be functioning as an extracellular latch in LAT1 that opens and closes during the transport cycle as observed in other transporters of the amino acid/polyamine/organocation (APC) family 67 . Hence, the rearrangement of EL4 triggers the broadening or narrowing of the substrate pathway leading to antiport flux of substrates. ...
L-type Amino acid Transporter 1 (LAT1) plays a significant role in the growth and propagation of cancer cells by facilitating the cross-membrane transport of essential nutrients, and is an attractive drug target. Several halogen-containing L-phenylalanine-based ligands display high affinity and high selectivity for LAT1; nonetheless, their molecular mechanism of binding remains unclear. In this study, a combined in silico strategy consisting of homology modeling, molecular docking, and Quantum Mechanics-Molecular Mechanics (QM-MM) simulation was applied to elucidate the molecular basis of ligand binding in LAT1. First, a homology model of LAT1 based on the atomic structure of a prokaryotic homolog was constructed. Docking studies using a set of halogenated ligands allowed for deriving a binding hypothesis. Selected docking poses were subjected to QM-MM calculations to investigate the halogen interactions. Collectively, the results highlight the dual nature of the ligand-protein binding mode characterized by backbone hydrogen bond interactions of the amino acid moiety of the ligands and residues I63, S66, G67, F252, G255, as well as hydrophobic interactions of the ligand’s side chains with residues I139, I140, F252, G255, F402, W405. QM-MM optimizations indicated that the electrostatic interactions involving halogens contribute to the binding free energy. Importantly, our results are in good agreement with the recently unraveled cryo-Electron Microscopy structures of LAT1.
... While the thermodynamic features of secondary active transport are well understood, intense study is currently focused on the identification of the specific sequence of substrate binding and release events, as well as the series of conformational changes that are coupled to, and facilitate, these processes (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). X-ray crystallographic studies have provided insight into the NSS molecular architecture, beginning with crystal structures of the Na + -coupled prokaryotic amino acid transporter LeuT (18,19), which allowed for existing mechanistic models to be put into a structural context (20). Single-molecule fluorescence resonance energy transfer (smFRET) studies have illuminated the dynamic sampling of functionally important intermediate states visited by the transporter during the transport cycle (21)(22)(23). ...
Significance
The combination of molecular dynamics simulation, single-molecule imaging, and functional assays described here reveals a mechanism of substrate selectivity in the SLC6 family of neurotransmitter transporters. We show that the rotameric state of a volumetric sensor in the substrate binding site is allosterically coupled to conformational changes necessary for transport, and as a consequence, upon binding large substrates, the transporter becomes stabilized in an inactive, nontransporting state upon binding larger substrates. This mechanistic insight suggests the possibility that medically relevant SLC6 transporters may be targeted by inhibitors that specifically modulate this sensor.
... Despite their evolutionary, structural and functional differences, all transporters appear to use an alternating-access mechanism where a substrate binding site, in "allosteric" cooperation with distinct gating domains, alternates between multiple conformations for receiving and delivering specific substrate(s) from one side of the membrane to the other. This basic mechanism, carried out by dynamic movements of the main transmembrane body and assisted by the flexibility of interconnecting hydrophilic loops, exists in different forms, of the so-called the rocker-switch, the rocking-bundle or the elevator sliding mechanisms [10,25,28,33]. ...
... In these structures, the α-amino group of the ligand forms hydrogen (H) bonds with residues of the unwound parts of TM1 and TM6 and the α-carboxyl group of the Arg participates in H bonds with the unwound segment of TM1 only ( Table 3). The discontinuity of these two TM helices has also been observed in other APC transporters (Krishnamurthy et al. 2009;Shi 2013). Residues in these helical discontinued portions form two motifs: "GSG" in TM1 (Gly25-Ser26-Gly27 in AdiC) and "GVSEA" in TM6 (Gly206-Val207-Glu208-Ser209-Ala210 in AdiC) in TM6 (Fig. 4b). ...
Bacterial pathogens are a major cause of foodborne diseases and food poisoning. To cope with the acid conditions encountered in different environments such as in fermented food or in the gastric compartment, neutralophilic bacteria have developed several adaptive mechanisms. One of those mechanisms, the amino acid dependent system, consumes intracellular protons in biochemical reactions. It involves an antiporter that facilitates the exchange of external substrate amino acid for internal product and a cytoplasmic decarboxylase that catalyzes a proton-consuming decarboxylation of the substrate. So far, four acid resistance antiporters have been discovered, namely the glutamate-γ-aminobutyric acid antiporter GadC, the arginine-agmatine antiporter AdiC, the lysine-cadaverine antiporter CadB, and the ornithine-putrescine antiporter PotE. The 3D structures of AdiC and GadC, reveal an inverted-repeat fold of two times 5 transmembrane helices, typical of the amino acid-polyamine-organocation (APC) superfamily of transporters. This review summarizes our current knowledge on the transport mechanism, the pH regulation and the selectivity of these four acid resistance antiporters. It also highlights that AdiC is a paradigm for eukaryotic amino acid transporters of the APC superfamily as structural models of several of these transporters built using AdiC structures were exploited to unveil their mechanisms of amino acid recognition and translocation.
... Specifically, we observed reduced HDX of TM1a and the connected residues of the N-terminal end (residue 28-43), TM6b, IL3 and the intracellular part of TM7 (residue 320-328, 330-347). These regions have all been proposed to participate in the opening and closing of the intracellular vestibule 9,22,24,42 , and the observed stabilization suggests isomerization of the inner-gate to a closed conformation as expected upon Na + -binding 43,44 . On the extracellular side, we observed increased HDX in the hinge region and TM1b (residue 45-56) together with large parts of TM7 (residue 349-357) and EL4 (residue 366-380) including the first helix of the loop (EL4a), while TM6a (residue 303-318) and TM2 (residue 58-73) showed decreased HDX upon ion binding. ...
The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.
... A possible deviation from the general model is that CD98hc-ED is on top of LAT1, and contacts EL4a. In most APC members, EL4 has been implicated in the latch function 41 , ...
The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1–CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.
Transporters mediate the uptake of solutes, metabolites and drugs across the cell membrane. The eukaryotic FurE nucleobase/H+ symporter of Aspergillus nidulans has been used as a model protein to address structure-function relationships in the APC transporter superfamily, members of which are characterized by the LeuT-fold and seem to operate by the so-called 'rocking-bundle' mechanism. In this study, we reveal the binding mode, translocation and release pathway of uracil/H+ by FurE, using path collective variable, funnel metadynamics and rational mutational analysis. Our study reveals a stepwise, induced-fit, mechanism of ordered sequential transport of proton and uracil, which in turn suggests that FurE, functions as a multi-step gated pore, rather than employing 'rocking' of compact domains, as often proposed for APC transporters. Finally, our work supports that specific residues of the cytoplasmic N-tail are involved in substrate translocation, in line with their essentiality for FurE function.
Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps.
The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na+ -dependent Cl- - and K+ -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+ , K+ , and 2 Cl- ) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- -ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl- -sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.
Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na ⁺ to H ⁺ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H ⁺ /K ⁺ pump, a strict H ⁺ -dependent electroneutral P-type ATPase, into a bona fide Na ⁺ -dependent electrogenic Na ⁺ /K ⁺ pump. Conversion of a H ⁺ -dependent primary-active transporter into a Na ⁺ -dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H ⁺ /K ⁺ pump, a suitable drug target to treat cystic fibrosis, and of its Na ⁺ /K ⁺ pump-mimicking mutant in two major conformations, providing insight on how Na ⁺ binding drives a concerted mechanism leading to Na ⁺ /K ⁺ pump phosphorylation.
Transport of ions and small molecules across the cell membrane against electrochemical gradients is catalyzed by integral membrane proteins that use a source of free energy to drive the energetically uphill flux of the transported substrate. Secondary active transporters couple the spontaneous influx of a "driving" ion such as Na+ or H+ to the flux of the substrate. The thermodynamics of such cyclical non-equilibrium systems are well understood, and recent work has focused on the molecular mechanism of secondary active transport. The fact that these transporters change their conformation between an inward-facing and outward-facing conformation in a cyclical fashion, called the alternating access model, is broadly recognized as the molecular framework in which to describe transporter function. However, only with the advent of high resolution crystal structures and detailed computer simulations, it has become possible to recognize common molecular-level principles between disparate transporter families. Inverted repeat symmetry in secondary active transporters has shed light onto how protein structures can encode a bi-stable two-state system. Based on structural data, three broad classes of alternating access transitions have been described as rocker-switch, rocking-bundle, and elevator mechanisms. More detailed analysis indicates that transporters can be understood as gated pores with at least two coupled gates. These gates are not just a convenient cartoon element to illustrate a putative mechanism but map to distinct parts of the transporter protein. Enumerating all distinct gate states naturally includes occluded states in the alternating access picture and also suggests what kind of protein conformations might be observable. By connecting the possible conformational states and ion/substrate bound states in a kinetic model, a unified picture emerges in which the symporter, antiporter, and uniporter functions are extremes in a continuum of functionality. As usual with biological systems, few principles and rules are absolute and exceptions are discussed as well as how biological complexity may be integrated in quantitative kinetic models that may provide a bridge from the structure to function.
Transporters mediate the uptake or efflux of solutes, metabolites and drugs across the cell membrane. The FurE symporter of uracil-allantoin-uric acid/H+ of Aspergillus nidulans has been used as a model eukaryotic transporter to address structure-function relationships and the mechanism of transport in the NCS1/APC family of transporters. Extensive genetic, functional and cellular studies, as well as homology modeling based on a prokaryotic transporter with a similar fold (Mhp1) have provided important information on specific structural elements of FurE. However, the exact mechanism by which substrates and proton or other cations are translocated by FurE or transporters with a similar fold remains elusive. In this study, we reveal the binding mode, translocation and release pathway of uracil/H+ from the extracellular space to the cytoplasm, using novel metadynamics calculations and rationally designed mutational analysis. In particular, Metadynamics Free Energy Surface maps provide the relative order of internalization of uracil and proton, permitting also selection of intermediate conformational states related to transport cycle. Funnel Metadynamics allow the sampling of specific interactions of both uracil and proton with residues during their internalization, generating a holistic model of the transport events in conjunction with experimental mutation studies. Our work not only complements and extends the existing knowledge on FurE same-fold transporters, but also challenges the so-called rocking bundle mechanism associated with biomedically interesting members of the APC superfamily of transporters.
The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure-function relationships of NSS family proteins. With hydrogen-deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperature (25°C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 – 95°C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45°C and 60°C). We have further compared the conformational impact of binding the efficiently transported substrate, alanine (Ala) to the much slower transported leucine (Leu). At elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25°C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. Furthermore, comparing the HDX signature of Ala vs. Leu we observe distinct differences that could correspond to the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of LeuT in the presence of Ala. Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.
Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl⁻ with K⁺ and/or Na⁺ in an electroneutral manner. They play important roles in myriad fundamental physiological processes––from cell volume regulation to transepithelial solute transport and intracellular ion homeostasis––and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacology of these transporters, there have been several breakthroughs in the structural determination of CCC transporters. The insights provided by these new structures for the Na⁺/K⁺/Cl⁻ cotransporter NKCC1 and the K⁺/Cl⁻ cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their molecular basis and transport function. This focused review discusses recent advances in our structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.
K⁺-Cl⁻ cotransporters (KCCs) play important roles in physiological processes such as inhibitory neurotransmission and cell volume regulation. KCCs exhibit significant variations in K⁺-affinities, yet recent atomic structures demonstrated that K⁺ and Cl⁻- binding sites are highly conserved, raising the question of whether additional structural elements may contribute to ion coordination. The termini and the large extracellular domain (ECD) of KCCs exhibit only low sequence identity and were already discussed as modulators of transport activity. Here, we used the extracellular loop 2 (EL2) which links transmembrane helices (TM) 3 and 4, as a mechanism to modulate ECD folding. We compared consequences of point mutations in the K⁺-binding site on the function of wild-type KCC2 (KCC2WT) and in a KCC2 variant, in which EL2 was structurally altered by insertion of an 3xHA-tag (KCC2HA). In KCC2wt, individual mutations of five residues in the K⁺-binding site resulted in a two to three-fold decreased transport rate. However, the same mutations in KCC2HA had no effect on transport activity. Homology models of mouse KCC2 with the 3xHA-tag inserted into EL2 using ab initio prediction were generated. The models suggest subtle conformational changes occur in the ECD upon EL2 modification. These data suggest that a conformational change in the ECD, e.g., by interaction with EL2, might be an elegant way to modulate the K⁺- affinity of the different isoforms in the KCC subfamily.
The thiazide-sensitive NaCl cotransporter (NCC, SLC12A3) is a member of the solute carrier 12 (SLC12) family of electroneutral cotransporters. NCC is expressed in the distal convoluted tubule (DCT) of the kidney, where it mediates the transport of sodium with chloride in 1:1 stoichiometry. NCC is responsible for reabsorbing approximately 5–10% of sodium from the glomerular filtrate. NCC plays a key role in determining the blood pressure set point and potassium balance. NCC is the major molecular target of thiazide-type diuretics, drugs commonly used as first-line agents in the treatment of essential hypertension and edema. Since its cloning decades ago, much has been learned about the physiological transport properties of NCC, its regulation, and its connections to human disease. In this chapter, I provide an overview of the biology of this important cotransporter.
NKCC and KCC transporters mediate coupled transport of Na⁺+K⁺+Cl⁻ and K⁺+Cl⁻ across the plasma membrane, thus regulating cell Cl⁻ concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational analysis and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.
Nucleosides play central roles in all facets of life, from metabolism to cellular signaling. Because of their physiochemical properties, nucleosides are lipid bilayer impermeable and thus rely on dedicated transport systems to cross biological membranes. In humans, two unrelated protein families mediate nucleoside membrane transport: the concentrative and equilibrative nucleoside transporter families. The objective of this review is to provide a broad outlook on the current status of nucleoside transport research. We will discuss the role played by nucleoside transporters in human health and disease, with emphasis placed on recent structural advancements that have revealed detailed molecular principles of these important cellular transport systems and exploitable pharmacological features.
The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis , the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.
Significance
Amino acids are involved in metabolism, protein biosynthesis, and signal transduction of humans. Heteromeric amino acid transporters maintain homeostasis of amino acid pools in various tissues and organs representing a unique group among members of the solute carrier superfamily. The neutral and basic amino acid transport complex (b [0,+] AT1-rBAT) is responsible for reabsorption of cystine and dibasic amino acids in kidney and intestine. Misguided trafficking, incomplete maturation, or defective transport activity result in hyperexcretion of cystine, causing cystinuria. While the function of this amino acid transport complex is known, details of its evolution, biogenesis, and mechanism of action have remained elusive. We determined the cryo-EM structure of this protein complex to gain a deeper understanding of its mode of action.
Cation chloride co-transporters (CCC) play an essential role for neuronal chloride homeostasis. CCC includes K⁺ Cl⁻ outward co-transporters (KCCs) and Na⁺ K⁺ Cl⁻ inward co-transporters (NKCCs). Although NKCCs and KCCs co-transporters have been studied intensively for several decades, a full picture of their common structural determinants and structure/function differences is still missing. A recent molecular architecture of KCC2 has been reported. This structure will be described and compared to high resolution structures of related co-transporters NKCC1 and KCC1 as well as previously reported biochemical and bioinformation information. Thus, the aim of this chapter is to provide a comprehensive overview of our current understanding of the architecture-function relationships of NKCCs and KCCs co-transporters. Differential cell expression profiles, sequence alignment, secondary structure prediction, structural comparison analysis, post-translational modification and oligomerization provide explanation of the subcellular and molecular organization and function of KCCs and NKCCs. This knowledge will enable structure-guided drug discovery allowing to meet many currently unmet medical needs related to neurological disorders and other human diseases.
The cellular mechanisms underpinning changes in metabolism during postnatal development in young mammals have not been extensively examined. This study examines changes in sodium pump capacity (Na⁺, K⁺-ATPase activity), number and molecular activity, as well as, Na⁺ flux, cholesterol level and fatty acid composition in a number of major organs during postnatal development in the rat. In liver, Na⁺ flux was highest (2.6 times) in the youngest rats (3-day old) and decreased with increasing age, whereas Na⁺, K⁺-ATPase activity increased with age (up to 9–28 days) in liver, kidney and brain, but not in heart. Increases in Na⁺, K⁺-ATPase activity where primarily driven by increases in molecular activity, 4-fold in brain and 7-fold in kidney, rather than by increases in sodium pump number. Membrane polyunsaturation increased in both kidney and brain during development, with kidney becoming increasingly dominated by omega-6 (18:2n-6 and 20:4n-6) and brain by omega-3 (22:6n-3) fatty acids. Membrane reconstitution experiments support the concept that changes in membrane composition might underpin higher sodium molecular activities in the adult. In conclusion, at birth rats possess high Na⁺ flux but a lower sodium pump capacity that increases with age being driven by increases in molecular activities associate with changes in membrane lipid composition.
Na+/H+ exchangers play pivotal roles in the control of cell and tissue pH by mediating the electroneutral exchange of Na+ and H+ across cellular membranes. They belong to an ancient family of highly evolutionarily conserved proteins, and they play essential physiological roles in all phyla. In this review, we focus on the mammalian Na+/H+ exchangers (NHEs), the solute carrier (SLC) 9 family. This family of electroneutral transporters constitutes three branches: SLC9A, -B, and -C. Within these, each isoform exhibits distinct tissue expression profiles, regulation, and physiological roles. Some of these transporters are highly studied, with hundreds of original articles, and some are still only rudimentarily understood. In this review, we present and discuss the pioneering original work as well as the current state-of-the-art research on mammalian NHEs. We aim to provide the reader with a comprehensive view of core knowledge and recent insights into each family member, from gene organization over protein structure and regulation to physiological and pathophysiological roles. Particular attention is given to the integrated physiology of NHEs in the main organ systems. We provide several novel analyses and useful overviews, and we pinpoint main remaining enigmas, which we hope will inspire novel research on these highly versatile proteins.
Cation–chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we determined the cryo-electron microscopy structure of the Na–K–Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiological functions of CCCs and interpreting disease-related mutations.
Neurotransmitter sodium symporters (NSS) belong to the SLC6 family of solute carriers and play an essential role in neurotransmitter homeostasis throughout the body. In the past decade, structural studies employing bacterial orthologs of NSSs have provided insight into the mechanism of neurotransmitter transport. While the overall architecture of SLC6 transporters is conserved among species, in comparison to the bacterial homologs, the eukaryotic SLC6 family members harbor differences in amino acid sequence and molecular structure, which underpins their functional and pharmacological diversity, as well as their ligand specificity. Here, we review the structures and mechanisms of eukaryotic NSSs, focusing on the molecular basis for ligand recognition and on transport mechanism.
The rat thyroid Na+/I- symporter (NIS) was expressed in Xenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+ > Li+ > H+). The apparent affinity constants for Na+ and I- were 28 +/- 3 mM and 33 +/- 9 microM, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I-, ClO3-, SCN-, SeCN-, NO3-, Br-, BF4-, IO4-, BrO3-, but perchlorate (ClO4-) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (approximately 35% of maximum substrate-induced current) with an apparent Na+ affinity of 74 +/- 14 mM and a Hill coefficient (n) of 1. In response to step voltage changes, NIS exhibited current transients that relaxed with a time constant of 8-14 ms. Presteady-state charge movements (integral of the current transients) versus voltage relations obey a Boltzmann relation. The voltage for half-maximal charge translocation (V0.5) was -15 +/- 3 mV, and the apparent valence of the movable charge was 1. Total charge was insensitive to [Na+]o, but V0.5 shifted to more negative potentials as [Na+]o was reduced. NIS charge movements are attributed to the conformational changes of the empty transporter within the membrane electric field. The turnover rate of NIS was >/=22 s-1 in the Na+ uniport mode and >/=36 s-1 in the Na+/I- cotransport mode. Transporter density in the plasma membrane was determined using freeze-fracture electron microscopy. Expression of NIS in oocytes led to a approximately 2. 5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles. On the basis of the kinetic results, we propose an ordered simultaneous transport mechanism in which the binding of Na+ to NIS occurs first.
The control by Na+/H+ antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na+/H+ antiporters. Their activity is tightly controlled by pH. Here we present the crystal structure of pH-downregulated NhaA, the main antiporter of Escherichia coli and many enterobacteria. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. We propose that the binding of charged substrates causes an electric imbalance, inducing movements, that permit a rapid alternating-access mechanism. This ion-exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the entry to the cytoplasmic funnel.
Crystal structures of LeuT, a bacterial homologue of mammalian neurotransmitter transporters, show a molecule of bound substrate that is essentially exposed to the extracellular space but occluded from the cytoplasm. Thus, there must exist an alternate conformation for LeuT in which the substrate is accessible to the cytoplasm and a corresponding mechanism that switches accessibility from one side of the membrane to the other. Here, we identify the cytoplasmic accessibility pathway of the alternate conformation in a mammalian serotonin transporter (SERT) (a member of the same transporter family as LeuT). We also propose a model for the cytoplasmic-facing state that exploits the internal pseudosymmetry observed in the crystal structure. LeuT contains two structurally similar repeats (TMs1–5 and TMs 6–10) that are inverted with respect to the plane of the membrane. The conformational differences between them result in the formation of the extracellular pathway. Our model for the cytoplasm-facing state exchanges the conformations of the two repeats and thus exposes the substrate and ion-binding sites to the cytoplasm. The conformational change that connects the two states primarily involves the tilting of a 4-helix bundle composed of transmembrane helices 1, 2, 6, and 7. Switching the tilt angle of this bundle is essentially equivalent to switching the conformation of the two repeats. Extensive mutagenesis of SERT and accessibility measurements, using cysteine reagents, are accommodated by our model. These observations may be of relevance to other transporter families, many of which contain internal inverted repeats.
• alternating access mechanism
• neurotransmitter:sodium symporters
• serotonin
• structural modeling
• structural repeats
Secondary glutamate transporters in neuronal and glial cells in the mammalian central nervous system remove the excitatory neurotransmitter glutamate from the synaptic cleft and prevent the extracellular glutamate concentration to rise above neurotoxic levels. Secondary structure prediction algorithms predict 6 transmembrane helices in the first half of the transporters but fail in the C-terminal half where no clear helix-loop-helix motif is resolved in the hydropathy profile of the primary sequences. A number of previous studies have emphasized the importance of the C-terminal half of the molecules for the function. Here we determine the membrane topology of the C-terminal half of the glutamate transporters by applying the phoA gene fusion technique to the homologous bacterial glutamate transporter of Bacillus stearothermophilus. High sequence conservation and very similar hydropathy profiles in the C-terminal half warrant a similar folding as in the glutamate transporters of the mammalian central nervous system. The C-terminal half contains four putative transmembrane helices. The strong hydrophobic moment and substitution moment of the most C-terminal helix X that point to opposite faces of the helix suggest that the helix faces the lipid environment with its least conserved, hydrophobic face and the interior of the protein with its well conserved, hydrophilic face. Residues that were shown before to be critical for function cluster in helix X and VII.
GAT-1 is a sodium- and chloride-coupled gamma-aminobutyric acid (GABA) transporter, which fulfills an essential role in the synaptic transmission by this neurotransmitter. Cysteine-399 is the major site of inhibition of GAT-1 by membrane-permeant sulfhydryl reagents. This cysteine residue was previously thought to reside on a cytoplasmic loop connecting transmembrane domains (TMs) 8 and 9. However, the crystal structure of LeuT, a bacterial homologue of the mammalian neurotransmitter:sodium symporters, revealed that the residue corresponding to Cys-399 is in fact located in the middle of TM 8. This residue is located to the cytoplasmic side of Asp-395 and Ser-396, whose side chains are thought to ligand one of the two cotransported sodium ions. To determine how the sulfhydryl reagents approach cysteine-399, a cysteine scan of all 35 residues of TM 8 was performed. Sulfhydryl reagents inhibited transport when a cysteine residue was present at either of the positions 399, 402, 406, and 410. SKF-89976A and other non-transportable analogues, which are expected to lock the transporter in a conformation facing the extracellular medium, protected against the sulfhydryl modification at positions 399, 402, and 406. Such a protection was not seen by GABA itself, which actually modestly potentiated the modification at positions 399 and 402. Our results point to an alpha-helical stripe on TM8 lining an aqueous access pathway from the cytoplasm into the binding pocket, which gets occluded in the conformation of the transporter where the binding pocket is exposed to the extracellular medium.
Secondary transporters are workhorses of cellular membranes, catalyzing the movement of small molecules and ions across the bilayer and coupling substrate passage to ion gradients. However, the conformational changes that accompany substrate transport, the mechanism by which a substrate moves through the transporter, and principles of competitive inhibition remain unclear. We used crystallographic and functional studies on the leucine transporter (LeuT), a model for neurotransmitter sodium symporters, to show that various amino acid substrates induce the same occluded conformational state and that a competitive inhibitor, tryptophan (Trp), traps LeuT in an open-to-out conformation. In the Trp complex, the extracellular gate residues arginine 30 and aspartic acid 404 define a second weak binding site for substrates or inhibitors as they permeate from the extracellular solution to the primary substrate site, which demonstrates how residues that participate in gating also mediate permeation.
The nucleobase–cation–symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related
metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures
of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes
upon binding of substrate. Comparisons with the leucine transporter LeuTAa and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite
sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices
3 and 8, providing the structural basis of the alternating access model for membrane transport.
The platelet plasma membrane serotonin transporter requires Na+ for two reactions, serotonin transport and imipramine binding. Although imipramine binding has been thought to reflect the same process required for serotonin binding prior to transport (Talvenheimo, J., Nelson, P.J., and Rudnick, G. (1979) J. Biol. Chem. 254, 4631-4635), binding and transport display markedly different responses to Na+. Imipramine binding (and competitive inhibition of transport) apparently requires two sodium ions which bind with a KD of 300 +/- 70 meq/liter. The total number of sites (Bmax) is the same at all Na+ concentrations, but the affinity for imipramine increases from 7.3 x 10(6) M-1 at 20 meq/liter to 110 x 10(6) M-1 at 200 meq/liter. Na+ acts, at least in part, by decreasing the rate of imipramine dissociation from its binding site. Serotonin binding displaces imipramine from its site on the membrane. In contrast to imipramine binding, this displacement is a simple, hyperbolic function of Na+ concentration with a KD for Na+ of 400 +/- 100 meq/liter, which suggests that only one Na+ is required. Serotonin transport is also much less responsive to Na+ concentration. Over the same concentration range in which the affinity for imipramine increases 15-fold, the affinity for serotonin increases only 2-fold. Despite the lack of Na+ effect on the Bmax for imipramine binding, the Vmax for serotonin transport increases as a simple saturable function of Na+ with a KM (Na+) of 52 meq/liter. Thus, substrate translocation as well as binding requires Na+. Since serotonin is cotransported with Na+, the serotonin gradient accumulated depends on the coupling stoichiometry and the magnitude of the Na+ gradient imposed. From the response of the serotonin gradient to imposed Na+ gradients, we calculated a serotonin:Na+ cotransport stoichiometry of 0.9. Taken together, the results suggest that serotonin and imipramine bind either to the same site or to mutually exclusive sites, but maximal imipramine binding requires two sodium ions, while maximal serotonin binding and translocation requires only one.
A pH difference (acid inside) across the platelet plasma membrane increases both the rate and extent of serotonin accumulation inside plasma membrane vesicles. Even in the absence of other transmembrane ion gradients, this pH difference (delta pH) serves as the sole driving force for serotonin accumulation, leading to a serotonin concentration 18-fold higher inside the vesicle. This process requires Na+ and is blocked by imipramine, indicating that it is mediated by the serotonin transporter. At physiological pH, internal K+ is counter-transported with serotonin, and high internal K+ stimulates transport maximally. Internal K+ also blocks the delta pH stimulation of serotonin transport. Conversely, low internal pH (5.6) inhibits the ability of internal K+ to stimulate transport. This apparent competition between K+ and protons suggests that delta pH drives serotonin accumulation through counter-transport with protons, and that serotonin is transported in its cationic form.
We prepared membrane vesicles from stable LLC-PK cells expressing serotonin (5-HT) -aminobutyric acid (GABA) and norepinephrine (NE) transporters (SERT, GAT-1, and NET). These vesicles accumulate transport
substrates when the appropriate transmembrane ion gradients are imposed. For NET, accumulation of [3H]dopamine (DA) was stimulated by imposition of Na and Cl gradients (out > in) and of a K gradient (in > out). The presence of Na or Cl, even in the absence of a gradient, stimulated DA accumulation by NET, but K had little or no effect in the absence of a K gradient. Stimulation by a K gradient was markedly enhanced by increasing the K permeability with valinomycin, suggesting that net positive charge is transported together with DA. Cationic DA is likely
to be the major substrate for NET, since varying pH did not affect K. We estimated the Na:DA stoichiometry by measuring the effect of the transmembrane Na gradient on peak DA accumulation. The results suggest a 1:1 cotransport of Na with DA. Taken together, the results suggest that NET catalyzes cotransport of one cationic substrate molecule with one Na ion, and one Cl ion, and that K does not participate directly in the transport process.
Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. After cotransport of glutamic acid with Na+, the cycle is completed by countertransport of K+. We have identified an amino acid residue (glutamate 404) influencing ion coupling in a domain of the transporter implicated previously in kainate binding. Mutation of this residue to aspartate (E404D) prevents both forward and reverse transport induced by K+. Sodium-dependent transmitter exchange and a transporter-mediated chloride conductance are unaffected by the mutation, indicating that this residue selectively influences potassium flux coupling. The results support a kinetic model in which sodium and potassium are translocated in distinct steps and suggest that this highly conserved region of the transporter is intimately associated with the ion permeation pathway.
Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters.
A neurotransmitter transporter can potentially mediate uptake or release of substrate, and its stoichiometry is a key factor that controls the driving force and thus the neurotransmitter flux direction. We have used a combination of electrophysiology and radio-tracing techniques to evaluate the stoichiometries of two glycine transporters involved in glycinergic or glutamatergic transmission. We show that GlyT2a, a transporter present in glycinergic boutons, has a stoichiometry of 3 Na+/Cl-/glycine, which predicts effective glycine accumulation in all physiological conditions. GlyT1b, a glial transporter, has a stoichiometry of 2 Na+/Cl-/ glycine, which predicts that glycine can be exported or imported, depending on physiological conditions. GlyT1b may thus modulate glutamatergic synapses by increasing or decreasing the glycine concentration around N-methyl-D-aspartate receptors (NMDARs).
Calcium ATPase is a member of the P-type ATPases that transport ions across the membrane against a concentration gradient. Here we have solved the crystal structure of the calcium ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) at 2.6 A resolution with two calcium ions bound in the transmembrane domain, which comprises ten alpha-helices. The two calcium ions are located side by side and are surrounded by four transmembrane helices, two of which are unwound for efficient coordination geometry. The cytoplasmic region consists of three well separated domains, with the phosphorylation site in the central catalytic domain and the adenosine-binding site on another domain. The phosphorylation domain has the same fold as haloacid dehalogenase. Comparison with a low-resolution electron density map of the enzyme in the absence of calcium and with biochemical data suggests that large domain movements take place during active transport.
In the brain, neurons and glial cells compete for the uptake of the fast neurotransmitters, glutamate, GABA and glycine, through specific transporters. The relative contributions of glia and neurons to the neurotransmitter uptake depend on the kinetic properties, thermodynamic coupling and density of transporters but also on the intracellular metabolization or sequestration of the neurotransmitter. In the case of glycine, which is both an inhibitory transmitter and a neuromodulator of the excitatory glutamatergic transmission as a co-agonist of N-methyl D-aspartate receptors, the glial (GlyT1b) and neuronal (GlyT2a) transporters differ at least in three aspects: (i) stoichiometries, (ii) reverse uptake capabilities and (iii) pre-steady-state kinetics. A 3 Na(+)/1 Cl(-)/gly stoichiometry was established for GlyT2a on the basis of a 2 charges/glycine flux ratio and changes in the reversal potential of the transporter current as a function of the extracellular glycine, Na(+) and Cl(-) concentrations. Therefore, the driving force available for glycine uphill transport in neurons is about two orders of magnitude larger than for glial cells. In addition, GlyT2a shows a severe limitation for reverse uptake, which suggests an essential role of GlyT2a in maintaining a high intracellular glycine pool, thus facilitating the refilling of synaptic vesicles by the low affinity, low specificity vesicular transporter VGAT/VIAAT. In contrast, the 2 Na(+)/1 Cl(-)/gly stoichiometry and bi-directional transport properties of GlyT1b are appropriate for the control of the extracellular glycine concentration in a submicromolar range that can modulate N-methyl D-aspartate receptors effectively. Finally, analysis of the pre-steady-state kinetics of GlyT1b and GlyT2a revealed that at the resting potential neuronal transporters are preferentially oriented outward, ready to bind glycine, which suggests a kinetic advantage in the uptake contest.
AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.
The electroneutral cation-chloride-coupled cotransporter gene family ( SLC12) was identified initially at the molecular level in fish and then in mammals. This nine-member gene family encompasses two major branches, one including two bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporters and the thiazide-sensitive Na(+):Cl(-) cotransporter. Two of the genes in this branch ( SLC12A1 and SLC12A3), exhibit kidney-specific expression and function in renal salt reabsorption, whereas the third gene ( SLC12A2) is expressed ubiquitously and plays a key role in epithelial salt secretion and cell volume regulation. The functional characterization of both alternatively-spliced mammalian Na(+)-K(+)-2Cl(-) cotransporter isoforms and orthologs from distantly related species has generated important structure-function data. The second branch includes four genes ( SLC12A4- 7) encoding electroneutral K(+)-Cl(-) cotransporters. The relative expression level of the neuron-specific SLC12A5 and the Na(+)-K(+)-2Cl(-) cotransporter SLC12A2 appears to determine whether neurons respond to GABA with a depolarizing, excitatory response or with a hyperpolarizing, inhibitory response. The four K(+)-Cl(-) cotransporter genes are co-expressed to varying degrees in most tissues, with further roles in cell volume regulation, transepithelial salt transport, hearing, and function of the peripheral nervous system. The transported substrates of the remaining two SLC12 family members, SLC12A8 and SLC12A9, are as yet unknown. Inactivating mutations in three members of the SLC12 gene family result in Mendelian disease; Bartter syndrome type I in the case of SLC12A1, Gitelman syndrome for SLC12A3, and peripheral neuropathy in the case of SLC12A6. In addition, knockout mice for many members of this family have generated important new information regarding their respective physiological roles.
The sodium/glucose cotransporter family (SLCA5) has 220 or more members in animal and bacterial cells. There are 11 human genes expressed in tissues ranging from epithelia to the central nervous system. The functions of nine have been revealed by studies using heterologous expression systems: six are tightly coupled plasma membrane Na(+)/substrate cotransporters for solutes such as glucose, myo-inositol and iodide; one is a Na(+)/Cl(-)/choline cotransporter; one is an anion transporter; and another is a glucose-activated ion channel. The exon organization of eight genes is similar in that each comprises 14-15 exons. The choline transporter (CHT) is encoded in eight exons and the Na(+)-dependent myo-inositol transporter (SMIT) in one exon. Mutations in three genes produce genetic diseases (glucose-galactose malabsorption, renal glycosuria and hypothyroidism). Members of this family are multifunctional membrane proteins in that they also behave as uniporters, urea and water channels, and urea and water cotransporters. Consequently it is a challenge to determine the role(s) of these genes in human physiology and pathology.
The major facilitator superfamily represents the largest group of secondary membrane transporters in the cell. Here we report
the 3.3 angstrom resolution structure of a member of this superfamily, GlpT, which transports glycerol-3-phosphate into the
cytoplasm and inorganic phosphate into the periplasm. The amino- and carboxyl-terminal halves of the protein exhibit a pseudo
two-fold symmetry. Closed off to the periplasm, a centrally located substrate-translocation pore contains two arginines at
its closed end, which comprise the substrate-binding site. Upon substrate binding, the protein adopts a more compact conformation.
We propose that GlpT operates by a single–binding site, alternating-access mechanism through a rocker-switch type of movement.
The highly evolutionarily conserved serotonin transporter (SERT) regulates the entire serotoninergic system and its receptors via modulation of extracellular fluid serotonin concentrations. Differences in SERT expression and function produced by three SERT genes and their variants show associations with multiple human disorders. Screens of DNA from patients with autism, ADHD, bipolar disorder, and Tourette's syndrome have detected signals in the chromosome 17q region where SERT is located. Parallel investigations of SERT knockout mice have uncovered multiple phenotypes that identify SERT as a candidate gene for additional human disorders ranging from irritable bowel syndrome to obesity. Replicated studies have demonstrated that the SERT 5'-flanking region polymorphism SS genotype is associated with poorer therapeutic responses and more frequent serious side effects during treatment with antidepressant SERT antagonists, namely, the serotonin reuptake inhibitors (SRIs).
Glutamate transporters are integral membrane proteins that catalyse the concentrative uptake of glutamate from the synapse to intracellular spaces by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function, and are implicated in stroke, epilepsy and neurodegenerative diseases. Here we present the crystal structure of a eukaryotic glutamate transporter homologue from Pyrococcus horikoshii. The transporter is a bowl-shaped trimer with a solvent-filled extracellular basin extending halfway across the membrane bilayer. At the bottom of the basin are three independent binding sites, each cradled by two helical hairpins, reaching from opposite sides of the membrane. We propose that transport of glutamate is achieved by movements of the hairpins that allow alternating access to either side of the membrane.
SLC5 is an ancient gene family with 11 members in the human genome. These membrane proteins have diverse, multiple functions ranging from actively transporting solutes, ions, and water, to channeling water and urea, to sensing glucose in cholinergic neurons. Metabolic disorders have been identified that are associated with congenital mutations in two of the human genes.
Methods developed originally to analyze domain motions from simulation [Proteins 27:425–437, 1997] are adapted and extended for the analysis of X-ray conformers and for proteins with more than two domains. The method can be applied as an automatic procedure to any case where more than one conformation is available. The basis of the methodology is that domains can be recognized from the difference in the parameters governing their quasi-rigid body motion, and in particular their rotation vectors. A clustering algorithm is used to determine clusters of rotation vectors corresponding to main-chain segments that form possible dynamic domains. Domains are accepted for further analysis on the basis of a ratio of interdomain to intradomain fluctuation, and Chasles' theorem is used to determine interdomain screw axes. Finally residues involved in the interdomain motion are identified. The methodology is tested on citrate synthase and the M6I mutant of T4 lysozyme. In both cases new aspects to their conformational change are revealed, as are individual residues intimately involved in their dynamics. For citrate synthase the beta sheet is identified to be part of the hinging mechanism. In the case of T4 lysozyme, one of the four transitions in the pathway from the closed to the open conformation, furnished four dynamic domains rather than the expected two. This result indicates that the number of dynamic domains a protein possesses may not be a constant of the motion. Proteins 30:144–154, 1998. © 1998 Wiley-Liss, Inc.
The Facilitation of Diffusion by Catalytic CarriersTranslocation Catalysis through Lipoprotein MembranesThe General Mechanisms of Translocation CatalysisSecondary TranslocationPrimary TranslocationThe Difference between Primary and Secondary Translocation and the “Driving” of Transport by Metabolism
The gate type non-carrier mechanism, an active transport model, is discussed. In this mechanism, the actively transported
particle passes through the gate itself by means of a series of reorganizations of the active transport mechanism. The net
rate of transport, the rate of transport in either direction, and the efficiency of this model are analyzed. It is shown that
on the basis of these analyses alone, this mechanism cannot be distinguished from a carrier mechanism.
Three generalizations which apply to many individual type active transport models are then discussed. These pertain to (1)
the dependency of the flow in one direction on the cencentration of the particles on the opposite side of the membrane, (2)
the possibility of very high efficiencies for these models independent of the rate of the active transport, and (3) the methods
whereby the energy expended in the active transport may be experimentally found.
The relationships between currents generated by the rabbit Na+/glucose cotransporter (SGLT1) and the fluxes of Na+ and sugar were investigated using Xenopus laevis oocytes expressing SGLT1. In individual voltage-clamped oocytes we measured: (i) the current evoked by 10 mM alpha MG and the 22Na+ uptake at 10 mM Na+; (ii) the currents evoked by 50 to 500 microM [14C]alpha MG and the [14C]alpha MG uptakes at 100 mM Na+; and (iii) phlorizin-sensitive leak currents in the absence of sugar and 22Na+ uptakes at 10 mM Na+. We demonstrate that the SGLT1 leak currents are Na+ currents, and that the sugar-evoked currents are directly proportional to both alpha MG and Na+ uptakes. The Na+/alpha MG coupling coefficients were estimated to be 1.6 at -70 mV and 1.9 at -110 mV. This suggests that the rabbit SGLT1 Na+/alpha MG stoichiometry for sugar uptake is 2 under fully saturating, zero-trans conditions. Coupling coefficients of less than 2 are expected under nonsaturating conditions due to uncoupled Na+ fluxes (slippage). The similarity between the Na+ Hill coefficients and the coupling coefficients suggests strong cooperativity between the two Na+ binding sites.
NADH-ubiquinone oxidoreductase (Complex I) is located at the entrance of the mitochondrial electron transfer chain and transfers electrons from NADH to ubiquinone with 10 isoprene units (Q(10)) coupled with proton pumping. The composition of Complex I, the largest and most complex proton pump in the mitochondrial electron transfer system, especially the contents of Q(10) and phospholipids, has not been well established. An improved purification method including solubilization of mitochondrial membrane with deoxycholate followed by sucrose gradient centrifugation and anion-exchange column chromatography provided reproducibly a heme-free preparation containing 1 Q(10), 70 phosphorus atoms of phospholipids, 1 zinc ion, 1 FMN, 30 inorganic sulfur ions, and 30 iron atoms as the intrinsic constituents. The rotenone-sensitive enzymatic activity of the Complex I preparation was comparable to that of Complex I in the mitochondrial membrane. It has been proposed that Complex I has two Q(10) binding sites, one involved in the proton pump and the other functioning as a converter between one and two electron transfer pathways [Ohnishi, T., Johnson, J. J. E., Yano, T., LoBrutto, R., and Widger, R. W. (2005) FEBS Lett. 579, 500-506]. The existence of one molecule of Q(10) in the fully oxidized Complex I suggests that the affinity of Q(10) to one of the two Q(10) sites is greatly dependent on the oxidation state and/or the membrane potential and that the Q(10) in the present preparation functions as the converter of the electron transfer pathways which should be present in any oxidation state.
Although several antidepressants (including fluoxetine, imipramine, citalopram, venlafaxine, and duloxetine) are known to inhibit the serotonin transporter (SERT), whether or not these molecules compete with 5-hydroxytryptamine (serotonin) (5-HT) for binding to SERT has remained controversial. We have performed radioligand competition binding experiments and found that all data can be fitted via a simple competitive interaction model, using Cheng-Prusoff analysis (Biochem Pharmacol 22:3099-3108, 1973). Two different SERT-selective radioligands, [(3)H]N,N-dimethyl-2-(2-amino-4-cyanophenyl thio)-benzylamine (DASB) and [(3)H]S-citalopram, were used to probe competitive binding to recombinantly expressed human SERT or native SERT in rat cortical membranes. All the SERT inhibitors that we tested were able to inhibit [(3)H]DASB and [(3)H]S-citalopram binding in a concentration-dependent manner, with unity Hill coefficient. In accordance with the Cheng-Prusoff relationship for a competitive interaction, we observed that test compound concentrations associated with 50% maximal inhibition of radiotracer binding (IC(50)) increased linearly with increasing radioligand concentration for all ligands: 5-HT, S-citalopram, R-citalopram, paroxetine, clomipramine, fluvoxamine, imipramine venlafaxine, duloxetine, indatraline, cocaine, and 2-beta-carboxy-3-beta-(4-iodophenyl)tropane. The equilibrium dissociation constant of 5-HT and SERT inhibitors were also derived using Scatchard analysis of the data set, and they were found to be comparable with the data obtained using the Cheng-Prusoff relationship. Our studies establish a reference framework that will contribute to ongoing efforts to understand ligand binding modes at SERT by demonstrating that 5-HT and the SERT inhibitors tested bind to the serotonin transporter in a competitive manner.
Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) play an important role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a high-resolution structure for a bacterial homolog of Na(+)/Cl(-)-dependent neurotransmitter transporters, LeuT, offers a unique opportunity to analyze the functional roles of the multi-ion binding sites within the binding pocket. The binding pocket of LeuT contains two metal binding sites. The first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in the neighboring site (NA2) only approximately 7 A away. Extensive, fully atomistic, molecular dynamics, and free energy simulations of LeuT in an explicit lipid bilayer are performed to evaluate substrate-binding affinity as a function of the ion load (single versus double occupancy) and occupancy by specific monovalent cations. It was shown that double ion occupancy of the binding pocket is required to ensure substrate coupling to Na(+) and not to Li(+) or K(+) cations. Furthermore, it was found that presence of the ion in site NA2 is required for structural stability of the binding pocket as well as amplified selectivity for Na(+) in the case of double ion occupancy.
Permeability coefficients for amino acid classes, including neutral, polar, hydrophobic, and charged species, were measured and compared with values for other ionic solutes such as phosphate. The rates of efflux of glycine, lysine, phenylalanine, serine and tryptophan were determined after they were passively entrapped in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (EPC) or dimyristoylphosphatidylcholine (DMPC). The following permeability coefficients were obtained for: glycine, 5.7 x 10(-12) cm s-1 (EPC), 2.0 x 10(-11) cm s-1 (DMPC); serine, 5.5 x 10(-12) cm s-1 (EPC), 1.6 x 10(-11) cm s-1 (DMPC); lysine, 5.1 x 10(-12) cm s-1 (EPC), 1.9 x 10(-11) cm s-1 (DMPC); tryptophan, 4.1 x 10(-10) cm s-1 (EPC); and phenylalanine, 2.5 x 10(-10) cm s-1 (EPC). Decreasing lipid chain length increased permeability slightly, while variations in pH had only minor effects on the permeability coefficients of the amino acids tested. Phosphate permeability was in the range of 10(-12)-10(-13) cm s-1 depending on the pH of the medium. The values for the polar and charged amino acids were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium, which are in the range of 10(-12)-10(-13) cm s-1, depending on conditions and the lipid species used. This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. The results are relevant to the permeation of certain peptides into lipid bilayers during protein translocation and membrane biogenesis.
Transport of gamma-aminobutyric acid (GABA) is electrogenic and completely depends on the presence of both sodium and chloride ions. These ions appear to be cotransported with gamma-aminobutyric acid through its transporter [reviewed in Kanner, B. I. (1983) Biochim. Biophys. Acta 726, 293-316]. Using proteoliposomes into which a partially purified gamma-aminobutyric acid transporter preparation was reconstituted, we have been able--for the first time--to provide direct evidence for sodium- and chloride-coupled gamma-aminobutyric acid transport. This has been done by measuring the fluxes of 22Na+, 36Cl-, and [3H]GABA. These fluxes have the following characteristics: There are components of the net fluxes of sodium and chloride that are gamma-aminobutyric acid dependent. The sodium flux is chloride dependent; i.e., when Cl- is replaced by inorganic phosphate or by SO4(2-), gamma-aminobutyric acid dependent sodium fluxes are abolished. The chloride flux is sodium dependent; i.e., when Na+ is replaced by Tris+ or by Li+, gamma-aminobutyric acid dependent chloride fluxes are abolished. Thus, the gamma-aminobutyric acid dependent sodium and chloride fluxes appear to be catalyzed by the transporter. Using these fluxes we have attempted to determine the stoichiometry of the process. We measured the initial rate of sodium-dependent gamma-aminobutyric acid fluxes and that of gamma-aminobutyric acid dependent sodium fluxes. This yields the stoichiometry between sodium and gamma-aminobutyric acid (2.58 +/- 0.99). Similarly, we measured the stoichiometry between chloride and gamma-aminobutyric acid, which is found to be 1.27 +/- 0.12.(ABSTRACT TRUNCATED AT 250 WORDS)
Two models for mediated transport are described. The “allosteric” model involves transition of a combining site between two alternate shapes. The “H bond” model involves transition of a combining site between two alternate hydrogen bonded states. In both cases, the alternate forms have different directional properties. These models are kinetically equivalent to a mobile carrier model.
THE list of plausible hypothetical schemes which have been advanced to account for active transport is so long that any hope of extending it might appear unwarranted. However, very few specific molecular mechanisms have been considered. It therefore appears timely to point out that transport contingent on a chemical reaction can be understood in terms of a very simple molecular model, for which a noteworthy precedent can be found among recent crystal structure determinations.
Methods developed originally to analyze domain motions from simulation [Proteins 27:425-437, 1997] are adapted and extended for the analysis of X-ray conformers and for proteins with more than two domains. The method can be applied as an automatic procedure to any case where more than one conformation is available. The basis of the methodology is that domains can be recognized from the difference in the parameters governing their quasi-rigid body motion, and in particular their rotation vectors. A clustering algorithm is used to determine clusters of rotation vectors corresponding to main-chain segments that form possible dynamic domains. Domains are accepted for further analysis on the basis of a ratio of interdomain to intradomain fluctuation, and Chasles' theorem is used to determine interdomain screw axes. Finally residues involved in the interdomain motion are identified. The methodology is tested on citrate synthase and the M6I mutant of T4 lysozyme. In both cases new aspects to their conformational change are revealed, as are individual residues intimately involved in their dynamics. For citrate synthase the beta sheet is identified to be part of the hinging mechanism. In the case of T4 lysozyme, one of the four transitions in the pathway from the closed to the open conformation, furnished four dynamic domains rather than the expected two. This result indicates that the number of dynamic domains a protein possesses may not be a constant of the motion.
In the central nervous system, (Na+ + K+)-coupled glutamate transporters restrict the neurotoxicity of this transmitter and limit the duration of synaptic excitation at some synapses. The various isotransporters exhibit a particularly high homology in an extended hydrophobic domain of ill-defined topology that contains several determinants involved in ion and transmitter binding. Here, we describe the determination of the membrane topology of the cloned astroglial glutamate transporter GLT-1. A series of functional transporters containing single cysteines was engineered. Their topological disposition was determined by using a biotinylated sulfhydryl reagent. The glutamate transporter has eight transmembrane domains long enough to span the membrane as et heiices. Strikingly, between the seventh and eighth domains, a structure reminiscent of a pore loop and an outward-facing hydrophobic linker are positioned.
To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.
A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional assignments of newly sequenced transport proteins that will result from future genome sequencing projects.
Sodium-glucose cotransporters (SGLTs) mediate active transport of sugar across cell membranes coupled to Na+, by using the electrochemical gradient as a driving force. In the kidney, there is evidence for two kinds of cotransporters, a high-affinity, low-capacity system, and a low-affinity, high-capacity system, with differences in substrate specificity and kinetics. Three renal SGLT clones have been identified: SGLT1 corresponding to the high-affinity system, and SGLT2 and SGLT3 with properties reminiscent of the low-affinity system. We have determined the stoichiometry of pig SGLT3 (pSGLT3) by using a direct method, comparing the substrate-induced inward charge to 22Na or [14C]alpha-methyl-D-glucopyranoside uptake in the same oocyte. pSGLT3 stoichiometry is 2 Na+:1 sugar, the same as that for SGLT1, but different from SGLT2 (1:1). The Na+ Hill coefficient for SGLT3 is approximately 1.5, suggesting low cooperativity between Na+ binding sites. Thus SGLT3 has functional characteristics intermediate between SGLT1 and SGLT2, so, whereas SGLT3 stoichiometry is the same as that for SGLT1 (2:1), sugar affinity and specificity are similar to SGLT2.
Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation.
The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude.
Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7×12Å. Important hydrogen bond interactions occur at five positions bordering this surface.
In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site ∼8Å from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1).
Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors.
British Journal of Pharmacology (2001) 134, 484–495; doi:10.1038/sj.bjp.0704274
The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 A, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with alpha-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.
The Transporter Classification (TC) system is a functional/phylogenetic system designed for the classification of all transmembrane transport proteins found in living organisms on Earth. It parallels but differs from the strictly functional EC system developed decades ago by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB) for the classification of enzymes. Recently, the TC system has been adopted by the IUBMB as the internationally acclaimed system for the classification of transporters. Here we present the characteristics of the nearly 400 families of transport systems included in the TC system and provide statistical analyses of these families and their constituent proteins. Specifically, we analyze the transporter types for size and topological differences and analyze the families for the numbers and organismal sources of their constituent members. We show that channels and carriers exhibit distinctive structural and topological features. Bacterial-specific families outnumber eukaryotic-specific families about 2 to 1, while ubiquitous families, found in all three domains of life, are about half as numerous as eukaryotic-specific families. The results argue against appreciable horizontal transfer of genes encoding transporters between the three domains of life over the last 2 billion years.
The SLC6 family is a diverse set of transporters that mediate solute translocation across cell plasma membranes by coupling solute transport to the cotransport of sodium and chloride down their electrochemical gradients. These transporters probably have 12 transmembrane domains, with cytoplasmic N- and C-terminal tails, and at least some may function as homo-oligomers. Family members include the transporters for the inhibitory neurotransmitters GABA and glycine, the aminergic transmitters norepinephrine, serotonin, and dopamine, the osmolytes betaine and taurine, the amino acid proline, and the metabolic compound creatine. In addition, this family includes a system B(0+) cationic and neutral amino acid transporter, and two transporters for which the solutes are unknown. In general, SLC6 transporters act to regulate the level of extracellular solute concentrations. In the central and the peripheral nervous system, these transporters can regulate signaling among neurons, are the sites of action of various drugs of abuse, and naturally occurring mutations in several of these proteins are associated with a variety of neurological disorders. For example, transgenic animals lacking specific aminergic transporters show profoundly disturbed behavioral phenotypes and probably represent excellent systems for investigating psychiatric disease. SLC6 transporters are also found in many non-neural tissues, including kidney, intestine, and testis, consistent with their diverse physiological roles. Transporters in this family represent attractive therapeutic targets because they are subject to multiple forms of regulation by many different signaling cascades, and because a number of pharmacological agents have been identified that act specifically on these proteins.
Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (co-transport) consistent with both the structure and a large body of experimental data.
The Human Genome Organisation (HUGO) Nomenclature Committee Database provides a list of transporter families of the solute carrier (SLC) gene series (see http://www.gene.ucl.ac.uk/nomenclature/). Currently, it includes 43 families and 298 transporter genes. This special issue features mini-reviews on each of these SLC families written by the experts in each field. A WEB site has been established (http://www.pharmaconference.org/slctable.asp) that gives the latest updates for the SLC families and their members as well as relevant links to gene databases and reviews in the literature. A list of all currently known SLC families, a discussion of additional SLC families and family members as well as a brief summary of non-SLC transporter genes is included in this introduction.