Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na⁺ and Cl^?-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na⁺-binding sites and a putative Cl^?-binding site. Mutation of all residues in the two proposed Na⁺-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na⁺-binding site 1) and mutants S20A, S20G, S20G/G405S (Na⁺-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na⁺-binding site 1 caused a decrease of the Na⁺-K_0.5, whereas mutations of site 2 increased the K_0.5. Mutation of Na⁺-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates ³⁶Cl^? demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na⁺/1Cl^?-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.     

Insights on Na+ binding and conformational dynamics in multidrug and toxic compound extrusion transporter NorM

MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+‐substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild‐type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na⁺ ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di‐Na⁺ binding state is likely more efficient for conformational changes of NorM_VC toward the inward‐facing conformation than single‐Na⁺ binding state. The observation of two Na⁺ binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na⁺ binding and enrich our knowledge of the transport mechanism of secondary transporters. Proteins 2014; 82:240–249. © 2013 Wiley Periodicals, Inc.     

A conserved asparagine residue in transmembrane segment 1 (TM1) of serotonin transporter dictates chloride-coupled neurotransmitter transport

Na(+)- and Cl(-)-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl(-) coordination of human serotonin transport have been identified, the role of Cl(-) in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl(-) binding to concentrative neurotransmitter uptake.     

A Conserved Na+ Binding Site of the Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2)

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na⁺ into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT_Aa and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na⁺ binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na⁺ binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na⁺ binding to SNAT2, but also dramatically lowers the Na⁺ affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na⁺ binding and which is also expected to form part of the Na⁺ binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na⁺ affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.The sodium-coupled neutral amino acid transporter, SNAT2,² belongs to the SLC38 gene family of solute carrier proteins (1). Together with SNAT1 and -4 (2), it is believed to mediate Na⁺-dependent amino acid transport activity that was classically assigned to System A transporters (3–8). In addition to SNAT1 and -2, the SLC38 family has four other known members, two of which predominantly mediate glutamine transport (SNAT3 and -5, System N (9–11)). SNAT2 is widely expressed in mammalian tissue (1, 7), but it may play a particularly critical role in the brain (12), where it may help shuttle glutamine from astrocytes to neurons via the glutamate-glutamine cycle (1). This process is essential for recycling the neurotransmitter glutamate (13). However, the exact contribution of SNAT2 to the glutamate-glutamine cycle is still controversially discussed (14).Despite this physiological importance, surprisingly little is known about the functional properties and the structural basis of amino acid transport by the SLC38 proteins. Although hydropathy analysis predicts 11 transmembrane helices (TMs), with an intracellular N terminus and an extracellular C terminus (1), it is not clear whether the transporters belong to a large superfamily of transporters, of which members have been characterized structurally through x-ray crystallography. At present, sequence homology has only been established with transporters of the mammalian SLC32 and SLC36 families as well as with the more distantly related plant auxin carriers and the bacterial amino acid-polyamine-organocation (APC) family (15, 16). High resolution crystal structures are not available for any of the transporters from these families, although low resolution projection structures were recently reported for the APC family members AdiC (17) and SteT (18). However, these structures do not allow the assignment of transmembrane helices. Thus, it remains unknown whether the SLC38 fold is similar to established transport protein folds, although homology to the major facilitator superfamily seems unlikely.We have recently identified a conserved amino acid residue in SNAT2, Asn-82, which is involved in controlling the Na⁺ affinity of the transporter (19). Interestingly, Asn-82 is localized in the predicted TM1 of SNAT2. This first transmembrane helix was recently found to contribute ligands to a Na⁺ binding site in several bacterial transporters, which are related to the SLC5 (sodium glucose symporter) and SLC6 (sodium- and chloride-dependent neurotransmitter transporter) family members (20–22), which also comprises bacterial members (23, 24). Although sequence similarity with SLC5 and -6 is not detectable, SLC38 may be a member of a possibly very large superfamily with the same general fold, which also contains many amino acid transport proteins.Here, we used a homology modeling approach based on profile-based sequence alignment (25, 26). A search against sequences deposited in the Protein Data Bank (PDB (27)) revealed that the transporters with the highest likelihood to share an analogous fold are a leucine transporter from Aquifex aeolicus, LeuT_Aa, and a homologous hydantoin transporter from Microbacterium liquefaciens, Mhp1. We established a homology model based on these structures, which predicts Asn-82 to be part of a Na⁺ binding site. Furthermore, another conserved hydrophilic amino acid residue in TM8, Thr-384, was predicted to be near this cation binding site. When Thr-384 was mutated to alanine, a dramatic loss of the affinity of SNAT2 for Na⁺ was observed, whereas mutations to other sites that were spatially removed from the predicted Na⁺ binding site had little or no effect on Na⁺ affinity. We hypothesize that the SLC38 family is a member of a large superfamily of cation/organic substrate transporters which includes the mammalian SLC5 and -6 proteins and which has a conserved cation binding site formed by TMs 1 and 8.     

Identification of the First Sodium Binding Site of the Phosphate Cotransporter NaPi-IIa (SLC34A1)

Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (P_i) in renal and intestinal epithelia. The transport cycle requires three Na⁺ ions and one divalent P_i to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na⁺ ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na⁺ ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li⁺ ions substitute for Na⁺ at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and P_i based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and ³²P uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li⁺ ions to substitute for Na⁺ ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.     

Mutation of Asparagine 76 in the Center of Glutamine Transporter SNAT3 Modulates Substrate-induced Conductances and Na+ Binding

The glutamine transporter SLC38A3 (SNAT3) plays an important role in the release of glutamine from brain astrocytes and the uptake of glutamine into hepatocytes. It is related to the vesicular GABA (γ-aminobutyric acid) transporter and the SLC36 family of proton-amino acid cotransporters. The transporter carries out electroneutral Na⁺-glutamine cotransport-H⁺ antiport. In addition, substrate-induced uncoupled cation currents are observed. Mutation of asparagine 76 to glutamine or histidine in predicted transmembrane helix 1 abolished all substrate-induced currents. Mutation of asparagine 76 to aspartate rendered the transporter Na⁺-independent and resulted in a gain of a large substrate-induced chloride conductance in the absence of Na⁺. Thus, a single residue is critical for coupled and uncoupled ion flows in the glutamine transporter SNAT3. Homology modeling of SNAT3 along the structure of the related benzyl-hydantoin permease from Microbacterium liquefaciens reveals that Asn-76 is likely to be located in the center of the membrane close to the translocation pore and forms part of the predicted Na⁺ -binding site.The amino acid and auxin permease superfamily comprises a wide variety of transport proteins. In mammals, three distinct solute carrier families (SLC) belong to this superfamily, namely SLC32, SLC36, and SLC38 (1). Despite belonging to the same superfamily, the three solute carrier families have different transport mechanisms. The SLC32 family has only one member, the vesicular inhibitory amino acid transporter, which supposedly carries out a H⁺-GABA (γ-aminobutyric acid) antiport (2). The SLC36 family comprises four members, two of which have been characterized in more detail. These are the proton amino acid cotransporters 1 and 2 (PAT1 and 2) that carry out glycine and proline uptake in kidney and intestine and are mutated in iminoglycinuria (3, 4). The SLC38 family is comprised of 11 members, 5 of which have been characterized in more detail (5). Two different transport mechanisms are found within this family, namely the Na⁺-amino acid cotransporters SNAT1, SNAT2, and SNAT4 and the Na⁺-amino acid cotransporters-H⁺-antiporters SNAT3 and SNAT5. Transporters of the superfamily play a key role in inhibitory and excitatory neurotransmission, metabolite absorption, and liver metabolism. Despite their important roles in mammalian physiology, relatively little is known about the structure and function of these transporters.The activity of ion-coupled membrane transporters is frequently associated with currents which de- or hyperpolarize the cell membrane. These currents may be due to electrogenic transport stoichiometry and/or to a non-stoichiometric ion conductance (6). Transport-associated ion conductances have been identified in a number of transporters but have been particularly well studied in several Na⁺-coupled neurotransmitter transporters (7–11). Transport-associated conductances have also been observed in electroneutral transporters that do not carry out net charge movement (8, 12–15). The glutamine transporter SNAT3, for instance, has a transport mechanism in which glutamine uptake is coupled to the cotransport of 1Na⁺ and the antiport of 1H⁺ and, hence, is unaffected by changes of the membrane potential (13, 16). Despite the electroneutral transport mechanism, substrate uptake is accompanied by inward currents, which are carried by cations below pH 7 and by protons at alkaline pH. In addition, a substrate-independent cation conductance and a Na⁺/H⁺ exchange activity has been observed (17). Non-stoichiometric currents can be mediated by the same ions that are involved in the coupled transport process, such as in the case of SNAT3, but may also be carried by different ions. Stoichiometric glutamate transport, for instance, involves Na⁺, H⁺, and K⁺ ions, whereas the glutamate transport-associated conductance is carried by chloride (18).A crucial question concerning transporter-associated ion conductances is whether the conducting pore coincides with the translocation pathway of the substrate and whether both use the same critical residues. In the case of the glutamate transporters, evidence has been presented suggesting that different residues are critical for the anion conductance than for substrate transport (19, 20) but that they all line the same pathway (21). Here we show that asparagine 76 of SNAT3 is critical for substrate-induced ion conductance and affects binding of the cosubstrate Na⁺. In addition we show that this residue is likely to be localized in the translocation pore in the center of the membrane.     

Identification of a 3rd Na+ Binding Site of the Glycine Transporter,GlyT2

The Na⁺/Cl^- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na⁺ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na⁺ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na⁺-dependent leucine transporter, LeuT_Aa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na⁺ binding sites in GlyT2, but the physical location of the third Na⁺ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuT_Aa. Although betaine transport by BetP is coupled to the co-transport of two Na⁺ ions, the first Na⁺ site is not conserved between BetP and LeuT_Aa, the so called Na1'' site. We hypothesized that the third Na⁺ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1'' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na⁺ placed at the location consistent with the Na1'' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na⁺ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.     

Electrical coupling between the human serotonin transporter and voltage-gated Ca channels

Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca²⁺ mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca²⁺ permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca²⁺ channels (Ca_V). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in Ca_V1.1-null muscle cells, thus implicating Ca²⁺ channels. Ca_V1.3 and Ca_V2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type Ca_V1.3 channel show Ca²⁺ transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and Ca_V1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca²⁺ channels such as Ca_V1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca²⁺-driven signals in excitable cells.     

Microseconds Simulations Reveal a New Sodium-binding Site and the Mechanism of Sodium-coupled Substrate Uptake by LeuT

The bacterial sodium-coupled leucine/alanine transporter LeuT is broadly used as a model system for studying the transport mechanism of neurotransmitters because of its structural and functional homology to mammalian transporters such as serotonin, dopamine, or norepinephrine transporters, and because of the resolution of its structure in different states. Although the binding sites (S1 for substrate, and Na1 and Na2 for two co-transported sodium ions) have been resolved, we still lack a mechanistic understanding of coupled Na⁺- and substrate-binding events. We present here results from extensive (>20 μs) unbiased molecular dynamics simulations generated using the latest computing technology. Simulations show that sodium binds initially the Na1 site, but not Na2, and, consistently, sodium unbinding/escape to the extracellular (EC) region first takes place at Na2, succeeded by Na1. Na2 diffusion back to the EC medium requires prior dissociation of substrate from S1. Significantly, Na⁺ binding (and unbinding) consistently involves a transient binding to a newly discovered site, Na1″, near S1, as an intermediate state. A robust sequence of substrate uptake events coupled to sodium bindings and translocations between those sites assisted by hydration emerges from the simulations: (i) bindings of a first Na⁺ to Na1″, translocation to Na1, a second Na⁺ to vacated Na1″ and then to Na2, and substrate to S1; (ii) rotation of Phe²⁵³ aromatic group to seclude the substrate from the EC region; and (iii) concerted tilting of TM1b and TM6a toward TM3 and TM8 to close the EC vestibule.     

Mechanism of Cation Binding to the Glutamate Transporter EAAC1 Probed with Mutation of the Conserved Amino Acid Residue Thr101

The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na⁺ ions, one H⁺ ion, and one glutamate molecule into the cell, in exchange for one K⁺ ion. Na⁺ binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na⁺] dependence of this conductance as a read-out of Na⁺ binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr¹⁰¹, in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na⁺ was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1_WT, in the T101A mutant this [Na⁺] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na⁺ ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na⁺ binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr¹⁰¹ and Asp³⁶⁷. A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.     

Type 1 Sodium-dependent Phosphate Transporter (SLC17A1 Protein) Is a Cl?-dependent Urate Exporter

SLC17A1 protein (NPT1) is the first identified member of the SLC17 phosphate transporter family and mediates the transmembrane cotransport of Na⁺/P_i in oocytes. Although this protein is believed to be a renal polyspecific anion exporter, its transport properties are not well characterized. Here, we show that proteoliposomes containing purified SLC17A1 transport various organic anions such as p-aminohippuric acid and acetylsalicylic acid (aspirin) in an inside positive membrane potential (Δψ)-dependent manner. We found that NPT1 also transported urate. The uptake characteristics were similar to that of SLC17 members in its Cl⁻ dependence and inhibitor sensitivity. When arginine 138, an essential amino acid residue for members of the SLC17 family such as the vesicular glutamate transporter, was specifically mutated to alanine, the resulting mutant protein was inactive in Δψ-dependent anion transport. Heterologously expressed and purified human NPT1 carrying the single nucleotide polymorphism mutation that is associated with increased risk of gout in humans exhibited 32% lower urate transport activity compared with the wild type protein. These results strongly suggested that NPT1 is a Cl⁻-dependent polyspecific anion exporter involved in urate excretion under physiological conditions.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.     

Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter

The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5-HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12-TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high-resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn2+-binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn2+-binding sites between V102 or W103 (TMH I) and I179-L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn2+ sensitivity was assessed. Dose-response assays suggest an approximately twofold increase in sensitivity to Zn2+ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn2+ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn2+-binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.     

Structural Fold and Binding Sites of the Human Na-Phosphate Cotransporter NaPi-II

Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in P_i homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na⁺-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na⁺ and P_i coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites.     

Structural Fold and Binding Sites of the Human Na+-Phosphate Cotransporter NaPi-II

Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in P_i homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na⁺-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na⁺ and P_i coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites.     

The mitochondrial transporter of ascorbic acid functions with high affinity in the presence of low millimolar concentrations of sodium and in the absence of calcium and magnesium

We recently reported that U937 cell mitochondria express a functional Na⁺-dependent ascorbic acid (AA) transporter recognised by anti-SVCT2 antibodies. The present study confirms and extends these observations by showing that this transporter is characterised by a Km and a pH-dependence comparable with that reported for the plasma membrane SVCT2. In isolated mitochondria, Na⁺ increased AA transport rate in a cooperative manner, revealed by a sigmoid curve and a Hill coefficient of 2, as also observed in intact Raw 264.7 cells (uniquely expressing SVCT2). There was however a striking difference on the Na⁺ concentrations necessary to reach saturation, i.e., 1 or 100 mM for the mitochondrial and plasma membrane transporters, respectively. Furthermore the mitochondrial, unlike the plasma membrane, transporter was fully active also in the absence of added Ca⁺⁺ and/or Mg⁺⁺.Taken together, the results presented in this study indicate that the U937 cell mitochondrial transporter of AA, because of its very low requirement for Na⁺ and independence for Ca⁺⁺ and Mg⁺⁺, displays kinetic characteristics surprisingly similar with those of the plasma membrane SVCT2.     

Unifying concept of serotonin transporter-associated currents

Serotonin (5-HT) uptake by the human serotonin transporter (hSERT) is driven by ion gradients. The stoichiometry of transported 5-HT and ions is predicted to result in electroneutral charge movement. However, hSERT mediates a current when challenged with 5-HT. This discrepancy can be accounted for by an uncoupled ion flux. Here, we investigated the mechanistic basis of the uncoupled currents and its relation to the conformational cycle of hSERT. Our observations support the conclusion that the conducting state underlying the uncoupled ion flux is in equilibrium with an inward facing state of the transporter with K+ bound. We identified conditions associated with accumulation of the transporter in inward facing conformations. Manipulations that increased the abundance of inward facing states resulted in enhanced steady-state currents. We present a comprehensive kinetic model of the transport cycle, which recapitulates salient features of the recorded currents. This study provides a framework for exploring transporter-associated currents.     

Mechanism and Putative Structure of B0-like Neutral Amino Acid Transporters

The Na⁺-dependent transport of neutral amino acids in epithelial cells and neurons is mediated by B⁰-type neutral amino acid transporters. Two B⁰-type amino acid transporters have been identified in the neurotransmitter transporter family SLC6, namely B⁰AT1 (SLC6A19) and B⁰AT2 (SLC6A15). In contrast to other members of this family, B⁰-like transporters are chloride-independent. B⁰AT1 and B⁰AT2 preferentially bind the substrate prior to the Na⁺-ion. The Na⁺-concentration affects the K _m of the substrate and vice versa. A kinetic scheme is proposed that is consistent with the experimental data. An overlapping binding site of substrate and cosubstrate has been demonstrated in the bacterial orthologue LeuT _Aa from Aquifex aeolicus, which elegantly explains the mutual effect of substrate and cosubstrate on each other’s K _m -value. LeuT _Aa is sequence-related to transporters of the SLC6 family, allowing homology modeling of B⁰-like transporters along its structure.     

Functional significance of channels and transporters expressed in the inner ear and kidney

A number of ion channels and transporters are expressed in both the inner ear and kidney. In the inner ear, K⁺ cycling and endolymphatic K⁺, Na⁺, Ca²⁺, and pH homeostasis are critical for normal organ function. Ion channels and transporters involved in K⁺ cycling include K⁺ channels, Na⁺-2Cl^–-K⁺ cotransporter, Na⁺/K⁺-ATPase, Cl^– channels, connexins, and K⁺/Cl^– cotransporters. Furthermore, endolymphatic Na⁺ and Ca²⁺ homeostasis depends on Ca²⁺-ATPase, Ca²⁺ channels, Na⁺ channels, and a purinergic receptor channel. Endolymphatic pH homeostasis involves H⁺-ATPase and Cl^–/HCO₃^– exchangers including pendrin. Defective connexins (GJB2 and GJB6), pendrin (SLC26A4), K⁺ channels (KCNJ10, KCNQ1, KCNE1, and KCNMA1), Na⁺-2Cl^–-K⁺ cotransporter (SLC12A2), K⁺/Cl^– cotransporters (KCC3 and KCC4), Cl^– channels (BSND and CLCNKA + CLCNKB), and H⁺-ATPase (ATP6V1B1 and ATPV0A4) cause hearing loss. All these channels and transporters are also expressed in the kidney and support renal tubular transport or signaling. The hearing loss may thus be paralleled by various renal phenotypes including a subtle decrease of proximal Na⁺-coupled transport (KCNE1/KCNQ1), impaired K⁺ secretion (KCNMA1), limited HCO₃^– elimination (SLC26A4), NaCl wasting (BSND and CLCNKB), renal tubular acidosis (ATP6V1B1, ATPV0A4, and KCC4), or impaired urinary concentration (CLCNKA). Thus, defects of channels and transporters expressed in the kidney and inner ear result in simultaneous dysfunctions of these seemingly unrelated organs. cochlea; vestibular labyrinth; stria vascularis; deafness; renal tubule