Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2

Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates—in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1—prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with K_D values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (k_a) in the range of 10³ M^-1s^-1 and an even slower dissociation rate (k_d) in the range of 10⁻⁴ s^-1 for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1—which is normally expressed in liver and other tissues—interacts with prostaglandin analogs. While it is not entirely clear from the published literature whether OCTs function as major prostaglandin transporters, the tight binding of the naturally occurring PGE2, as well as the slow dissociation rate, could conceivably affect the transport of lower affinity substrates such as drugs and metabolites by SLC22 transporters. More research is necessary to establish the extent to which individual SLC22 family members actually function as PG transporters in vitro and in vivo and to investigate whether PGs can, independent of being directly transported, alter the ability of SLC22 transporters to handle drugs and other substrates.     

The large extracellular loop of organic cation transporter 1 influences substrate affinity and is pivotal for oligomerization

Polyspecific organic anion transporters (OATs) and organic cation transporters (OCTs) of the SLC22 transporter family play a pivotal role in absorption, distribution, and excretion of drugs. Polymorphisms in these transporters influence therapeutic effects. On the basis of functional characterizations, homology modeling, and mutagenesis, hypotheses for how OCTs bind and translocate structurally different cations were raised, assuming functionally competent monomers. However, homo-oligomerization has been described for OATs and OCTs. In the present study, evidence is provided that the large extracellular loops (EL) of rat Oct1 (rOct1) and rat Oat1 (rOat1) mediate homo- but not hetero-oligomerization. Replacement of the cysteine residues in the EL of rOct1 by serine residues (rOct1(6ΔC-l)) or breaking disulfide bonds with dithiothreitol prevented oligomerization. rOct1 chimera containing the EL of rOat1 (rOct1(rOat1-l)) showed oligomerization but reduced transporter amount in the plasma membrane. For rOct1(6ΔC-l) and rOct1(rOat1-l), similar K(m) values for 1-methyl-4-phenylpyridinium(+) (MPP(+)) and tetraethylammonium(+) (TEA(+)) were obtained that were higher compared with rOct1 wild type. The increased K(m) of rOct1(rOat1-l) indicates an allosteric effect of EL on the cation binding region. The similar substrate affinity of the oligomerizing and non-oligomerizing loop mutants suggests that oligomerization does not influence transport function. Independent transport function of rOct1 monomers was also demonstrated by showing that K(m) values for MPP(+) and TEA(+) were not changed after treatment with dithiothreitol and that a tandem protein with two rOct1 monomers showed about 50% activity with unchanged K(m) values for MPP(+) and TEA(+) when one monomer was blocked. The data help to understand how OCTs work and how mutations in patients may affect their functions.     

Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3

Organic cation transporter 3 (OCT3, SLC22A3) is a polyspecific, facilitative transporter expressed in astrocytes and in placental, intestinal, and blood-brain barrier epithelia, and thus elucidating the molecular mechanisms underlying OCT3 substrate recognition is critical for the rational design of drugs targeting these tissues. The pharmacology of OCT3 is distinct from that of other OCTs, and here we investigated the role of a hydrophobic cavity tucked within the translocation pathway in OCT3 transport properties. Replacement of an absolutely conserved Asp by charge reversal (D478E), neutralization (D478N), or even exchange (D478E) abolished MPP⁺ uptake, demonstrating this residue to be obligatory for OCT3-mediated transport. Mutations at non-conserved residues lining the putative binding pocket of OCT3 to the corresponding residue in OCT1 (L166F, F450L, and E451Q) reduced the rate of MPP⁺ transport, but recapitulated the higher sensitivity pharmacological profile of OCT1. Thus, interactions of natural polyamines (putrescine, spermidine, spermine) and polyamine-like potent OCT1 blockers (1,10-diaminodecane, decamethonium, bistriethylaminodecane, and 1,10-bisquinuclidinedecane) with wild-type OCT3 were weak, but were significantly potentiated in the mutant OCT3s. Conversely, a reciprocal mutation in OCT1 (F161L) shifted the polyamine-sensitivity phenotype toward that of OCT3. Further analysis indicated that OCT1 and OCT3 can recognize essentially the same substrates, but the strength of substrate-transporter interactions is weaker in OCT3, as informed by the distinct makeup of the hydrophobic cleft. The residues identified here are key contributors to both the observed differences between OCT3 and OCT1 and to the mechanisms of substrate recognition by OCTs in general.     

Inhibitory Effects of Green Tea and (–)-Epigallocatechin Gallate on Transport by OATP1B1, OATP1B3, OCT1, OCT2, MATE1, MATE2-K and P-Glycoprotein

Green tea catechins inhibit the function of organic anion transporting polypeptides (OATPs) that mediate the uptake of a diverse group of drugs and endogenous compounds into cells. The present study was aimed at investigating the effect of green tea and its most abundant catechin epigallocatechin gallate (EGCG) on the transport activity of several drug transporters expressed in enterocytes, hepatocytes and renal proximal tubular cells such as OATPs, organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and P-glycoprotein (P-gp). Uptake of the typical substrates metformin for OCTs and MATEs and bromosulphophthalein (BSP) and atorvastatin for OATPs was measured in the absence and presence of a commercially available green tea and EGCG. Transcellular transport of digoxin, a typical substrate of P-gp, was measured over 4 hours in the absence and presence of green tea or EGCG in Caco-2 cell monolayers. OCT1-, OCT2-, MATE1- and MATE2-K-mediated metformin uptake was significantly reduced in the presence of green tea and EGCG (P < 0.05). BSP net uptake by OATP1B1 and OATP1B3 was inhibited by green tea [IC₅₀ 2.6% (v/v) and 0.39% (v/v), respectively]. Green tea also inhibited OATP1B1- and OATP1B3-mediated atorvastatin net uptake with IC₅₀ values of 1.9% (v/v) and 1.0% (v/v), respectively. Basolateral to apical transport of digoxin was significantly decreased in the presence of green tea and EGCG. These findings indicate that green tea and EGCG inhibit multiple drug transporters in vitro. Further studies are necessary to investigate the effects of green tea on prototoypical substrates of these transporters in humans, in particular on substrates of hepatic uptake transporters (e.g. statins) as well as on P-glycoprotein substrates.     

Molecular mechanisms of organic cation transport in OCT2-expressing Xenopus oocytes

The molecular mechanisms of organic cation transport by rat OCT2 was examined in the Xenopus oocyte expression system. When extracellular Na+ ions were replaced with K+ ions, uptake of tetraethylammonium (TEA) by OCT2-expressing oocytes was decreased, suggesting that TEA uptake by OCT2 is dependent on membrane potential. Kinetic analysis revealed that the decreased TEA uptake by ion substitution was caused at least in part by decreased substrate affinity. Acidification of extracellular buffer resulted in decreased uptake of TEA, whereas TEA efflux from OCT1- and OCT2-expressing oocytes was not stimulated by inward proton gradient, in consistent with basolateral organic cation transport in the kidney. Inhibition of TEA uptake by various organic cations revealed that apparent substrate spectrum of OCT2 was similar with that of OCT1. However, the affinity of procainamide to OCT1 was higher than that to OCT2. Uptake of 1-methyl-4-phenylpyridinium was stimulated by OCT2 as well as OCT1, but uptake of levofloxacin, a zwitterion, was not stimulated by both OCTs. These results suggest that OCT2 is a multispecific organic cation transporter with the characteristics comparable to those of the basolateral organic cation transporter in the kidney.     

Unbiased simulations reveal the inward-facing conformation of the human serotonin transporter and Na(+) ion release

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 μs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na(+)-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport.     

Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs)

Metformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT) 1, 2, and 3 (encoded by SLC22A1, SLC22A2, or SLC22A3), which are tissue specifically expressed at significant levels in various organs such as liver, muscle, and kidney. Because metformin does not undergo hepatic metabolism, drug-drug interaction by inhibition of OCT transporters may be important. So far, comprehensive data on the interaction of proton pump inhibitors (PPIs) with OCTs are missing although PPIs are frequently used in metformin-treated patients. Using in silico modeling and computational analyses, we derived pharmacophore models indicating that PPIs (i.e. omeprazole, pantoprazole, lansoprazole, rabeprazole, and tenatoprazole) are potent OCT inhibitors. We then established stably transfected cell lines expressing the human uptake transporters OCT1, OCT2, or OCT3 and tested whether these PPIs inhibit OCT-mediated metformin uptake in vitro. All tested PPIs significantly inhibited metformin uptake by OCT1, OCT2, and OCT3 in a concentration-dependent manner. Half-maximal inhibitory concentration values (IC(50)) were in the low micromolar range (3-36 μM) and thereby in the range of IC(50) values of other potent OCT drug inhibitors. Finally, we tested whether the PPIs are also transported by OCTs, but did not identify PPIs as OCT substrates. In conclusion, PPIs are potent inhibitors of the OCT-mediated metformin transport in vitro. Further studies are needed to elucidate the clinical relevance of this drug-drug interaction with potential consequences on metformin disposition and/or efficacy.     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

Two amino acid residues determine the low substrate affinity of human cationic amino acid transporter-2A

Mammalian cationic amino acid transporters (CAT) differ in their substrate affinity and sensitivity to trans-stimulation. The apparent Km values for cationic amino acids and the sensitivity to trans-stimulation of CAT-1, -2B, and -3 are characteristic of system y+. In contrast, CAT-2A exhibits a 10-fold lower substrate affinity and is largely independent of substrate at the trans-side of the membrane. CAT-2A and -2B demonstrate such divergent transport properties, even though their amino acid sequences differ only in a stretch of 42 amino acids. Here, we identify two amino acid residues within this 42-amino acid domain of the human CAT-2A protein that are responsible for the apparent low affinity of both the extracellular and intracellular substrate-binding sites. These residues are located in the fourth intracellular loop, suggesting that they are not part of the translocation pathway. Rather, they may be responsible for the low affinity conformation of the substrate-binding sites. The sensitivity to trans-stimulation is not determined by the same amino acid residues as the substrate affinity and must involve a more complex interaction between individual amino acid residues. In addition to the 42-amino acid domain, the adjacent transmembrane domain X seems to be involved in this function.     

Docking and homology modeling explain inhibition of the human vesicular glutamate transporters

As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.     

Structural insights into ABC transporter mechanism

ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.     

Molecular and kinetic characterization of histamine transport into adult rat cultured astrocytes

Astrocytes have a key role in the clearance and inactivation of histamine in the adult central nervous system, but transporters which mediate histamine uptake into astrocytes have not been fully characterized. We therefore investigated the kinetic and molecular characteristics of histamine uptake into cultured adult rat astrocytes. [(3)H]-histamine was taken up by astrocytes in a temperature-, time- and concentration-dependent manner and was inhibited up to 60-70% by 1mM ouabain or by substitution of NaCl with choline chloride. Specific [(3)H]-histamine uptake, determined as the difference between transport at 37 and 4°C, displayed saturation kinetics with the apparent Michaelis-Menten constant (K(m)) of 141 and 101μM and the apparent maximal uptake rate (V(max)) of 22.5 and 17.8pmol/min/mg protein, as estimated from the Woolf and the Eadie-Hofstee plots, respectively. Since our data suggested the presence of a carrier-operated histamine uptake system, we assessed the possible involvement of the organic cation transporters (OCT) 1, 2 and 3, which have been previously described to play a role in histamine transport in the central nervous system. Low level mRNA expression of all OCT isoforms was detected, but in contrast to rat brain cortex homogenate, where OCT3 was the most prominently expressed OCT isoform, OCT2 mRNA was the predominant OCT species in cultured astrocytes. However, OCT inhibitors corticosterone and decynium 22 (D22) had no effect or only modestly reduced [(3)H]-histamine uptake. Thus, our data indicate that adult rat astrocytes possess an efficient high-capacity, low-affinity carrier-operated histamine uptake system, which does not seem to involve OCTs.     

Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3

Organic anion transporters (OATs) and organic cation transporters (OCTs) mediate the flux of xenobiotics across the plasma membranes of epithelia. Substrates of OATs generally carry negative charge(s) whereas substrates of OCTs are cations. The goal of this study was to determine the domains and amino acid residues essential for recognition and transport of organic anions by the rat organic anion transporter, rOAT3. An rOAT3/rOCT1 chimera containing transmembrane domains 1-5 of rOAT3 and 6-12 of rOCT1 retained the specificity of rOCT1, suggesting that residues involved in substrate recognition reside within the carboxyl-terminal half of these transporters. Mutagenesis of a conserved basic amino acid residue, arginine 454 to aspartic acid (R454D), revealed that this amino acid is required for organic anion transport. The uptakes of p-aminohippurate (PAH), estrone sulfate, and ochratoxin A were approximately 10-, approximately 48-, and approximately 32-fold enhanced in oocytes expressing rOAT3 and were only approximately 2-, approximately 6-, and approximately 5-fold enhanced for R454D. Similarly, mutagenesis of the conserved lysine 370 to alanine (K370A) suggested that K370 is important for organic anion transport. Interestingly, the charge specificity of the double mutant, R454DK370A, was reversed in comparison to rOAT3-R454DK370A preferentially transported the organic cation, MPP(+), in comparison to PAH (MPP(+) uptake/PAH uptake = 3.21 for the double mutant vs 0.037 for rOAT3). These data indicate that arginine 454 and lysine 370 are essential for the anion specificity of rOAT3. The studies provide the first insights into the molecular determinants that are critical for recognition and translocation of organic anions by a member of the organic anion transporter family.     

Properties of an Inward-Facing State of LeuT: Conformational Stability and?Substrate Release

The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters, which are important pharmaceutical targets. There are no high-resolution structures of the human transporters available; however, LeuT has been crystallized in several different conformational states. Recently, an inward-facing conformation of LeuT was solved revealing an unexpectedly large movement of transmembrane helix 1a (TM1a). We have performed molecular dynamics simulations of the mutated and wild-type transporter, with and without the cocrystallized Fab antibody fragment, to investigate the properties of this inward-facing conformation in relation to transport by LeuT within the membrane environment. In all of the simulations, local conformational changes with respect to the crystal structure are consistently observed, especially in TM1a. Umbrella sampling revealed a soft potential for TM1a tilting. Furthermore, simulations of inward-facing LeuT with Na⁺ ions and substrate bound suggest that one of the Na⁺ ion binding sites is fully disrupted. Release of alanine and the second Na⁺ ion is also observed, giving insight into the final stage of the translocation process in atomistic detail.     

Putative drug binding conformations of monoamine transporters

Structural information about monoamine transporters and their interactions with psychotropic drugs is important for understanding their molecular mechanisms of action and for drug development. The crystal structure of a Major Facilitator Superfamily (MFS) transporter, the lactose permease symporter (lac permease), has provided insight into the three-dimensional structure and mechanisms of secondary transporters. Based on the hypothesis that the 12 transmembrane alpha-helix (TMH) secondary transporters belong to a common folding class, the lac permease structure was used for molecular modeling of the serotonin transporter (SERT), the dopamine transporter (DAT), and the noradrenaline transporter (NET). The molecular modeling methods used included amino acid sequence alignment, homology modeling, and molecular mechanical energy calculations. The lac permease crystal structure has an inward-facing conformation, and construction of outward-facing SERT, DAT, and NET conformations allowing ligand binding was the most challenging step of the modeling procedure. The psychomotor stimulants cocaine and S-amphetamine, and the selective serotonin reuptake inhibitor (SSRI) S-citalopram, were docked into putative binding sites on the transporters to examine their molecular binding mechanisms. In the inward-facing conformation of SERT the translocation pore was closed towards the extracellular side by hydrophobic interactions between the conserved amino acids Phe105, Pro106, Phe117, and Ala372. An unconserved amino acid, Asp499 in TMH10 in NET, may contribute to the low affinity of S-citalopram to NET.     

Sulfenamide derivatives can improve transporter-mediated cellular uptake of metformin and induce cytotoxicity in human breast adenocarcinoma cell lines

Metformin, the most frequently administered oral anti-diabetic drug, is a substrate for organic cation transporters (OCTs). This determines not only its pharmacokinetic properties but also its biochemical effects in humans, including its recently-discovered antiproliferative properties. The aim of the study was to verify the hypothesis whether chemical modification of its biguanide backbone may increase the cellular uptake and antiproliferative efficacy of metformin.The study examines five sulfenamide derivatives of metformin with differing lengths of alkyl chains. It determines their cellular uptake and the role of OCTs in their transport in human breast adenocarcinoma cells (epithelial-like MCF-7, and MDA-MB-231). It also evaluates whether increased cellular uptake of metformin derivatives is associated with their cytotoxic properties.Sulfenamide derivatives were characterized by a greater ability to bind to OCTs than metformin. Compound 2 with n-octyl alkyl chain was found to possess the greatest affinity towards OCTs, as measured by determination of [¹⁴C]choline uptake inhibition (IC₅₀ = 236.1 ± 1.28 μmol/L, and 217.4 ± 1.33 μmol/L, for MCF-7 and MDA-MB-231 respectively). Sulfenamides were also found to exhibit better cellular uptake in comparison with the parent drug, metformin. For instance, the uptake of cyclohexyl derivative 1 was 1.28 ± 0.19 nmol/min/mg of proteins and thus was 12-fold higher than the metformin in MCF-7 cells. Furthermore, higher uptake was associated with the greatest antiproliferative properties expressed as the lowest IC₅₀ value i.e. inhibiting the growth of 50% of the cells (IC₅₀ = 0.72 ± 1.31 μmol/L).Collectively, chemical modification of metformin into sulfenamides with different alkyl substituents obtains better substrates for OCTs, and subsequently higher cellular uptake in MCF-7 and MDA-MB-231 cells. Additionally, the length of alkyl chain introduced to the sulfenamides was found to influence selectivity and transport efficiency via OCT1 compared to other possible transporters, as well as potential intracellular activity and cytotoxicity.     

A new chemotype inhibitor for the human organic cation transporter 3 (hOCT3)

Human organic cation transporters (OCTs) represent an understudied neurotransmitter uptake mechanism for which no selective agents have yet been identified. Several neurotransmitters (e.g. serotonin, norepinephrine) are low-affinity substrates for these transporters, but possess higher affinity for other transporters (e.g. the serotonin or norepinephrine transporters; SERT and NET, respectively). We have identified a new class of OCT inhibitors with a phenylguanidine structural scaffold. Here, we examine the actions of a series of such compounds and report preliminary structure–activity relationships (SARs) – the first dedicated SAR study of OCT3 action. Initial results showed that the presence of a substituent on the phenyl ring, as well as its position, contributes to the phenylguanidines’ inhibitory potency (IC₅₀ values ranging from 2.2 to >450 μM) at hOCT3. There is a trend towards enhanced inhibitory potency of phenylguanidines with increased lipophilic character and the size of the substituent at the phenyl 4-position, with the latter reaching a ceiling effect. The first PiPT-based hOCT3 homology models were generated and are in agreement with our biological data.     

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

Dynamic interplay of membrane‐proximal POTRA domain and conserved loop L6 in Omp85 transporter FhaC

Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16‐stranded transmembrane β barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner β‐barrel wall. The membrane‐proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the β barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle.