Mutations in the carboxyl-terminal SEC24 binding motif of the serotonin transporter impair folding of the transporter

The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG(601,602), RII(607-609)) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI(607,608) is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG(601,602) and RI(607,608) was impaired folding, because mutated transporters failed to bind the inhibitor [(3)H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [(3)H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI(607,608)AA and SERT-RII(607-609)AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters.     

A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter

The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people. Here we examined the contribution of the C terminus in supporting folding of the serotonin transporter (SERT; SLC6A4). Our working hypothesis posited that the amphipathic nature of the C-terminal α-helix (Thr⁶⁰³–Thr⁶¹³) was important for folding of SERT. Accordingly, we disrupted the hydrophobic moment of the α-helix by replacing Phe⁶⁰⁴, Ile⁶⁰⁸, or Ile⁶¹² by Gln. The bulk of the resulting mutants SERT-F604Q, SERT-I608Q, and SERT-I612Q were retained in the endoplasmic reticulum, but their residual delivery to the cell surface still depended on SEC24C. This indicates that the amphipathic nature of the C-terminal α-helix was dispensable to endoplasmic reticulum export. The folding trajectory of SERT is thought to proceed through the inward facing conformation. Consistent with this conjecture, cell surface expression of the misfolded mutants was restored by (i) introducing second site suppressor mutations, which trap SERT in the inward facing state, or (ii) by the pharmacochaperone noribogaine, which binds to the inward facing conformation. Finally, mutation of Glu⁶¹⁵ at the end of the C-terminal α-helix to Lys reduced surface expression of SERT-E615K. A charge reversal mutation in intracellular loop 1 restored surface expression of SERT-R152E/E615K to wild type levels. These observations support a mechanistic model where the C-terminal amphipathic helix is stabilized by an intramolecular salt bridge between residues Glu⁶¹⁵ and Arg¹⁵². This interaction acts as a pivot in the conformational search associated with folding of SERT.     

Synthesis and monoamine transporter affinity of new 2beta-carbomethoxy-3beta-[aryl or heteroaryl]phenyltropanes

A series of 16 new 2beta-carbomethoxy-3beta-[aryl or heteroaryl]phenyltropane derivatives was synthesized and evaluated for binding to monoamine transporters. Most of the compounds exhibited nanomolar affinity for the serotonin transporter (SERT). Four compounds presented a particularly attractive pharmacological profile, with very high SERT affinity (K(i) 0.15-0.5 nM) and selectivity versus the dopamine transporter of 25- to 77-fold.     

A Conserved Salt Bridge between Transmembrane Segments 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter

Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the “thin gate” formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [³H]CFT. Also, the coordination of Zn²⁺ between introduced histidines (R85H/D476H) caused a ∼2.5-fold increase in [³H]CFT binding (B_max). Importantly, Zn²⁺ also inhibited [³H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [³H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.     

Characterization of an allosteric citalopram-binding site at the serotonin transporter

The serotonin transporter (SERT), which belongs to a family of sodium/chloride-dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. In the present study we show that the dissociation rate, of [3H]S-citalopram from human SERT, is retarded by the presence of serotonin, as well as by several antidepressants, when present in the dissociation buffer. Dissociation of [3H]S-citalopram from SERT is most potently inhibited by S-citalopram followed by R-citalopram, sertraline, serotonin and paroxetine. EC50 values for S- and R-citalopram are 3.6 +/- 0.4 microm and 19.4 +/- 2.3 microm, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [3H]S-citalopram. Allosteric modulation of dissociation is independent of temperature, or the presence of Na+ in the dissociation buffer. Dissociation of [3H]S-citalopram from a complex with the SERT double-mutant, N208Q/N217Q, which has been suggested to be unable to self-assemble into oligomeric complexes, is retarded to an extent similar to that found with the wild-type, raising the possibility that the allosteric mechanism is mediated within a single subunit. A species-scanning mutagenesis study comparing human and bovine SERT revealed that Met180, Tyr495 and Ser513 are important residues in mediating the allosteric effect, as well as contributing to high-affinity binding at the primary site.     

The role of zinc ions in reverse transport mediated by monoamine transporters

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.     

Interaction of the C-terminal region of the rat serotonin transporter with MacMARCKS modulates 5-HT uptake regulation by protein kinase C

The serotonin transporter (SERT) mediates the re-uptake of released serotonin into presynaptic nerve terminals. Its activity is regulated by different mechanisms including protein kinase C (PKC) triggered internalization. Here, we used yeast 2-hybrid screening and cotransfection into 293 cells to identify a homologue of the myristoylated alanine-rich C kinase substrate (MARCKS), MacMARCKS, as a C-terminally interacting protein of SERT. Upon cotransfection with SERT, MacMARCKS caused a reduction in the maximal rate of [(3)H]serotonin uptake and reduced its down-regulation elicited by activation of PKC. Our data are consistent with MARCKS proteins regulating the plasma membrane dynamics of neurotransmitter transporters.     

Cysteine-scanning mutagenesis of serotonin transporter intracellular loop 2 suggests an alpha-helical conformation

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.     

alpha-Latrotoxin affects mitochondrial potential and synaptic vesicle proton gradient of nerve terminals

Ca(2+)-independent [(3)H]GABA release induced by alpha-latrotoxin was found to consist of two sequential processes: a fast initial release realized via exocytosis and more delayed outflow through the plasma membrane GABA transporters [Linetska, M.V., Storchak, L.G., Tarasenko, A.S., Himmelreich, N.H., 2004. Involvement of membrane GABA transporters in alpha-latrotoxin-stimulated [(3)H]GABA release. Neurochem. Int. 44, 303-312]. To characterize the toxin-stimulated events attributable to the transporter-mediated [(3)H]GABA release from rat brain synaptosomes we studied the effect of alpha-latrotoxin on membrane potentials and generation of the synaptic vesicles proton gradient, using fluorescent dyes: potential-sensitive rhodamine 6G and pH-sensitive acridine orange. We revealed that alpha-latrotoxin induced a progressive dose-dependent depolarization of mitochondrial membrane potential and an irreversible run-down of the synaptic vesicle proton gradient. Both processes were insensitive to the presence of cadmium, a potent blocker of toxin-formed transmembrane pores, indicating that alpha-latrotoxin-induced disturbance of the plasma membrane permeability was not responsible to these effects. A gradual dissipation of the synaptic vesicle proton gradient closely coupled with lowering the vesicular GABA transporter activity results in a leakage of the neurotransmitter from synaptic vesicles to cytoplasm. As a consequence, there is an essential increase in GABA concentration in a soluble cytosolic pool that appears to be critical parameter for altering the mode of the plasma membrane GABA transporter operation from inward to outward. Thus, our data allow clarifying what cell processes underlain a recruitment of the plasma membrane transporter-mediated pathway in alpha-LTX-stimulated secretion.     

The design and synthesis of a potent Angiotensin II cyclic analogue confirms the ring cluster receptor conformation of the hormone Angiotensin II

The novel amide linked Angiotensin II potent cyclic analogue, c-[Sar1,Lys3,Glu5] ANG II 19 has been designed and synthesized in an attempt to test the aromatic ring clustering and the charge relay bioactive conformation we have recently suggested for ANG II. This constrained cyclic analogue was synthesized by connecting the Lys3 amino and Glu5 carboxyl side chain groups, and it was found to be potent in the rat uterus assay and in anesthetized rabbits. The central part of the molecule is fixed covalently in the conformation predicted according to the backbone bend conformational model proposed for Angiotensin II. The obtained results using a combination of 2D NMR, 1D NOE spectroscopy and molecular modeling revealed a similar Tyr4-Ile5-His6 bend, a His6-Pro7 trans configuration and a side chain aromatic ring cluster of the key aminoacids Tyr4, His6, Phe8 for c-[Sar1,Lys3,Glu5] ANG II as it has been found for ANG II (Matsoukas, J. H.; Hondrelis, J.; Keramida, M.; Mavromoustakos, T.; Markriyannis, A.; Yamdagni, R.; Wu, Q.; Moore, G. J. J. Biol. Chem. 1994, 269, 5303). Previous study of the conformational properties of the Angiotensin II type I antagonist [Hser(gamma-OMe)8] ANG II (Matsoukas, J. M.; Agelis, G.; Wahhab, A.; Hondrelis, J.; Panagiotopoulos. D.; Yamdagni, R.; Wu, Q.; Mavromoustakos, T.; Maia, H.; Ganter, R.; Moore, G. J. J. Med. Chem. 1995, 38, 4660) using 1-D NOE spectroscopy coupled with the present study of the same type of lead antagonist Sarilesin revealed that the Tyr4-Ile5-His6 bend, a conformational property found in Angiotensin II is not present in type I antagonists. The obtained results provide an important conformational difference between Angiotensin II agonists and type I antagonists. It appears that our synthetic attempt to further support our proposed model was successful and points out that the charge relay system and aromatic ring cluster are essential stereoelectronic features for Angiotensin II to exert its biological activity.     

Conformationally Sensitive Proximity of Extracellular Loops 2 and 4 of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Inferred from Paired Cysteine Mutagenesis

The sodium- and chloride-coupled GABA transporter GAT-1 is a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. Structural work on the bacterial homologue LeuT suggests that extracellular loop 4 closes the extracellular solvent pathway when the transporter becomes inward-facing. To test whether this model can be extrapolated to GAT-1, cysteine residues were introduced at positions 359 and 448 of extracellular loop 4 and transmembrane helix 10, respectively. Treatment of HeLa cells, expressing the double cysteine mutant S359C/K448C with the oxidizing reagent copper(II)(1,10-phenantroline)₃, resulted in a significant inhibition of [³H]GABA transport. However, transport by the single cysteine mutant S359C was also inhibited by the oxidant, whereas its activity was almost 4-fold stimulated by dithiothreitol. Both effects were attenuated when the conserved cysteine residues, Cys-164 and/or Cys-173, were replaced by serine. These cysteines are located in extracellular loop 2, the role of which in the structure and function of the eukaryotic neurotransmitter:sodium:symporters remains unknown. The inhibition of transport of S359C by the oxidant was markedly reduced under conditions expected to increase the proportion of inward-facing transporters, whereas the reactivity of the mutants to a membrane-impermeant sulfhydryl reagent was not conformationally sensitive. Our data suggest that extracellular loops 2 and 4 come into close proximity to each other in the outward-facing conformation of GAT-1.     

Synthesis and monoamine transporter affinity of front bridged tricyclic 3beta-(4'-halo or 4'-methyl)phenyltropanes bearing methylene or carbomethoxymethylene on the bridge to the 2beta-position

A series of front bridged tricyclic 3beta-(4'-halo or 4'-methyl)phenyltropanes bearing methylene or carbomethoxymethylene on the bridge to the 2beta-position was synthesized, and their binding affinities were determined in cells transfected to express human norepinephrine transporter (NET), serotonin transporter (SERT), and dopamine transporter (DAT) via competition binding assays. All compounds studied in this series exhibit a moderate to high potency at all three transporters with SERT or DAT selectivity. 3beta-(4'-iodo)phenyltropane bearing methylene on the bridge to the 2beta-position (24) presents a particularly attractive pharmacological profile, with very high SERT affinity (K(i) = 0.09 nM) and selectivity versus NET (65-fold) and DAT (94-fold).     

The Transport Systems for Selenomethionine/Methionine and Selenocystine/Cystine in Escherichia Coli K-12 Appear to be Cooperative

Dopamine transporters of bovine and rat striata were identified by their specific [³H]cocaine binding and cocaine-sensitive [³H]dopamine ([³H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes

Inorganic and organic mercury reagents inhibited both [³H]DA uptake and [³H]cocaine binding, though they were all more potent inhibitors of the former, n- Ethylmaleimide inhibited [³H]DA uptake totally but [³H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [³H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [³H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (< 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (>10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation     

A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin

Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT.     

Synthesis and monoamine transporter binding properties of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes

A series of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes were prepared and evaluated for affinities at dopamine, serotonin, and norepinephrine transporters using competitive radioligand binding assays. The 2beta-[3'-(substituted benzyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (3a-h) showed high binding affinities for the dopamine transporter (DAT). The IC(50) values ranged from 5.9 to 22nM. On the other hand, the 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (4a-h), with IC(50) values ranging from 65 to 173nM, were approximately 3- to 25-fold less potent than the corresponding 2beta-[3'-(substituted benzyl)isoxazol]tropanes. All tested compounds were selective for the DAT relative to the norepinephrine transporter (NET) and serotonin transporter (5-HTT). 3Beta-(4-Methylphenyl)-2beta-[3'-(4-fluorobenzyl)isoxazol-5-yl]tropane (3b) with IC(50) of 5.9nM at the DAT and K(i)s of 454 and 113nM at the NET and 5-HTT, respectively, was the most potent and DAT-selective analog. Molecular modeling studies suggested that the rigid conformation of the isoxazole side chain in 4a-h might play an important role on their low DAT binding affinities.     

Substrate specificity and ion coupling in the Na+/betaine symporter BetP

BetP is an Na(+)-coupled betaine-specific transporter of the betaine-choline-carnitine (BCC) transporter family involved in the response to hyperosmotic stress. The crystal structure of BetP revealed an overall fold of two inverted structurally related repeats (LeuT-fold) that BetP shares with other sequence-unrelated Na(+)-coupled symporters. Numerous structures of LeuT-fold transporters in distinct conformational states have contributed substantially to our understanding of the alternating access mechanism of transport. Nevertheless, coupling of substrate and co-transported ion fluxes has not been structurally corroborated to the same extent. We converted BetP by a single-point mutation--glycine to aspartate--into an H(+)-coupled choline-specific transporter and solved the crystal structure of this mutant in complex with choline. The structure of BetP-G153D demonstrates a new inward-facing open conformation for BetP. Choline binding to a location close to the second, low-affinity sodium-binding site (Na2) of LeuT-fold transporters is facilitated by the introduced aspartate. Our data confirm the importance of a cation-binding site in BetP, playing a key role in a proposed molecular mechanism of Na(+) and H(+) coupling in BCC transporters.     

Norepinephrine transporter (NET) knock-out upregulates dopamine and serotonin transporters in the mouse brain

The noradrenaline, serotonin and dopamine transporters are three main transporters, which are the target of the antidepressant drugs. In the present study we demonstrate that the life-long deletion of the noradrenaline transporter (NET) induced up-regulation of two other monoamine transporters, dopamine and serotonin (DAT and SERT, respectively). An increase in the binding of [³H]paroxetine to the SERT and [³H]GBR12935 to the DAT was observed in various brain regions of NET-KO mice, without alterations of mRNA encoding these transporters, as measured by in situ hybridization. This important finding impacts the interpretation of previous data indicating the supersensitizity of NET-KO mice for psychostimulants or stronger effect of citalopram in behavioral tests. While using the NET-KO mice in various psychopharmacological studies is very important, one has to be aware that these mice lack NET from the earliest period of their existence, thus compensatory alterations do take place and have to be considered when it comes to interpretation of the obtained results.     

The SLC6/NSS family members KAAT1 and CAATCH1 have weak chloride dependence

KAAT1 and CAATCH1 are amino acid transporters cloned from the intestine of the lepidoptera Manduca sexta.1,2 They are members of the SLC6/NSS family, which groups membrane proteins that use Na+, K+, and Cl- gradients for the coupled transport of amines and amino acids. The report of the atomic-resolution x-ray crystal structure of the eubacterium Aquifex aeolicus leucine transporter (AaLeuT)3 has contributed significantly to understanding of the structure–function relationship in NSS proteins. Transport by AaLeuT is Cl- independent, whereas many neurotransmitter:sodium symporters like serotonin transporter (SERT), GABA transporter (GAT1), dopamine transporter, and norephinephrine transporter, among others, are strongly Cl- dependent.4 A single Cl- ion is found bound to one of the extracellular loops, EL2 in AaLeuT. The Cl- is 20 Ã… away from the Na and leucine binding sites, and thus it is unclear whether this Cl- binding site is physiologically important. The nature of the association of Cl- ions with these proteins during transport remains to be resolved. The Cl- binding site of two members of the family, the serotonin transporter SERT 4 and the GABA transporter GAT1 5, has been recently modelled on the basis of their functional properties and by structural homology to AaLeuT. The analyses have highlighted the role of a serine residue, that in the Cl--independent AaLeuT corresponds to Glu 290, and of an asparagine (Asn 286) that also contributes to the coordination of Na+ in the Na1 binding site of AaLeuT. KAAT1 and CAATCH1 are able to transport different amino acids depending on the contransported cation (Na+ or K+) but their Cl- dependence is not completely defined yet. With the aim to clarify the role exerted by chloride in SLC6/NSS transporters, the Cl--dependence of KAAT1 and CAATCH1 have been investigated by the expression in Xenopus laevis oocytes and the measurement of induced amino acid uptakes. Despite KAAT1 and CAATCH1 posses the same residue of serine (Ser342, KAAT1 numbering) present in strictly chloride dependent transporters, their transport activities resulted weakly Cl--dependent compared to GAT1. By analysis of the pH dependence of the KAAT1 and CAATCH1 transport activity, we obtained more information to define their (particular) peculiar Cl- dependence.     

Putative cocaine receptor in striatum is a glycoprotein with active thiol function

Dopamine transporters of bovine and rat striata were identified by their specific [3H]cocaine binding and cocaine-sensitive [3H]dopamine [( 3H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes. Inorganic and organic mercury reagents inhibited both [3H]DA uptake and [3H]cocaine binding, though they were all more potent inhibitors of the former. n-Ethylmaleimide inhibited [3H]DA uptake totally but [3H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [3H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [3H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (less than 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (greater than 10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation.     

Synthesis and monoamine transporter affinity of new 2beta-carbomethoxy-3beta-[4-(substituted thiophenyl)]phenyltropanes: discovery of a selective SERT antagonist with picomolar potency

Preparation of cocaine analogues has been aimed largely at development of stable compounds with high affinity and selectivity for the dopamine transporter (DAT). We now report the synthesis and monoamine transporter affinity of 10 new 2beta-carbomethoxy-3beta-[4-(substituted thiophenyl)]phenyltropanes. Among these, compound 4b exhibited very high affinity for the serotonin transporter (SERT: K(i)=17 pM) and good selectivity over dopamine (DAT: 710-fold) and norepinephrine transporters (NET: 11,100-fold).