In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [ 125 I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/ I172M had a synergistic impact on (RS)-CIT recognition (ϳ10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/ I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT. SERT,2 like several other members of the SLC6A gene family, acts to remove neurotransmitter from the synapse following neurotransmission (1) and is a major target for treatment of mood disorders, including major depression, anxiety, post-traumatic stress, and obsessive-compulsive disorders (2). Agents that target SERT include tricyclic antidepressants and serotonin-specific reuptake inhibitors (SSRIs) that block 5HT binding and uptake. Despite its clinical significance, very little is understood concerning the structural aspects of SERT and how they relate to its function and antagonist recognition. The current data predict that SERT proteins are composed of 12 transmembrane-spanning segments, intracellular NH 2 and COOH termini and a large second extracellular...

The SLC6 family of secondary active transporters are integral membrane solute carrier proteins characterized by the Na+-dependent translocation of small amino acid or amino acid-like substrates. SLC6 transporters, which include the serotonin, dopamine, norepinephrine, GABA, taurine, creatine, as well as amino acid transporters, are associated with a number of human diseases and disorders making this family a critical target for therapeutic development. In addition, several members of this family are directly involved in the action of drugs of abuse such as cocaine, amphetamines, and ecstasy. Recent advances providing structural insight into this family have vastly accelerated our ability to study these proteins and their involvement in complex biological processes.

Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission, including ADHD, bipolar disorder, and now ASD. These findings provide valuable insight into a new cellular phenotype (altered hDAT trafficking) supporting dysregulated DA function in these disorders. They also provide a novel potential target (PKCβ) for therapeutic interventions in individuals with ASD.

To explore aqueous accessibility and functional contributions of transmembrane domain (TM) 1 in human serotonin transporter (hSERT) proteins, we utilized the largely methanethiosulfonate (MTS) insensitive hSERT C109A mutant and mutated individual residues of hSERT TM1 to Cys followed by tests of MTS inactivation of 5-hydroxytryptamine (5-HT) transport. Residues in TM1 cytoplasmic to Gly-94 were largely unaffected by Cys substitution, whereas the mutation of residues extracellular to Ile-93 variably diminished transport activity. TM1 Cys substitutions displayed differential sensitivity to MTS reagents, with residues more cytoplasmic to Asp-98 being largely insensitive to MTS inactivation. Aminoethylmethanethiosulfonate (MTSEA), [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET), and sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) similarly and profoundly inactivated 5-HT transport by SERT mutants D98C, G100C, W103C, and Y107C. MTSEA uniquely inactivated transport activity of S91C, G94C, Y95C but increased activity at I108C. MTSEA and MTSET, but not MTSES, inactivated transport function at N101C. Notably, 5-HT provided partial to complete protection from MTSET inactivation for D98C, G100C, N101C, and Y107C. Equivalent blockade of MTSET inactivation at N101C was observed with 5-HT at both room temperature and at 4°C, inconsistent with major conformational changes leading to protection. Notably, cocaine also protected MTSET inactivation of G100C and N101C, although MTS incubations with N101C that eliminate 5-HT transport do not preclude cocaine analog binding nor its inhibition by 5-HT. 5-HT modestly enhanced the inactivation by MTSET at I93C and Y95C, whereas cocaine significantly enhanced MTSET sensitivity at Y107C and I108C. In summary, our studies reveal physical differences in TM1 accessibility to externally applied MTS reagents and reveal sites supporting substrate and antagonist modulation of MTS inactivation. Moreover, we identify a limit to accessibility for membrane-impermeant MTS reagents that may reflect aspects of an occluded permeation pathway.Clearance of extracellular 5-HT 1 by SERT proteins constitutes the major pathway for inactivation of the neurotransmitter in the brain and periphery (1). SERT proteins are also targets for psychoactive agents, including 3,4-methylenedioxymethamphetamine ("ecstasy"), cocaine, and antidepressants (2, 3). Although SERT cDNAs and genes have been identified in multiple species (2, 4 -11), fundamental aspects of the mechanism of 5-HT transport remain ill-defined. SERTs are members of a family of transport proteins (in humans designated SLC6A, SERT is SLC6A4) that support high affinity Na ϩ -and Cl Ϫ -dependent uptake of amino acids and amino acid derivatives, and thus, it is likely that members of the family utilize common structural elements to catalyze transport. The 12 putative transmembrane domains (TMs) of SERT (4) are likely to contribute to a permeation pathway through the lipid bilayer, facilitating the coordinated movement of 5-HT and cou...

We have analyzed the role of chitin, a cellwall polysaccharide, in the virulence of Candida albicans. Mutants with a 5-fold reduction in chitin were obtained in two ways: (i) by selecting mutants resistant to Calcofluor, a fluorescent dye that binds to chitin and inhibits growth, and(ii) by disrupting CHS3, the C. albicans homolog of CSD2/ CALJ/DITIOI/KT12, a Saccharomyces cereviswe gene required for synthesis of '90% of the cell-wall chitin. A key step in the development of an antifungal drug is the determination of its efficacy. For practical reasons, much of the initial testing is done in culture (in vitro), where the test conditions differ markedly from the conditions encountered by a fungal pathogen during infection (in vivo). The limited ability of in vitro testing to accurately predict in vivo efficacy is illustrated by the azoles, which are highly effective in vivo despite modest fungicidal activity in vitro (1). Thus, it is possible that some fungal components are required for pathogenicity in vivo but not for growth in vitro. Possible virulence determinants include factors required for recognition and invasion of the host and for protection against host defense systems. Because the cell wall is involved in these processesthe wall protects the fungal cell from external injury and cell-wall components mediate adherence (2-4) and immune response (5-8)-changes in cell-wall structure and/or composition may affect virulence.We have begun to investigate the role of the cell-wall polysaccharide chitin in the virulence of Candida albicans. Chitin is found in all true fungi; therefore agents that inhibit chitin synthesis are potential broad-spectrum antifungal drugs. In the past decade, many of the genes involved in chitin synthesis have been isolated in Saccharomyces cerevisiae, and, more recently, the homologs of these genes have been identified in C. albicans (9-11).In S. cerevisiae, three chitin synthases have been detected (for review, see refs. 12 and 13). One of them, chitin synthase III, makes 90% of the cell-wall chitin. At least three genes, CSD2/CALl/DIT101/KT12, CSD4/CAL2, and CAL3, are required for this activity. Mutants lacking this enzyme are chitin-deficient and, consequently, are resistant to Calcofluor, a fluorescent dye that binds to chitin and inhibits growth by disrupting microfibril assembly (14, 15).Like S. cerevisiae, C. albicans has at least three chitin synthases (9-11). Recently, the C. albicans homolog of CSD2, designated CHS3, was cloned and sequenced (11). By analogy to S. cerevisiae, C. albicans CHS3 mutants should be chitindeficient and Calcofluor-resistant. In the present report, we show that chitin-deficient mutants of C. albicans obtained by two methods are significantly less virulent than the wild-type strain. . Media were supplemented with uridine at 0.1 mg/ml as required. Agar (2%, Difco) was added for solid media. Urdauxotrophs were selected on medium containing 5-fluoroorotic acid (5-FOA) as described (16). Germ-tube formation was induced in 20% fetal bovine serum ...