Autism is a spectrum of neurodevelopmental disorders with a primarily genetic etiology exhibiting deficits in (1) development of language and (2) social relationships and (3) patterns of repetitive, restricted behaviors or interests and resistance to change. Elevated platelet serotonin (5-HT) in 20%-25% of cases and efficacy of selective 5-HT reuptake inhibitors (SSRIs) in treating anxiety, depression, and repetitive behaviors points to the 5-HT transporter (5-HTT; SERT) as a strong candidate gene. Association studies involving the functional insertion/deletion polymorphism in the promoter (5-HTTLPR) and a polymorphism in intron 2 are inconclusive, possibly because of phenotypic heterogeneity. Nonetheless, mounting evidence for genetic linkage of autism to the chromosome 17q11.2 region that harbors the SERT locus (SLC6A4) supports a genetic effect at or near this gene. We confirm recent reports of sex-biased genetic effects in 17q by showing highly significant linkage driven by families with only affected males. Association with common alleles fails to explain observed linkage; therefore, we hypothesized that preferential transmission of multiple alleles does explain it. From 120 families, most contributing to linkage at 17q11.2, we found four coding substitutions at highly conserved positions and 15 other variants in 5' noncoding and other intronic regions transmitted in families exhibiting increased rigid-compulsive behaviors. In the aggregate, these variants show significant linkage to and association with autism. Our data provide strong support for a collection of multiple, often rare, alleles at SLC6A4 as imposing risk of autism.
Attention-Deficit Hyperactivity Disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at pre- and post-synaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a human DAT (hDAT) coding variant, R615C, located in the transporter’s distal C-terminus, a region previously implicated in constitutive and regulated transporter trafficking. Here we demonstrate that whereas wildtype DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively, and demonstrate insensitivity to the endocytic effects of AMPH and protein kinase C (PKC) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered calcium/calmodulin-dependent protein kinase II (CaMKII) and flotillin-1 interactions. Using C-terminal peptides derived from wildtype DAT and the R615C variant, we establish that the DAT 615C C-terminus can act dominantly to preclude AMPH regulation of wildtype DAT. Mutagenesis of DAT C-terminal sequences suggest that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.
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...
Background: Reuptake of synaptic norepinephrine (NE) via the antidepressant-sensitive NE transporter (NET) supports efficient noradrenergic signaling and presynaptic NE homeostasis. Limited, and somewhat contradictory, information currently describes the axonal transport and localization of NET in neurons.
The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies. Loss of function mutations in the gene encoding the Caenhorhabditis elegans DAT (dat-1) produces rapid immobility when animals are placed in water, a phenotype termed Swimming-induced paralysis (Swip). The ability of a DA neuron-expressed, GFP-tagged DAT-1 fusion protein (GFP::DAT-1) to localize to synapses and rescue Swip in these animals provides a facile approach to define sequences supporting DAT somatic export and function in vivo. In prior studies, we found that truncation of the last 25 amino acids of the DAT-1 C-terminus (Δ25) precludes Swip rescue, supported by a deficit in GFP::DAT-1 synaptic localization. Here, we further defined the elements within Δ25 required for DAT-1 export and function in vivo. We identified two conserved motifs (584KW585 and 591PYRKR595) where mutation results in a failure of GFP::DAT-1 to be efficiently exported to synapses and restore DAT-1 function. The 584KW585 motif conforms to a sequence proposed to support SEC24 binding, ER export from the endoplasmic reticulum (ER), and surface expression of mammalian DAT proteins, whereas the 591PYRKR595 sequence conforms to a 3R motif identified as a SEC24 binding site in vertebrate G-protein coupled receptors. Consistent with a potential role of SEC24 orthologs in DAT-1 export, we demonstrated DA neuron-specific expression of a sec-24.2 transcriptional reporter. Mutations of the orthologous C-terminal sequences in human DAT (hDAT) significantly reduced transporter surface expression and DA uptake, despite normal hDAT protein expression. Although, hDAT mutants retained SEC24 interactions, as defined in co-immunoprecipitation studies. However, these mutations disrupted the ability of SEC24D to enhance hDAT surface expression. Our studies document an essential role of conserved DAT C-terminal sequences in transporter somatic export and synaptic localization in vivo, that add further support for important roles for SEC24 family members in efficient transporter trafficking.
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