Transporter Cell Surface Expression Research Articles

Choline transporter mutations in severe congenital myasthenic syndrome disrupt transporter localization.

The presynaptic, high-affinity choline transporter is a critical determinant of signalling by the neurotransmitter acetylcholine at both central and peripheral cholinergic synapses, including the neuromuscular junction. Here we describe an autosomal recessive presynaptic congenital myasthenic syndrome presenting with a broad clinical phenotype due to homozygous choline transporter missense mutations. The clinical phenotype ranges from the classical presentation of a congenital myasthenic syndrome in one patient (p.Pro210Leu), to severe neurodevelopmental delay with brain atrophy (p.Ser94Arg) and extend the clinical outcomes to a more severe spectrum with infantile lethality (p.Val112Glu). Cells transfected with mutant transporter construct revealed a virtually complete loss of transport activity that was paralleled by a reduction in transporter cell surface expression. Consistent with these findings, studies to determine the impact of gene mutations on the trafficking of the Caenorhabditis elegans choline transporter orthologue revealed deficits in transporter export to axons and nerve terminals. These findings contrast with our previous findings in autosomal dominant distal hereditary motor neuropathy of a dominant-negative frameshift mutation at the C-terminus of choline transporter that was associated with significantly reduced, but not completely abrogated choline transporter function. Together our findings define divergent neuropathological outcomes arising from different classes of choline transporter mutation with distinct disease processes and modes of inheritance. These findings underscore the essential role played by the choline transporter in sustaining acetylcholine neurotransmission at both central and neuromuscular synapses, with important implications for treatment and drug selection.

Extreme Population Differences in the Human Zinc Transporter ZIP4 (SLC39A4) Are Explained by Positive Selection in Sub-Saharan Africa

Extreme differences in allele frequency between West Africans and Eurasians were observed for a leucine-to-valine substitution (Leu372Val) in the human intestinal zinc uptake transporter, ZIP4, yet no further evidence was found for a selective sweep around the ZIP4 gene (SLC39A4). By interrogating allele frequencies in more than 100 diverse human populations and resequencing Neanderthal DNA, we confirmed the ancestral state of this locus and found a strong geographical gradient for the derived allele (Val372), with near fixation in West Africa. In extensive coalescent simulations, we show that the extreme differences in allele frequency, yet absence of a classical sweep signature, can be explained by the effect of a local recombination hotspot, together with directional selection favoring the Val372 allele in Sub-Saharan Africans. The possible functional effect of the Leu372Val substitution, together with two pathological mutations at the same codon (Leu372Pro and Leu372Arg) that cause acrodermatitis enteropathica (a disease phenotype characterized by extreme zinc deficiency), was investigated by transient overexpression of human ZIP4 protein in HeLa cells. Both acrodermatitis mutations cause absence of the ZIP4 transporter cell surface expression and nearly absent zinc uptake, while the Val372 variant displayed significantly reduced surface protein expression, reduced basal levels of intracellular zinc, and reduced zinc uptake in comparison with the Leu372 variant. We speculate that reduced zinc uptake by the ZIP4-derived Val372 isoform may act by starving certain pathogens of zinc, and hence may have been advantageous in Sub-Saharan Africa. Moreover, these functional results may indicate differences in zinc homeostasis among modern human populations with possible relevance for disease risk.

Nongenomic, glucocorticoid receptor-mediated regulation of serotonin transporter cell surface expression in embryonic stem cell derived serotonergic neurons

Depressive disorders have been linked to the combined dysregulation of the hypothalamus–pituitary–adrenal (HPA)-axis and the serotonergic system. The HPA-axis and serotonergic (5-HT) neurons exert reciprocal regulatory actions. It has been reported that glucocorticoid-glucocorticoid receptor (GR) signaling influences serotonin transporter (5-HTT) transcription but data also points to the fact that 5-HTT expression is regulated nongenomically via redistribution of 5-HTT from the cell surface into intracellular compartments. In order to analyze the acute effects of glucocorticoids on 5-HTT cell surface localization we differentiated serotonergic neurons from mouse embryonic stem (ES) cells derived from the C57BL/6N blastocysts. These postmitotic 5-HT neurons express all relevant serotonergic markers following the application of a growth factor-based differentiation protocol. Increasing concentrations of the GR agonist dexamethasone (Dex) resulted in enhanced, dose-dependent 5-HTT cell surface localization in the presence of the protein synthesis inhibitor cycloheximide already 1h after incubation. Inhibition of GR function by the specific GR-antagonist mifepristone abolished the increase in 5-HTT cell surface localization. Hence, our data account for a nongenomic upregulation of 5-HTT cell surface expression by glucocorticoid–GR interaction which likely constitutes a rapid physiological response to increased levels of glucocorticoids as seen during stress. Taken together, we provide a cellular model to analyze and dissect glucocorticoid–5HTT interactions on a molecular level that corresponds to in vivo animal models using C57BL/6N mice.

Differential regulation of serotonin transporter cell surface expression

AbstractIn the central nervous system the efficiency of serotonergic signaling is regulated by the release of serotonin (5‐hydroxytryptamine, 5‐HT), the activity of the neurotransmitter synthesizing enzyme, tryptophane hydroxylase 2, the negative‐modulatory autoreceptors 5HT1ARand 5HT1BR, and a selective re‐uptake system which transports released serotonin back into serotonergic neurons, the serotonin transporter (SERT). Because SERT directly controls the concentration of extracellular free 5‐HT, drugs as well as intracellular signaling pathways which regulate SERT activity play an important role in modulating serotonergic neurotransmission. SERT activity can be regulated by two different means: (1) regulation of SERT acute activity, that is, substrate affinity and/or maximal transport rate, or (2) by modulation the concentration of SERT molecules on the cell surface of neurons. This can be achieved by translocating SERT molecules from intracellular stores to the cell membrane or by inducing an internalization of SERT from the cell surface SERT to the cell interior (‘transporter trafficking’). In recent years evidence has accumulated that some antidepressants but also drugs of abuse such as cocaine or amphetamines not only block SERT mediated re‐uptake, but also impact on SERT cellular trafficking. In addition, several pathways have been identified, which modulate SERT cell surface expression by influencing the cellular distribution of SERT. This involves kinase/phosphatase‐activated pathways as well as intracellular proteins which upon direct interaction with the transporter promote its internalization from or export to the cell surface.WIREs Membr Transp Signal2012,1:259–268. doi: 10.1002/wmts.10For further resources related to this article, please visit theWIREs website.

Antagonists and substrates differentially regulate serotonin transporter cell surface expression in serotonergic neurons

The serotonin transporter (SERT) terminates serotonergic neurotransmission by the rapid removal of serotonin (5-hydroxytryptamine, 5-HT) from the extracellular space back into serotonergic neurons. SERT therefore controls the concentration of extracellular 5-HT, and thus one mechanism to regulate the efficacy of serotonergic neurotransmission is via modulation of the density of SERT molecules on the cell membrane. We have studied the effects of prolonged exposure to various selective serotonin re-uptake inhibitors (SSRIs), as well as cocaine and the transport substrates 5-HT and 3,4-methylenedioxy-methamphetamine (MDMA), on SERT cell surface expression in cultured serotonergic neurons. This was achieved via quantification of the amount of cell surface-expressed SERT molecules using antibody detection combined with confocal laser scanning microscopy. Our results show that exposure to the SSRIs citalopram, fluoxetine, sertraline and paroxetine all induced SERT internalization, but with different efficacies. The substrates 5-HT and MDMA also induced SERT internalization, while cocaine elevated SERT cell surface expression.

Regulation of Dopamine Transporter Function and Cell Surface Expression by D3 Dopamine Receptors

D(3) dopamine receptors are expressed by dopamine neurons and are implicated in the modulation of presynaptic dopamine neurotransmission. The mechanisms underlying this modulation remain ill defined. The dopamine transporter, which terminates dopamine transmission via reuptake of released neurotransmitter, is regulated by receptor- and second messenger-linked signaling pathways. Whether D3 receptors regulate dopamine transporter function is unknown. We addressed this issue using a fluorescent imaging technique that permits real time quantification of dopamine transporter function in living single cells. Accumulation of the fluorescent dopamine transporter substrate trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium (ASP(+)) in human embryonic kidney cells expressing human dopamine transporter was saturable and temperature-dependent. In cells co-expressing dopamine transporter and D3 receptors, the D2/D3 agonist quinpirole produced a rapid, concentration-dependent, and pertussis toxin-sensitive increase of ASP(+) uptake. Similar agonist effects were observed in Neuro2A cells and replicated in human embryonic kidney cells using a radioligand uptake assay in which binding to and activation of D3 receptors by [(3)H]dopamine was prevented. D3 receptor stimulation activated phosphoinositide 3-kinase and MAPK. Inhibition of either kinase prevented the quinpirole-induced increase in uptake. D3 receptor activation differentially affected dopamine transporter function and subcellular distribution depending on the duration of agonist exposure. Biotinylation experiments revealed that the rapid increase of uptake was associated with increased cell surface and decreased intracellular expression and increased dopamine transporter exocytosis. In contrast, prolonged agonist exposure reduced uptake and transporter cell surface expression. These results demonstrate that D3 receptors regulate dopamine transporter function and identify a novel mechanism by which D3 receptors regulate extracellular dopamine concentrations.

Direct Protein Kinase C-dependent Phosphorylation Regulates the Cell Surface Stability and Activity of the Potassium Chloride Cotransporter KCC2

The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of gamma-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser(940)) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser(940) is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser(940) increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser(940) increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition.

Rapid regulation of dopamine transporters by tyrosine kinases in rat neuronal preparations

Termination of dopamine neurotransmission is primarily controlled by the plasma membrane-localized dopamine transporter. In this study, we investigated how this transporter is regulated by tyrosine kinases in neuronal preparations. In rat dorsal striatal synaptosomes, inhibition of tyrosine kinases by genistein or tyrphostin 23 resulted in a rapid (5-15 min), concentration-dependent decrease in [(3)H]dopamine uptake because of a reduction in maximal [(3)H]dopamine uptake velocity and dopamine transporter cell surface expression. The reduced transporter activity was associated with a decrease in phosphorylated p44/p42 mitogen-activated protein kinases. In primary rat mesencephalic neuronal cultures, the tyrosine kinase inhibitors similarly reduced [(3)H]dopamine uptake. When cultures were serum-deprived, acute activation of tyrosine kinase-coupled TrkB receptors by 100 ng/mL brain-derived neurotrophic factor significantly increased [(3)H]dopamine uptake; the effects were complex with increased maximal velocity but reduced affinity. The facilitatory effect of brain-derived neurotrophic factor on dopamine transporter activity depended on both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. Taken together, our results suggest that striatal dopamine transporter function and cell surface expression is constitutively up-regulated by tyrosine kinase activation and that brain-derived neurotrophic factor can mediate this type of rapid regulation.

Electrolysis stimulates creatine transport and transporter cell surface expression in incubated mouse skeletal muscle: potential role of ROS

Electrical field stimulation of isolated, incubated rodent skeletal muscles is a frequently used model to study the effects of contractions on muscle metabolism. In this study, this model was used to investigate the effects of electrically stimulated contractions on creatine transport. Soleus and extensor digitorum longus muscles of male NMRI mice (35-50 g) were incubated in an oxygenated Krebs buffer between platinum electrodes. Muscles were exposed to [(14)C]creatine for 30 min after either 12 min of repeated tetanic isometric contractions (contractions) or electrical stimulation of only the buffer before incubation of the muscle (electrolysis). Electrolysis was also investigated in the presence of the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase (SOD) and catalase. Both contractions and (to a lesser degree) electrolysis stimulated creatine transport severalfold over basal. The amount of electrolysis, but not contractile activity, induced (determined) creatine transport stimulation. Incubation with SOD and catalase at 100 and 200 U/ml decreased electrolysis-induced creatine transport by approximately 50 and approximately 100%, respectively. The electrolysis effects on creatine uptake were completely inhibited by beta-guanidino propionic acid, a competitive inhibitor of (creatine for) the creatine transporter (CRT), and were accompanied by increased cell surface expression of CRT. Muscle glucose transport was not affected by electrolysis. The present results indicate that electrical field stimulation of incubated mouse muscles, independently of contractions per se, stimulates creatine transport by a mechanism that depends on electrolysis-induced formation of ROS in the incubation buffer. The increased creatine uptake is paralleled by an increased cell surface expression of the creatine transporter.

Erratum: A dopamine transporter mutation associated with bipolar affective disorder causes inhibition of transporter cell surface expression

Correction to: Molecular psychiatry (2005) 10, 1104–1109. doi: 10.1038/sj.mp.4001730 Two rare missense substitutions (A559V and E602G) of the human dopamine transporter (hDAT) have been identified. The characterization of these mutants in a heterologous cellular system stably expressing the respective transporter proteins was described in Molecular Psychiatry (2005) 10, 1104–1109.

A dopamine transporter mutation associated with bipolar affective disorder causes inhibition of transporter cell surface expression

The dopamine transporter (DAT) plays a crucial role in dopaminergic neurotransmission as it clears the extracellular space of dopamine (DA) and thus controls the concentration of active neurotransmitter. Genetic association studies have reported a variable number of tandem repeat polymorphisms in the 3'-noncoding region of the DAT gene implicating this protein in the development of various psychiatric disorders. In a sample of bipolar patients, two rare missense substitutions (A559V and E602G) have been identified, one of which (E602G) was inherited by the patient from her affected father. None of these mutations had been identified in any control subjects of this survey. Using a heterologous cellular expression system, we have analysed possible consequences of these mutations on functional properties of the encoded DAT protein. DA transport measurements and antagonist binding revealed that the A559V mutant protein is fully functional, whereas the E602G mutant is not. Further analyses by confocal microscopy showed that the E602G protein is transcribed and translated but not delivered to the cell surface. Taken together, our results suggest that this missense mutation has functional consequences thus supporting the need to screen larger samples of patients and their relatives for this rare but bipolar disorder-associated mutation in the DAT gene.

Phosphorylation and regulation of psychostimulant-sensitive neurotransmitter transporters.

The neuronal transporters for the monoamines dopamine, serotonin, and norepinephrine are plasma membrane proteins that serve vital functions in the reuptake and control of synaptic neurotransmitter levels. They are also targets for abused and therapeutic drugs and play pivotal roles in neurological disorders such as depression, schizophrenia, and Parkinson's disease. There is increasing evidence that some activities of these carriers are subject to acute control by treatments that affect phosphorylation pathways, but the molecular basis for this is not understood. Recent work suggests that these regulatory processes may involve phosphorylation of the transporters by protein kinase C and other kinases, and may occur by affecting intrinsic transport activity or by controlling transporter cell surface expression. Phosphorylation-mediated regulation of monoamine transporters provides the potential for acute presynaptic control of neurotransmitter levels during normal neurophysiologic events, and dysregulation of these processes may lead to inappropriate transmitter clearance that contributes to the etiology of neurological disorders.

1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP +) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP +-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP + produced a concentration- and time-dependent reduction in the uptake of [ 3H]dopamine. We observed a significant decrease in [ 3H]WIN 35428 binding in the intact cells with MPP +. The saturation analysis of the [ 3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density ( B max) with an increase in the dissociation equilibrium constant ( K d) after MPP + treatment. Furthermore, biotinylation assays confirmed that MPP + reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP + may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.

Insulin-like growth factor-1 increases activity and surface levels of the GLAST subtype of glutamate transporter

Glutamate uptake systems are the primary mechanisms involved in excitatory amino acids clearance, their regulation is extremely important for proper neuronal function. Using cultured chick cerebellar Bergmann glia cells, the involvement of receptor tyrosine kinases in glutamate uptake was studied. Treatment of the cells with insulin-like growth factor-1 but not epidermal growth factor or neuronal growth factor, induces a dose and time dependent increase in [ 3 H ]- d-aspartate uptake that is sensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Saturation experiments show a significant increase in V max, suggesting that the amount of transporter molecules at the cell membrane under insulin-like growth factor-1 treatment is augmented. This interpretation was strengthen by equilibrium-binding experiments and by the fact that the increase in [ 3 H ]- d-aspartate uptake was not dependent on protein synthesis. The present studies suggest that insulin-like growth factor-1 signaling is involved in modulation of glutamate transporter cell surface expression.

Pharmacology of monoamine neurotransmitter transporters

Following exocytotic release, the biogenic amine neurotransmitters, norepinephrine, dopamine, and serotonin are removed from the synaptic cleft by the respective transporter, NET, DAT, and SERT, located on the plasma membrane and then re-stored into synaptic vesicles by vesicular monoamine transporter, VMAT. The molecular cloning of these transporters revealed that NET, DAT, and SERT are members of a sodium-dependent neurotransmitter transporter gene family, while VMATs arise from proton-dependent transporter gene family. Structural features common to NET, DAT, and SERT reveal a putative 12 transmembrane-spanning domain structure with cytosolic N- and C-terminal regions. Recent evidence suggest the regulation of the functional expression of these transporters via phosphorylation, which include direct phosphorylation of transporter proteins and/or of associated proteins that may control transporter function/expression. In addition, the substrates and inhibitors for these transporters appear capable of regulating transporter cell surface expression, thereby suggesting both activity-dependent and pharmacological regulatory mechanisms for transporter expression. Analyses of the genes provide new insight into their relation to neuronal diseases since NET, DAT and SERT are the molecular targets for many antidepressants as well as drugs of abuse such as cocaine and amphetamine. The availability of cDNAs of these and vesicular transporters has permitted detailed pharmacological studies in heterologous expression systems, and thus would promise the development of novel drugs with diverse chemical structures.