The mechanistic basis for noncompetitive ibogaine inhibition of serotonin and dopamine transporters

Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.     

Design, synthesis, and characterization of a novel, 4-[2-(diphenylmethoxy)ethyl]-1-benzyl piperidine-based, dopamine transporter photoaffinity label

The dopamine transporter (DAT) has been implicated strongly in cocaine's reinforcing effects. Many derivatives of piperidine analogs of GBR 12909 have been developed and were found to be quite potent and selective for the DAT. In this regard, most of these derivatives were found to be much more selective for the DAT than conventional GBR compounds e.g. GBR 12909 when their selectivity was compared with the serotonin transporter (SERT). A brief structure-activity relationship (SAR) study has been carried out in the development of a novel photoaffinity ligand which illustrated the effect of the presence of a sterically bulky iodine atom next to the azido group in activity and selectivity for the DAT. This SAR study also led to the development of the compound 4 which is one of the most potent and selective blockers for the DAT known today. The photoaffinity ligand [125I]AD-96-129 was incorporated into the DAT molecule as was demonstrated by immunoprecipitation with serum 16 which is specific for DAT. This photolabeling was antagonized by DAT-specific blockers and was unaffected by specific SERT and norepinephrine transporter (NET) blockers indicating interaction of this novel ligand with the DAT.     

Modulation of the dopamine transporter by interaction with Secretory Carrier Membrane Protein 2

The monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT) facilitate the homeostatic balance of neurotransmitters in the synaptic cleft and thus, play a fundamental role in regulating neuronal activity. Despite the importance of these monoamine transporters in controlling brain function, only relatively little information is available regarding the cellular and molecular regulation of these proteins. The monoamine transporters have been found to associate with a number of different proteins that regulate the function and subcellular localization of the transporters. We recently reported a functional interaction between SERT and the Secretory Carrier Membrane Protein 2 (SCAMP2). Here, we demonstrate that SCAMP2 also plays a role in the functional regulation of DAT. DAT and SCAMP2 interaction is here verified by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) microscopy. Moreover, co-expression of DAT and SCAMP2 results in a decrease in DAT-mediated dopamine uptake caused by reduced levels of DAT molecules on the cell surface. Our finding that SCAMP2 interacts with and regulates the subcellular distribution of both DAT and SERT suggests that interaction with SCAMP2 may constitute an important mechanism for coordinating cell surface expression of monoamine transporters.     

Association of attention-deficit disorder and the dopamine transporter gene.

Attention-deficit hyperactivity disorder (ADHD) has been shown to be familial and heritable, in previous studies. As with most psychiatric disorders, examination of pedigrees has not revealed a consistent Mendelian mode of transmission. The response of ADHD patients to medications that inhibit the dopamine transporter, including methylphenidate, amphetamine, pemoline, and bupropion, led us to consider the dopamine transporter as a primary candidate gene for ADHD. To avoid effects of population stratification and to avoid the problem of classification of relatives with other psychiatric disorders as affected or unaffected, we used the haplotype-based haplotype relative risk (HHRR) method to test for association between a VNTR polymorphism at the dopamine transporter locus (DAT1) and DSM-III-R-diagnosed ADHD (N = 49) and undifferentiated attention-deficit disorder (UADD) (N = 8) in trios composed of father, mother, and affected offspring. HHRR analysis revealed significant association between ADHD/UADD and the 480-bp DAT1 allele (chi 2 7.51, 1 df, P = .006). When cases of UADD were dropped from the analysis, similar results were found (Chi 2 7.29, 1 df, P = .007). If these findings are replicated, molecular analysis of the dopamine transporter gene may identify mutations that increase susceptibility to ADHD/UADD. Biochemical analysis of such mutations may lead to development of more effective therapeutic interventions.     

The VNTR polymorphism in the human dopamine transporter gene: improved detection and absence of association of VNTR alleles with attention-deficit hyperactivity disorder

The human dopamine transporter (DAT1) gene contains a variable number tandem repeat (VNTR) in its 3'-untranslated region because of repetition of a 40-bp core sequence. Methods available for the diagnosis of this polymorphism are limited in number. We have developed a new polymerase chain reaction (PCR) test, which is similar to that described originally by Vandenbergh's group, but provides a better detection of the VNTR alleles in the human DAT1 gene. Using two independent PCR methods, we have determined the distribution of VNTR alleles in 110 healthy Omani subjects, and in 92 children with attention-deficit hyperactivity disorder (ADHD). The frequency of the risk allele (DAT1*10) was similar in the healthy subjects and ADHD cases, indicating absence of association of this allele with ADHD in Oman.     

Structure-activity studies of 3'-4'-dichloro-meperidine analogues at dopamine and serotonin transporters

The structure-activity relationships of 3',4'-dichloro-meperidine were investigated at dopamine (DAT) and serotonin transporters (SERT). Large ester substituents and lipophilic groups at the 4-position favored molecular recognition at the SERT. The benzyl ester of 3',4'-dichloro-meperidine exhibited high potency and high selectivity for the SERT (DAT/SERT=760). Chemical modification of the ester group and N-substitution generally led to compounds with decreased DAT affinity. Only small esters and alkyl groups were tolerated at the 4-position of the meperidine ring system by the DAT. Overall, the meperidine analogues were generally more selective for the SERT than for the DAT.     

Des-keto lobeline analogs with increased potency and selectivity at dopamine and serotonin transporters

A series of des-keto lobeline analogs has been synthesized and evaluated for their ability to inhibit the dopamine transporter (DAT) and serotonin transporter (SERT) function and for their affinity for the synaptic vesicle monoamine transporter (VMAT2), as well as for alpha4beta2( *) and alpha7( *) neuronal nicotinic acetylcholine receptors (nAChRs). The enantiomers 8R-hydroxylobel-9-ene (3a) and 10S-hydroxylobel-7-ene (3c) exhibited high potency and selectivity at SERT and DAT, respectively.     

Distribution of the 3' VNTR polymorphism in the human dopamine transporter gene in world populations

A polymorphism with a variable number of tandem repeats (VNTR) found in the 3' untranslated region of the human dopamine transporter gene (DAT1) was scored in unrelated individuals drawn from 10 geographically widely dispersed populations in order to assess this marker's usefulness in human population genetics. The populations that were analyzed in this study included 4 indigenous groups of Siberia, natives of North and South America, as well as Caucasian and Oceanic groups, most of which represented small-scale societies. A total of 5 DAT1 alleles were seen overall, but only in one Siberian population, the Altai-Kizhi, were all 5 present, and in the Native Americans of Colombia the locus was monomorphic. The most common allele, DAT1*10, ranged in frequency from 52% in Greeks to 100% in South Americans. The high frequency of the DAT1*10 allele (approximately 90%) among Mongoloid groups of north and east Asia distinguishes them from most Caucasian groups. The presence of the rare DAT1*7 allele in relatively high frequency (approximately 5%) among all Siberian groups suggests a close affinity with north Asian groups, especially Mongolians. The presence of the even rarer DAT1*13 allele in one Siberian population, the Altai-Kizhi, reflects this group's long historical contact with Mongolians. The results demonstrated that the DAT1 VNTR polymorphism is useful in investigating population relationships, and that rare alleles at this locus may be particularly valuable in understanding the extent of genetic affinity between neighboring groups and in situations where admixture is suspected. However, because of both the association and linkage of this VNTR locus with attention-deficit hyperactivity disorder (ADHD) in children, and its highly restricted polymorphism (usually 3 alleles) in most human groups, the possibility of selection constraints on the DAT1 gene cannot be ignored.     

Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters

Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers.     

Parkin increases dopamine uptake by enhancing the cell surface expression of dopamine transporter

Mutations of parkin, a protein-ubiquitin E3 ligase, are linked to Parkinson's disease (PD). Although a variety of parkin substrates have been identified, none of these is selectively expressed in dopaminergic neurons, whose degeneration plays a critical role in PD. Here we show that parkin significantly increased dopamine uptake in the human dopaminergic neuroblastoma cell line SH-SY5Y. This effect was accompanied by increased V(max) of dopamine uptake and unchanged K(m). Consistent with this, increased binding sites for dopamine transporter (DAT) ligand were observed in SH-SY5Y cells overexpressing parkin. The results were confirmed when parkin was transfected in HEK293 cells stably expressing DAT. In these cells, parkin enhanced the ubiquitination and degradation of DAT, increased its cell surface expression, and augmented dopamine uptake. The effects of parkin were significantly abrogated by its PD-causing mutations. Because the cell surface expression of functional DAT requires its oligomerization, misfolded DAT, induced either by the protein glycosylation inhibitor tunicamycin or by its C-terminal truncation, significantly attenuated cell surface expression of native DAT and reduced dopamine uptake. Expression of parkin, but not its T240R mutant, significantly alleviated these detrimental effects of misfolded DAT. Thus, our studies suggest that parkin increases dopamine uptake by enhancing the ubiquitination and degradation of misfolded DAT, so as to prevent it from interfering with the oligomerization and cell surface expression of native DAT. This function of parkin would enhance the precision of dopaminergic transmission, increase the efficiency of dopamine utilization, and reduce dopamine toxicity on neighboring cells.     

Ethanol potentiates the function of the human dopamine transporter expressed in Xenopus oocytes

Ethanol alters a variety of properties of brain dopaminergic neurons including firing rate, synthesis, release, and metabolism. Recent studies suggest that ethanol's action on central dopamine systems may also involve modulation of dopamine transporter (DAT) activity. The human DAT was expressed in Xenopus oocytes to examine directly the effects of ethanol on transporter function. [3H]Dopamine (100 nM) accumulation into DAT-expressing oocytes increased significantly in response to ethanol (10 min; 10-100 mM). In two-electrode voltage-clamp experiments, DAT-mediated currents were also enhanced significantly by ethanol (10-100 mM). The magnitude of the ethanol-induced potentiation of DAT function depended on ethanol exposure time and substrate concentration. Cell surface DAT binding ([3H]WIN 35,428; 4 nM) also increased as a function of ethanol exposure time. Thus, the increase in dopamine uptake was associated with a parallel increase in the number of DAT molecules expressed at the cell surface. These experiments demonstrate that DAT-mediated substrate translocation and substrate-associated ionic conductances are sensitive to intoxicating concentrations of ethanol and suggest that DAT may represent an important site of action for ethanol's effects on central dopaminergic transmission. A potential mechanism by which ethanol acts to enhance DAT function may involve regulation of DAT expression on the cell surface.     

VNTR polymorphisms of the serotonin transporter and dopamine transporter genes in male opiate addicts

VNTR polymorphisms of the serotonin transporter (hSERT) and dopamine transporter (DAT1) gene were studied in male opiate addicts. Samples of ethnic Russians and ethnic Tatars did not differ in genotype and allele frequencies. Homozygosity at hSERT (especially 10/10) was associated with early opiate addiction, while genotype 12/10 proved to be protective. In the case of DAT1, genotype 9/9 was associated with early opiate addiction. The combination of hSERT genotype 10/10 with DAT1 genotype 10/10 was shown to be a risk factor of opiate abuse under 16 years of age.     

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.     

Global Down-Regulation of Gene Expression in the Brain Using RNA Interference,with Emphasis on Monoamine Transporters and GPCRs: Implications for Target Characterization in Psychiatric and Neurological Disorders

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.     

N-8-Substituted benztropinamine analogs as selective dopamine transporter ligands

A series of N-8-substituted benztropinamines was synthesized and evaluated for binding at the dopamine (DAT), serotonin (SERT), norepinephrine (NET) transporters, and muscarinic M1 receptors. In general, the isosteric replacement of the C-3 benzhydrol ether of benztropine by a benzhydryl amino group was well tolerated at the DAT. However, for certain N-8 substituted derivatives, selectivity over muscarinic M1 receptor affinity was reduced.     

Global down-regulation of gene expression in the brain using RNA interference, with emphasis on monoamine transporters and GPCRs: implications for target characterization in psychiatric and neurological disorders

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.