Studies of the biogenic amine transporters. VII. Characterization of a novel cocaine binding site identified with [125I]RTI-55 in membranes prepared from human, monkey and guinea pig caudate
Richard B. RothmanM L SilverthornJohn R. GlowaDorota MateckaKenner C. RiceF. Ivy CarrollJohn S. PartillaGeorge R. UhlDavid J. VandenberghChristina M. Dersch
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Abstract:
[125I]RTI-55 is a cocaine analog with high affinity for dopamine (DA) and serotonin (5-HT) transporters. Quantitative ligand binding studies revealed a novel high affinity [125I]RTI-55 binding site assayed under 5-HT transporter (SERT) conditions which has low affinity for almost all classic biogenic amine transporter ligands, including high affinity 5-HT transporter inhibitors such as paroxetine, but which retains high affinity for cocaine analogs. This site, termed SERT(site2) for its detection under 5-HT transporter conditions (not for an association with the SERT) occurs in monkey caudate, human caudate, and guinea pig caudate membranes, but not in rat caudate membranes. SERT(site2) is distinguished from the DA transporter (DAT) and SERT by several criteria, including a distinct ligand-selectivity profile, the inability to detect SERT(site2) in cells stably expressing the cloned human DAT, and insensitivity to irreversible ligands which inhibit [125I]RTI-55 binding to the DAT and SERT. Perhaps the most striking finding about SERT(site2) is that a wide range of representative antidepressant agents have very low affinity for SERT(site2). The affinity of cocaine for this site is not very different from the concentration cocaine achieves in the brain at pharmacological doses. Viewed collectively with the observation that ligands with high affinity for SERT(site2) are mostly cocaine analogs, these data lead us to speculate that actions of cocaine which differ from those of classic biogenic amine uptake inhibitors may be mediated in part via SERT(site2).Keywords:
Biogenic amine
The neurotransmitter dopamine is cleared from synapses by presynaptic dopamine transporters, which thus play a key role in regulating dopaminergic signaling. Mazei-Robison et al ., who previously identified a mutant form of the dopamine transporter in two brothers with attention deficit hyperactivity disorder (ADHD), investigated the effects of this mutation on transporter function. When transfected into human embryonic kidney (HEK) 293T cells, the total and surface abundance of the mutant transporter [in which an alanine at residue 559 is substituted with valine (hDAT A559V)] was similar to that of the transfected wild-type human dopamine transporter (hDAT), as was dopamine uptake. Amperometric recordings of cells preloaded with dopamine, however, revealed that, whereas cells expressing hDAT showed little dopamine efflux, hDAT A559V cells exhibited pronounced dopamine efflux currents. Whole-cell patch clamp analysis combined with experiments in which the intracellular concentrations of Na + or dopamine were varied indicated that hDAT A559V showed an exaggerated increase in dopamine efflux in response to depolarization compared with hDAT and an increased sensitivity to intracellular Na + . Intriguingly, amphetamine, which is used to treat ADHD, enhanced hDAT-mediated dopamine efflux (as it typically does) but inhibited hDAT A559V–mediated efflux. The authors thus propose that alterations in dopamine efflux through hDAT may play a role in ADHD and perhaps other brain disorders associated with altered dopamine signaling. M. S. Mazei-Robison, E. Bowton, M. Holy, M. Schmudermaier, M. Freissmuth, H. H. Sitte, A. Galli, R. D. Blakely, Anomalous dopamine release associated with a human dopamine transporter coding variant. J. Neurosci . 28 , 7040-7046 (2008). [Abstract] [Full Text]
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Escitalopram
Sertraline
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OBJECTIVE: Evidence of a relationship between genotype and binding availability was assessed for the dopamine and serotonin transporter genes. METHOD: The authors assessed dopamine transporter genotype at the SLC6A3 3′ variable number of tandem repeats (VNTR) polymorphism and serotonin transporter genotype at the SLC6A4 promotor VNTR polymorphism in 30 healthy subjects who also underwent single photon emission computed tomography with [123I]β-CIT. RESULTS: Subjects homozygous for the 10-repeat allele at the SLC6A3 locus demonstrated significantly lower dopamine transporter binding than carriers of the nine-repeat allele. There was no effect of SLC6A4 genotype upon serotonin transporter binding. CONCLUSIONS: These findings suggest that genetic variation at the SLC6A3 3′ VNTR polymorphism may modify dopamine transporter function.
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Although our understanding of the actions of cocaine in the brain has improved, an effective drug treatment for cocaine addiction has yet to be found. Methylphenidate binds the dopamine transporter and increases extracellular dopamine levels in mammalian central nervous systems similar to cocaine, but it is thought to elicit fewer addictive and reinforcing effects owing to slower pharmacokinetics for different routes of administration between the drugs. This study utilizes the fruit fly model system to quantify the effects of oral methylphenidate on dopamine uptake during direct cocaine exposure to the fly CNS. The effect of methylphenidate on the dopamine transporter has been explored by measuring the uptake of exogenously applied dopamine. The data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter and the inhibition is concentration dependent. The peak height increased to 150% of control when cocaine was used to block the dopamine transporter for untreated flies but only to 110% for methylphenidate-treated flies. Thus, the dopamine transporter is mostly inhibited for the methylphenidate-fed flies before the addition of cocaine. The same is true for the rate of the clearance of dopamine measured by amperometry. For untreated flies the rate of clearance changes 40% when the dopamine transporter is inhibited with cocaine, and for treated flies the rate changes only 10%. The results were correlated to the in vivo concentration of methylphenidate determined by CE-MS. Our data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter for cocaine uptake, and the inhibition is concentration dependent.
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Using radiation inactivation, we have estimated the target size for the neuronal dopamine transporter protein. The specific binding of several radioligands previously shown to label the dopamine transporter was determined in an irradiated striatal membrane preparation. The apparent target size of the 1-[1-(2-[3H]benzo[b]thienyl)cyclohexyl]piperidine site was approximately 98 kDa. However, the apparent target size of the "cocaine binding site," as measured with the cocaine analogue 2 beta-[3H]carbomethoxy-3 beta-(4-fluorophenyl)tropane in the same assays, was approximately 140 kDa. Radiation inactivation of the binding of other ligands (GBR-12935 and mazindol) led to target size estimates in the same range (94 kDa and 133 kDa, respectively). All of these target sizes are significantly larger than the estimate of 70 kDa derived from the deduced amino acid sequence for the cloned dopamine reuptake transporter cDNA. Larger target sizes than expected have also been reported for ligand binding to the sodium-dependent serotonin transporter and glucose transporter. The estimated sizes for the ligand binding site(s) associated with the dopamine transporter protein are difficult to reconcile with a single transporter protein of 70 kDa. We conclude that the dopamine transporter protein is a homo- or hetero-oligomer when occupied in situ by uptake-blocking drugs like cocaine.
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