SERT Substrates Research Articles

5-MeO-MET, 5-MeO-DET and 5-MeO-pyr-T strongly bind to 5-HT1A and 5-HT2A receptors and act as partial SERT substrates

5-MeO-MET, 5-MeO-DET and 5-MeO-pyr-T strongly bind to 5-HT1A and 5-HT2A receptors and act as partial SERT substrates

Structure–Activity Relationship of Novel Second-Generation Synthetic Cathinones: Mechanism of Action, Locomotion, Reward, and Immediate-Early Genes

Several new synthetic cathinones, which mimic the effect of classical psychostimulants such as cocaine or MDMA, have appeared in the global illicit drug market in the last decades. In fact, the illicit drug market is continually evolving by constantly adding small modifications to the common chemical structure of synthetic cathinones. Thus, the aim of this study was to investigate the in vitro and in vivo structure–activity relationship (SAR) of six novel synthetic cathinones currently popular as recreational drugs, pentedrone, pentylone, N-ethyl-pentedrone (NEPD), N-ethyl-pentylone (NEP), 4-methyl-pentedrone (4-MPD), and 4-methyl-ethylaminopentedrone (4-MeAP), which structurally differ in the absence or presence of different aromatic substituents and in their amino terminal group. Human embryonic kidney (HEK293) cells expressing the human isoforms of SERT and DAT were used for the uptake inhibition and release assays. Moreover, Swiss CD-1 mice were used to investigate the psychostimulant effect, rewarding properties (3, 10, and 30 mg/kg, i.p.), and the induction of immediate-early genes (IEGs), such as Arc and c-fos in the dorsal striatum (DS) and ventral striatum (VS) as well as bdnf in the medial prefrontal cortex (mPFC), of the test compounds. Our results demonstrated that all tested synthetic cathinones are potent dopamine (DA) uptake inhibitors, especially the N-ethyl analogs, while the ring-substituted cathinones tested showed higher potency as SERT inhibitors than their no ring-substituted analogs. Moreover, unlike NEP, the remaining test compounds showed clear “hybrid” properties, acting as DAT blockers but SERT substrates. Regarding the locomotion, NEP and NEPD were more efficacious (10 mg/kg) than their N-methyl analogs, which correlates with their higher potency inhibiting the DAT and an overexpression of Arc levels in the DS and VS. Furthermore, all compounds tested induced an increase in c-fos expression in the DS, except for 4-MPD, the least effective compound in inducing hyperlocomotion. Moreover, NEP induced an up-regulation of bdnf in the mPFC that correlates with its 5-HTergic properties. Finally, the present study demonstrated for the first time that NEP, 4-MPD, and 4-MeAP induce reward in mice. Altogether, this study provides valuable information about the mechanism of action and psychostimulant and rewarding properties as well as changes in the expression of IEGs related to addiction induced by novel second-generation synthetic cathinones.

The synthetic cathinones, butylone and pentylone, are stimulants that act as dopamine transporter blockers but 5-HT transporter substrates

Synthetic cathinones continue to emerge in recreational drug markets worldwide. 1-(1,3-Benzodioxol-5-yl)-2-(methylamino)butan-1-one (butylone) and 1-(1,3-benzodioxol-5-yl)-2-(methylamino)pentan-1-one (pentylone) are derivatives of the cathinone compound, 1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one (methylone), that are being detected in drug products and human casework. The purpose of the present study was to examine the neuropharmacology of butylone and pentylone using in vitro and in vivo methods. In vitro uptake and release assays were carried out in rat brain synaptosomes and in cells expressing human dopamine transporters (DAT) and 5-HT transporters (SERT). In vivo microdialysis was performed in the nucleus accumbens of conscious rats to assess drug-induced changes in neurochemistry. Butylone and pentylone were efficacious uptake blockers at DAT and SERT, though pentylone was more DAT-selective. Both drugs acted as transporter substrates that evoked release of [3H]5-HT at SERT, while neither evoked release at DAT. Consistent with the release data, butylone and pentylone induced substrate-associated inward currents at SERT but not DAT. Administration of butylone or pentylone to rats (1 and 3mg/kg, i.v.) increased extracellular monoamines and motor activity, but pentylone had weaker effects on 5-HT and stronger effects on motor stimulation. Our data demonstrate that increasing the α-carbon chain length of methylone creates "hybrid" transporter compounds which act as DAT blockers but SERT substrates. Nevertheless, butylone and pentylone elevate extracellular dopamine and stimulate motor activity, suggesting both drugs possess significant risk for abuse.

Effects of repeated treatment with methcathinone, mephedrone, and fenfluramine on intracranial self-stimulation in rats.

Synthetic cathinones constitute a class of abused drugs that can act at dopamine, norepinephrine, and serotonin transporters (DAT, NET, and SERT, respectively). Intracranial self-stimulation (ICSS) is a preclinical procedure that can be used to evaluate abuse potential of drugs, and prior studies have indicated that abuse-related ICSS effects of monoamine-transporter substrates, including some synthetic cathinones, are positively correlated with drug selectivity for DAT vs. SERT. Abuse potential of drugs can also be influenced by regimens of repeated drug exposure, but the role of repeated exposure on abuse-related ICSS effects of synthetic cathinones has not been examined. This study used ICSS to evaluate effects of repeated treatment with the DAT>SERT substrate methcathinone, the DAT<SERT substrate fenfluramine, and the DAT≈SERT substrate mephedrone. Male Sprague-Dawley rats were trained in a frequency-rate ICSS procedure, and different groups were used to evaluate effects of methcathinone, mephedrone, and fenfluramine before, during, and after regimens of repeated treatment with the designated drug. Before repeated treatment, methcathinone produced dose-dependent and abuse-related ICSS facilitation, fenfluramine produced dose-dependent ICSS depression, and mephedrone produced mixed effects that included both facilitation and depression. Chronic treatment produced no change in effects of methcathinone, but complete tolerance to effects of fenfluramine. For mephedrone, chronic treatment produced partial tolerance to ICSS depression and enhanced expression of ICSS facilitation. Repeated exposure to mixed-action DAT≈SERT substrates such as mephedrone can result in increased abuse potential due to sustained expression of DAT-mediated abuse-related effects and tolerance to SERT-mediated abuse-limiting effects.

N-Alkylated Analogs of 4-Methylamphetamine (4-MA) Differentially Affect Monoamine Transporters and Abuse Liability.

Clandestine chemists synthesize novel stimulant drugs by exploiting structural templates known to target monoamine transporters for dopamine, norepinephrine, and serotonin (DAT, NET, and SERT, respectively). 4-Methylamphetamine (4-MA) is an emerging drug of abuse that interacts with transporters, but limited structure-activity data are available for its analogs. Here we employed uptake and release assays in rat brain synaptosomes, voltage-clamp current measurements in cells expressing transporters, and calcium flux assays in cells coexpressing transporters and calcium channels to study the effects of increasing N-alkyl chain length of 4-MA on interactions at DAT, NET, and SERT. In addition, we performed intracranial self-stimulation in rats to understand how the chemical modifications affect abuse liability. All 4-MA analogs inhibited uptake at DAT, NET, and SERT, but lengthening the amine substituent from methyl to ethyl, propyl, and butyl produced a stepwise decrease in potency. N-methyl 4-MA was an efficacious substrate-type releaser at DAT that evoked an inward depolarizing current and calcium influx, whereas other analogs did not exhibit these effects. N-methyl and N-ethyl 4-MA were substrates at NET, whereas N-propyl and N-butyl 4-MA were not. All analogs acted as SERT substrates, though N-butyl 4-MA had very weak effects. Intracranial self-stimulation in rats showed that elongating the N-alkyl chain decreased abuse-related effects in vivo that appeared to parallel reductions in DAT activity. Overall, converging lines of evidence show that lengthening the N-alkyl substituent of 4-MA reduces potency to inhibit transporters, eliminates substrate activity at DAT and NET, and decreases abuse liability of the compounds.

Dopamine and 5HT Transporter Amino And Carboxy Termini Contribute To Substrate Affinity And Psychostimulant Activity

Determining how psychoactive drugs interact with their molecular targets is essential for understanding psychostimulant abuse and pharmacotherapies. The SLC6 carriers, DAT and SERT, are the primary targets for both addictive and therapeutic psychostimulants. Recent SLC6 structures have shed light on these molecular interactions. However, these structures lack much of the carriers' N‐ and C‐termini, raising questions regarding their potential contributions to the transport mechanism. To address this question, we generated DAT/SERT chimeras that exchange the N‐, C‐, or both termini, and tested their effect on substrate uptake, ligand binding and efflux kinetics. The SERT N‐terminus decreased DAT's affinity for DA and AMPH affinity two‐fold, whereas substituting the SERT C‐terminus had no effect on DAT substrate affinities. Moreover, replacing both DAT intracellular termini with SERT domains rescued DAT substrate affinity. In contrast, substituting the DAT N‐terminus had no effect on SERT substrate affinities, whereas the DAT C‐terminus decreased SERT substrate affinities and this was not rescued when both DAT termini were substituted onto SERT. Chimeric DAT and SERT protein affinities for cocaine were unaffected by interchanging domains, suggesting that DAT and SERT tail domains contribute in substrate translocation but not ligand binding. Ongoing studies test whether these intracellular domains contribute to AMPH‐stimulated substrate efflux and catalytic turnover rates and should shed considerable light onto the differential contribution of these domains to the DA vs. 5HT transport processes.

'Second-generation' mephedrone analogs, 4-MEC and 4-MePPP, differentially affect monoamine transporter function.

The nonmedical use of synthetic cathinones is increasing on a global scale. 4-Methyl-N-methylcathinone (mephedrone) is a popular synthetic cathinone that is now illegal in the United States and other countries. Since the legislative ban on mephedrone, a number of 'second-generation' analogs have appeared in the street drug marketplace, including 4-methyl-N-ethylcathinone (4-MEC) and 4'-methyl-α-pyrrolidinopropiophenone (4-MePPP). Here we characterized the interactions of 4-MEC and 4-MePPP with transporters for 5-HT (SERT) and dopamine (DAT) using molecular, cellular, and whole-animal methods. In vitro transporter assays revealed that 4-MEC displays unusual 'hybrid' activity as a SERT substrate (ie, 5-HT releaser) and DAT blocker, whereas 4-MePPP is a blocker at both transporters but more potent at DAT. In vivo microdialysis experiments in rat brain demonstrated that 4-MEC (1-3 mg/kg, i.v.) produced large increases in extracellular 5-HT, small increases in dopamine, and minimal motor stimulation. In contrast, 4-MePPP (1-3 mg/kg, i.v.) produced selective increases in dopamine and robust motor stimulation. Consistent with its activity as a SERT substrate, 4-MEC evoked inward current in SERT-expressing Xenopus oocytes, whereas 4-MePPP was inactive in this regard. To examine drug-transporter interactions at the molecular level, we modeled the fit of 4-MEC and 4-MePPP into the binding pockets for DAT and SERT. Subtle distinctions in ligand-transporter binding were found that account for the differential effects of 4-MEC and 4-MePPP at SERT. Collectively, our results provide key information about the pharmacology of newly emerging mephedrone analogs, and give clues to structural requirements that govern drug selectivity at DAT vs SERT.

Probing the Selectivity of Monoamine Transporter Substrates by Means of Molecular Modeling

Probing the Selectivity of Monoamine Transporter Substrates by Means of Molecular Modeling

Methylene blue inhibits function of the 5‐HT transporter

Methylene blue (MB) is commonly employed as a treatment for methaemoglobinaemia, malaria and vasoplegic shock. An increasing number of studies indicate that MB can cause 5-HT toxicity when administered with a 5-HT reuptake inhibitor. MB is a potent inhibitor of monoamine oxidases, but other targets that may contribute to MB toxicity have not been identified. Given the role of the 5-HT transporter (SERT) in the regulation of extracellular 5-HT concentrations, the present study aimed to characterize the effect of MB on SERT. Live cell imaging, in conjunction with the fluorescent SERT substrate 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP(+) ), [(3) H]5-HT uptake and whole-cell patch-clamp techniques were employed to examine the effects of MB on SERT function. In EM4 cells expressing GFP-tagged human SERT (hSERT), MB concentration-dependently inhibited ASP(+) accumulation (IC(50) : 1.4 ± 0.3 µM). A similar effect was observed in N2A cells. Uptake of [(3) H]5-HT was decreased by MB pretreatment. Furthermore, patch-clamp studies in hSERT expressing cells indicated that MB significantly inhibited 5-HT-evoked ion currents. Pretreatment with 8-Br-cGMP did not alter the inhibitory effect of MB on hSERT activity, and intracellular Ca(2+) levels remained unchanged during MB application. Further experiments revealed that ASP(+) binding to cell surface hSERT was reduced after MB treatment. In whole-cell radioligand experiments, exposure to MB (10 µM; 10 min) did not alter surface binding of the SERT ligand [(125) I]RTI-55. MB modulated SERT function and suggested that SERT may be an additional target upon which MB acts to produce 5-HT toxicity.

Effects of MDMA and related analogs on plasma 5-HT: Relevance to 5-HT transporters in blood and brain

(±)-3,4-Methylenedioxymethamphetamine (MDMA) is an illicit drug that evokes transporter-mediated release of serotonin (5-HT) in the brain. 5-HT transporter (SERT) proteins are also expressed in non-neural tissues (e.g., blood), and evidence suggests that MDMA targets platelet SERT to increase plasma 5-HT. Here we tested two hypotheses related to the effects of MDMA on circulating 5-HT. First, to determine if MDMA metabolites might contribute to actions of the drug in vivo, we used in vitro microdialysis in rat blood specimens to examine the effects of MDMA and its metabolites on plasma 5-HT. Second, to determine whether effects of MDMA on plasma 5-HT might be used as an index of central SERT activity, we carried out in vivo microdialysis in blood and brain after intravenous MDMA administration. The in vitro results show that test drugs evoke dose-related increases in plasma 5-HT ranging from two- to sevenfold above baseline, with MDMA and its metabolite, (±)-3,4-methylenedioxyamphetamine (MDA), producing the largest effects. The ability of MDMA and related analogs to elevate plasma 5-HT is correlated with their potency as SERT substrates in rat brain synaptosomes. The in vivo results reveal that MDMA causes concurrent increases in extracellular 5-HT in blood and brain, but there are substantial individual differences in responsiveness to the drug. Collectively, our findings indicate that MDMA and its metabolites increase plasma 5-HT by a SERT-dependent mechanism, and suggest the possibility that measures of evoked 5-HT release in blood may reflect central SERT activity.

RE: Pulmonary Hypertension Associated with Use of Phentermine?

Dear Sir, Recently Bang, et al.1 reported a case of a 29 year-old obese woman who was hospitalized with pulmonary arterial hypertension (PAH) and a history of having taken phentermine hydrochloride 37.5 milligrams daily for 35 days. Since common etiologies of PAH were excluded by the patient's history, examination procedures, and hospital course, the authors suggest this is the first case of phentermine-induced PAH. We suggest instead this is a case of idiopathic pulmonary arterial hypertension (IPAH) rather than PAH secondary to therapeutic phentermine. The authors suggest, based on one case report, that phentermine mono-therapy produces the same increase in incidence of PAH previously seen in patients treated with fenfluramine mono-therapy and in patients treated with a combination of fenfluramine and phentermine. However, a survey of 95 European cases of PAH by Abenhaim, et al.2 found that of the 30 patients that had a history of taking anorexic agents, 24 had taken a fenfluramine, but none had taken phentermine. Moreover, Rich, et al.,3 in a North American prospective study in which 579 patients with pulmonary hypertension were studied, found that the odds ratios for an association of pulmonary hypertension and fenfluramine to be 7.5, an odds ratio for a similar association for phentermine of 0.6, and concluded that fenfluramines, but not phentermine are risk factors for PAH. Since fenfluramines were taken off-market in 1997 there have been no subsequent reports linking PAH or IPAH to anorectic medicines. Hence the literature does not support the contention of Bang, et al. that phentermine mono-therapy has caused an increased incidence of secondary PAH similar to that caused by fenfluramine. Physicians in other countries may not be aware that phentermine has long been the most widely used anti-obesity medicine in the United States. A recent survey of United States obesity treatment specialists found that for 97%, phentermine was their first choice among the available anti-obesity drugs.4 The majority of physicians surveyed reported they prescribed phentermine long-term in any patient provided the medicine was effective and produced no intolerable side effects. The longest duration of phentermine therapy reported in the literature has been forty years with no serious adverse effects.5 The survey confirmed that most specialists have employed doses higher than the maximum dose of 37.5 milligrams daily recommended by the U.S. Food and Drug Administration (FDA)-approved label. There are other reports of the safe use of higher doses.4,6-9 Thus, in spite of continued widespread use of phentermine for longer durations and higher doses than recommended by the FDA-approved label, there is no report of an increased incidence of pulmonary hypertension due to phentermine in the U.S. The incidence of IPAH is approximately one case per million per year in the general population. Since phentermine is so widely used, it is reasonable to expect an occasional patient diagnosed with IPAH will coincidently have taken phentermine. PAH and IPAH are characterized by an increase in pulmonary vascular resistance due to progressive obliteration of pulmonary arterioles, which in turn are induced by pulmonary arteriole smooth muscle cell proliferation and arteriolar vasoconstriction with accompanying inflammation and thrombosis. One hypothesis to explain fenfluramine-induced PAH is that fenfluramine releases 5-hydroxytryptophan (5-HT) via the 5-HT transporter (SERT), and that the increased concentration of plasma 5-HT provokes pulmonary vasoconstriction and pulmonary arterial smooth muscle cell proliferation.10 However, recent data raises questions about this hypothesis.11 5-HT is also thought to play a central role in the pathogenesis of IPAH. Bang, et al. imply that phentermine is active as a serotonin transporter producing increases in 5-HT levels, and therefore has the potential to cause PAH. However, Rothman, et al.12 have shown previously that phentermine has considerably less potent SERT substrate activity, and is considerably less effective in elevating plasma 5-HT compared to fenfluramine, and compared to the commonly abused stimulants methamphetamine and 3,4-methylenedioxymethamphetamine. In another study Rothman, et al. found that phentermine produced minimal increases in 5HT, insufficient to provoke pulmonary arteriolar smooth muscle proliferation.13 These studies suggest that it is unlikely phentermine could induce PAH by the mechanism proposed by Bang, et al. In summary we suggest that phentermine, after 52 years of widespread use has proven to be both effective and safe. Historically, an association between phentermine mono-therapy and PAH has been suggested but confirming evidence has not appeared. Currently, while we have few effective pharmacologic agents to include in obesity treatment, patients and physicians are fortunate to have phentermine available. Bang, et al. are correct in noting the absence of 870long-term, dose ranging, clinical trials with phentermine; we concur that such trials are needed and should be conducted. Meanwhile, while we await trials, phentermine treatment should not be withheld from the obese because of fear of theoretical but unproven adverse effects.

Serotonin (5‐HT) Transporter Ligands Affect Plasma 5‐HT in Rats

Dual dopamine (DA)/serotonin (5-HT)-releasing agents are promising candidate medications for stimulant addiction and other disorders. However, certain 5-HT transporter (SERT) substrates are associated with development of idiopathic pulmonary arterial hypertension (IPAH) and valvular heart disease (VHD). According to the "5-HT hypothesis," SERT substrates increase the risk for developing IPAH and VHD by increasing plasma 5-HT. To test this hypothesis directly, we determined the effects of acute and chronic fenfluramine, and other SERT ligands, on plasma 5-HT in male rats. For acute treatments, rats received i.v. vehicle or test drug (0.3 and 1.0 mg/kg), and serial blood samples were withdrawn. For chronic treatments, vehicle or test drug was infused via osmotic minipump (3 and 10 mg/kg/d) for 2 weeks. On the last day of infusion, rats received i.v. fenfluramine challenge (1 mg/kg), and serial blood samples were withdrawn. Plasma 5-HT was measured using ex vivo microdialysis in whole-blood samples. Baseline plasma 5-HT was <1.0 nM. Acute injection of fenfluramine or other SERT substrates caused large (up to 24-fold) dose-dependent increases in plasma 5-HT. Chronic fenfluramine at 3 and 10 mg/kg/d produced 1.7- and 3.5-fold increases in baseline plasma 5-HT, while chronic fluoxetine had no effect. Chronic infusions of fenfluramine or fluoxetine diminished the ability of acute fenfluramine to elevate dialysate 5-HT, and both drugs markedly reduced whole-blood 5-HT. Acute fenfluramine increases plasma 5-HT to concentrations that are below the micromolar levels necessary to produce adverse cardiovascular effects. Chronic fenfluramine and fluoxetine have minimal effects on plasma 5-HT, suggesting that the increased risk for IPAH associated with fenfluramine does not depend upon elevations in plasma 5-HT.

Distinct recognition of substrates by the human and Drosophila serotonin transporters.

The human and Drosophila serotonin transporters (hSERT and dSERT, respectively) were used to explore differences in substrate properties. hSERT and dSERT showed similar Km values for 5-hydroxytryptamine (5-HT; serotonin) transport (1.2 and 0.9 micro M, respectively), suggesting similar recognition of 5-HT by the two species variants. Although dSERT cell surface expression was approximately 8-fold lower than that of hSERT, dSERT does appear to have a 2-fold faster turnover number for inward transport of 5-HT. Interestingly, another substrate, N-methyl-4-phenylpyridinium (MPP+), was transported only by hSERT. However, MPP+ inhibited 5-HT uptake in both species variants with similar potencies. Two cross-species chimeras, H1-118D119-627 and H1-281D282-476H477-638, were also unable to transport MPP+, implicating the role of transmembrane domains V to IX in the substrate permeation pathway. Based on exchange experiments, certain substituted-amphetamines also appear to be poor substrates at dSERT. Two-electrode voltage-clamp studies in oocytes confirmed that the amphetamines do not possess substrate-like properties for dSERT. Our data suggest distinct molecular recognition among SERT substrate classes that influence translocation mechanisms.

Psychostimulants Differentially Regulate Serotonin Transporter Expression in Thalamocortical Neurons

5-HT transporters (SERTs) are transiently expressed in thalamocortical neurons during development, permitting these glutamatergic neurons to co-release 5-HT as a "borrowed" transmitter. The high level of SERT expression in these neurons is likely important in the serotonergic modulation of neocortical circuits and provides a system for examining endogenous SERT regulation. We tested the hypothesis that developmental expression of SERT in thalamocortical neurons is regulated by psychostimulants that are agonists and antagonists of SERT. Cultured thalamocortical neurons from embryonic day 18 rats were examined for SERT expression until P15. In untreated cultures, SERT protein levels peaked at postnatal day 3 (P3) and were absent by P10. Chronic treatment with SERT substrates (5-HT, 3,4-methylenedioxymethamphetamine) increased both peak SERT protein levels (fourfold) and the time course of SERT expression. SERT substrates also shifted the relative functional expression of SERT by redistributing intracellular SERT protein to the plasma membrane. The subcellular redistribution was prevented by PKC activators. SERT antagonists (e.g., fluoxetine, cocaine) reduced total SERT expression levels and the time course of SERT expression. These data (1) show that endogenous SERT is differentially regulated by 5-HT and psychostimulants, (2) indicate that SERT modulation occurs via changes in both total SERT protein levels and subcellular redistribution of the transporter, and (3) suggest that some of the actions of drugs of abuse in neocortical development may be attributable to alterations in SERT expression and concomitant changes in 5-HT signaling.

Cocaine and Antidepressant-Sensitive Biogenic Amine Transporters Exist in Regulated Complexes with Protein Phosphatase 2A

Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.