The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.

While classical cathinones, such as methcathinone, have been shown to be monoamine releasing agents at human monoamine transporters, the subgroup of α-pyrrolidinophenones has thus far solely been characterized as monoamine transporter reuptake inhibitors. Herein, we report data from previously undescribed α-pyrrolidinopropiophenone (α-PPP) derivatives and compare them with the pharmacologically well-researched α-PVP (α-pyrrolidinovalerophenone). Radiotracer-based in vitro uptake inhibition assays in HEK293 cells show that the investigated α-PPP derivatives inhibit the human high-affinity transporters of dopamine (hDAT) and norepinephrine (hNET) in the low micromolar range, with α-PVP being ten times more potent. Similar to α-PVP, no relevant pharmacological activity was found at the human serotonin transporter (hSERT). Unexpectedly, radiotracer-based in vitro release assays reveal α-PPP, MDPPP and 3Br-PPP, but not α-PVP, to be partial releasing agents at hNET (EC50 values in the low micromolar range). Furthermore, uptake inhibition assays at low-affinity monoamine transporters, i.e., the human organic cation transporters (hOCT) 1-3 and human plasma membrane monoamine transporter (hPMAT), bring to light that all compounds inhibit hOCT1 and 2 (IC50 values in the low micromolar range) while less potently interacting with hPMAT and hOCT3. In conclusion, this study describes (i) three new hybrid compounds that efficaciously block hDAT while being partial releasers at hNET, and (ii) highlights the interactions of α-PPP-derivatives with low-affinity monoamine transporters, giving impetus to further studies investigating the interaction of drugs of abuse with OCT1-3 and PMAT.

Various psychostimulants targeting monoamine neurotransmitter transporters (MATs) have been shown to rescue cognition in patients with neurological disorders and improve cognitive abilities in healthy subjects at low doses. Here, we examined the effects upon cognition of a chemically synthesized novel MAT inhibiting compound 2-(benzhydrylsulfinylmethyl)-4-methylthiazole (named as CE-104). The efficacy of CE-104 in blocking MAT [dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter] was determined using in vitro neurotransmitter uptake assay. The effect of the drug at low doses (1 and 10 mg/kg) on spatial memory was studied in male rats in the radial arm maze (RAM). Furthermore, the dopamine receptor and transporter complex levels of frontal cortex (FC) tissue of trained and untrained animals treated either with the drug or vehicle were quantified on blue native PAGE (BN-PAGE). The drug inhibited dopamine (IC50: 27.88 μM) and norepinephrine uptake (IC50: 160.40 μM), but had a negligible effect on SERT. In the RAM, both drug-dose groups improved spatial working memory during the performance phase of RAM as compared to vehicle. BN-PAGE Western blot quantification of dopamine receptor and transporter complexes revealed that D1, D2, D3, and DAT complexes were modulated due to training and by drug effects. The drug's ability to block DAT and its influence on DAT and receptor complex levels in the FC is proposed as a possible mechanism for the observed learning and memory enhancement in the RAM.

Psychostimulants such as amphetamine and cocaine are illicitly used drugs that act on neurotransmitter transporters for dopamine, serotonin or norepinephrine. These drugs can by themselves already cause severe neurotoxicity. However, an additional health threat arises from adulterant substances which are added to the illicit compound without declaration. One of the most frequently added adulterants in street drugs sold as cocaine is the anthelmintic drug levamisole. We tested the effects of levamisole on neurotransmitter transporters heterologously expressed in HEK293 cells. Levamisole was 100 and 300-fold less potent than cocaine in blocking norepinephrine and dopamine uptake, and had only very low affinity for the serotonin transporter. In addition, levamisole did not trigger any appreciable substrate efflux. Because levamisole and cocaine are frequently co-administered, we searched for possible allosteric effects; at 30μM, a concentration at which levamisole displayed already mild effects on norepinephrine transport it did not enhance the inhibitory action of cocaine. Levamisole is metabolized to aminorex, a formerly marketed anorectic drug, which is classified as an amphetamine-like substance. We examined the uptake-inhibitory and efflux-eliciting properties of aminorex and found it to exert strong effects on all three neurotransmitter transporters in a manner similar to amphetamine. We therefore conclude that while the adulterant levamisole itself has only moderate effects on neurotransmitter transporters, its metabolite aminorex may exert distinct psychostimulant effects by itself. Given that the half-time of levamisole and aminorex exceeds that of cocaine, it may be safe to conclude that after the cocaine effect "fades out" the levamisole/aminorex effect "kicks in".

The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) is thought to be fueled by the transmembrane Na+ gradient, but it is conceivable that they can also tap other energy sources, for example, membrane voltage and/or the transmembrane K+ gradient. We have addressed this by recording uptake of endogenous substrates or the fluorescent substrate APP+(4-(4-dimethylamino)phenyl-1-methylpyridinium) under voltage control in cells expressing DAT, NET, or SERT. We have shown that DAT and NET differ from SERT in intracellular handling of K+. In DAT and NET, substrate uptake was voltage-dependent due to the transient nature of intracellular K+ binding, which precluded K+ antiport. SERT, however, antiports K+ and achieves voltage-independent transport. Thus, there is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power, which we conclude to occur due to subtle differences in the kinetics of co-substrate ion binding in closely related transporters.

The illicit consumption of psychoactive compounds may cause short and long-term health problems and addiction. This is also true for amphetamines and cocaine, which target monoamine transporters. In the recent past, an increasing number of new compounds with amphetamine-like structure such as mephedrone or 3,4-methylenedioxypyrovalerone (MDPV) entered the market of illicit drugs. Subtle structural changes circumvent legal restrictions placed on the parent compound. These novel drugs are effectively marketed "designer drugs" (also called "research chemicals") without any knowledge of the underlying pharmacology, the potential harm or a registration of the manufacturing process. Accordingly new entrants and their byproducts are identified postmarketing by chemical analysis and their pharmacological properties inferred by comparison to compounds of known structure. However, such a heuristic approach fails, if the structures diverge substantially from a known derivative. In addition, the understanding of structure-activity relations is too rudimentary to predict detailed pharmacological activity. Here, we tested a combined approach by examining the composition of street drugs using mass spectrometry and by assessing the functional activity of their constituents at the neuronal transporters for dopamine, serotonin, and norepinephrine. We show that this approach is superior to mere chemical analysis in recognizing novel and potentially harmful street drugs.

Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP(+) accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP(+)). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signalling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists.

Addiction to psychostimulants (ie, amphetamines and cocaine) imposes a major socioeconomic burden. Prevention and treatment represent unmet medical needs, which may be addressed, if the mechanisms underlying psychostimulant action are understood. Cocaine acts as a blocker at the transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET), but amphetamines are substrates that do not only block the uptake of monoamines but also induce substrate efflux by promoting reverse transport. Reverse transport has been a focus of research for decades but its mechanistic basis still remains enigmatic. Recently, transporter-interacting proteins were found to regulate amphetamine-triggered reverse transport: calmodulin kinase IIα (αCaMKII) is a prominent example, because it binds the carboxyl terminus of DAT, phosphorylates its amino terminus, and supports amphetamine-induced substrate efflux in vitro. Here, we investigated whether, in vivo, the action of amphetamine was contingent on the presence of αCaMKII by recording the behavioral and neurochemical effects of amphetamine. Measurement of dopamine efflux in the dorsal striatum by microdialysis revealed that amphetamine induced less dopamine efflux in mice lacking αCaMKII. Consistent with this observation, the acute locomotor responses to amphetamine were also significantly blunted in αCaMKII-deficient mice. In addition, while the rewarding properties of amphetamine were preserved in αCaMKII-deficient mice, their behavioral sensitization to amphetamine was markedly reduced. Our findings demonstrate that amphetamine requires the presence of αCaMKII to elicit a full-fledged effect on DAT in vivo: αCaMKII does not only support acute amphetamine-induced dopamine efflux but is also important in shaping the chronic response to amphetamine.

A series of drugs have been reported to increase memory performance modulating the dopaminergic system and herein modafinil was tested for its working memory (WM) enhancing properties. Reuptake inhibition of dopamine, serotonin (SERT) and norepinephrine (NET) by modafinil was tested. Sixty male Sprague-Dawley rats were divided into six groups (modafinil-treated 1-5-10 mg/kg body weight, trained and untrained and vehicle treated trained and untrained rats; daily injected intraperitoneally for a period of 10 days) and tested in a radial arm maze (RAM), a paradigm for testing spatial WM. Hippocampi were taken 6 h following the last day of training and complexes containing the unphosphorylated or phosphorylated dopamine transporter (DAT-CC and pDAT-CC) and complexes containing the D1-3 dopamine receptor subunits (D1-D3-CC) were determined. Modafinil was binding to the DAT but insignificantly to SERT or NET and dopamine reuptake was blocked specifically (IC50 = 11.11 μM; SERT 1547 μM; NET 182 μM). From day 8 (day 9 for 1 mg/kg body weight) modafinil was decreasing WM errors (WMEs) in the RAM significantly and remarkably at all doses tested as compared to the vehicle controls. WMEs were linked to the D2R-CC and the pDAT-CC. pDAT and D1-D3-CC levels were modulated significantly and modafinil was shown to enhance spatial WM in the rat in a well-documented paradigm at all the three doses and dopamine reuptake inhibition with subsequent modulation of D1-3-CC is proposed as a possible mechanism of action.

The plasmalemmal monoamine transporters for dopamine, norepinephrine, and serotonin (SERT) are targets for amphetamines. In vivo, amphetamines elicit most, if not all, of their actions by triggering monoamine efflux. This is thought to be accomplished by an amphetamine-induced switch from the forward-transport to the substrate-exchange mode. The mechanism underlying this switch has remained elusive; available kinetic models posit that substrates and cosubstrate Na+ ions bind either in a random or in a sequential order. Neither can account for all reported experimental observations. We used electrophysiological recordings to interrogate crucial conformational transitions associated with the binding of five different substrates (serotonin, para-chloroamphetamine, and the high-affinity naphthyl-propan-amines PAL-287, PAL-1045, and PAL-1046) to human SERT expressed in HEK293 cells; specifically, we determined the relaxation kinetics of SERT from a substrate-loaded to a substrate-free state at various intracellular and extracellular Na+ concentrations. These rates and their dependence on intracellular and extracellular Na+ concentrations differed considerably between substrates. We also examined the effect of K+ on substrate affinity and found that K+ enhanced substrate dissociation. A kinetic model was developed, which allowed for random, but cooperative, binding of substrate and Na+ (or K+). The synthetic data generated by this model recapitulated the experimental observations. More importantly, the cooperative binding model accounted for the releasing action of amphetamines without any digression from alternating access. To the best of our knowledge, this model is the first to provide a mechanistic framework for amphetamine-induced monoamine release and to account for the findings that some substrates are less efficacious than others in promoting the substrate-exchange mode.

A series of compounds have been reported to enhance memory via the DA system and herein a heterocyclic compound was tested for working memory (WM) enhancement. 2-((benzhydrylsulfinyl)methyl)thiazole (CE-103) was synthesized in a six-step synthesis. Binding of CE-103 to the dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters and dopamine reuptake inhibition was tested as well as blood brain permeation and a screen for GPCR targets. 60 male Sprague Dawley rats were divided into six groups: CE-103 treated 1-10 mg/kg body weight, trained (TDI) and yoked (YDI) and vehicle treated, trained (TVI) and yoked (YVI) rats. Daily single intraperitoneal injections for a period of 10 days were administered and rats were tested in a radial arm maze (RAM). Hippocampi were taken 6 h following the last day of training and complexes containing the unphosphorylated or phosphorylated dopamine transporter (DAT) and complexes containing the D1-3 dopamine receptor subunits were determined. CE-103 was binding to the DAT but insignificantly to SERT or NET and dopamine reuptake was blocked specifically (IC50 = 14.73 μM). From day eight the compound was decreasing WM errors in the RAM significantly at both doses tested as compared to the vehicle controls. In the trained CE-103-treated group levels of the complex containing the phosphorylated dopamine transporter (pDAT) as well as D1R were decreased while levels of complexes containing D2R and D3R were significantly increased. CE-103 was shown to enhance spatial WM and DA reuptake inhibition with subsequent modulation of D1-3 receptors is proposed as a possible mechanism of action.

Mephedrone is a largely abused psychostimulant. It elicits the release of monoamines via the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Stereoselective metabolic reactions are involved in the inactivation and the elimination of its chemical structure. However, during these processes, several structures are generated and some of them have been reported to be still pharmacologically active. In this study 1) we have newly synthetized several putative mephedrone metabolites, 2) compared their activity at monoamine transporters, 3) generated quantitative structure activity relationships, and 4) exploited the chemical structure of the putative metabolites to screen a urine sample from a drug user and dissect mephedrone metabolism. We have found that most of the tested metabolites are weak inhibitors of monoamine transporters and that all of them are more potent at DAT and NET in comparison to SERT. The only exception is represented by the COOH-metabolite which shows no pharmacological activity at all three monoamine transporters. The enantioselectivity of mephedrone and its metabolites is present mainly at SERT, with only minor effects at DAT and NET being introduced when the β-keto group is reduced to an OH-group. Importantly, while at DAT the putative metabolites did not show changes in inhibitory potencies, but rather changes in their substrate/blocker profile, at SERT they showed mainly changes in inhibitory potencies. Molecular modeling suggests that the hydrophobic nature of a specific SERT subpocket may be involved in such loss of affinity. Finally, the assessment of the putative metabolites in one urine sample of mephedrone user displayed two previously uncharacterized metabolites, 4-COOH-nor-mephedrone (4-COOH-MC) and dihydro-4- nor-mephedrone (dihydro-4-MC). These results confirm and expand previous studies highlighting the importance of the stereochemistry in the pharmacodynamics of phase-1 metabolites of mephedrone, established their structure-activity relationships at DAT, NET and SERT and pave the way for a systematic dissection of mephedrone metabolic routes. Given the number of structures found having residual and modified pharmacological profiles, these findings may help in understanding the complex subjective effects of administered mephedrone. Moreover, the dissection of mephedrone metabolic routes may help in developing new therapies for treating psychostimulants acute intoxications.