Multi-modal antidepressant-like action of 6- and 7-chloro-2-aminodihydroquinazolines in the mouse tail suspension test.

RATIONALE: 2-Amino-6-chloro-3,4-dihydroquinazoline (e.g., A6CDQ) represents a novel putative antidepressant originally thought to act through a 5-HT3 serotonin receptor antagonist mechanism. Here, we investigated this further by examining a positional isomer of A6CDQ (i.e., A7CDQ). MATERIALS AND METHODS: 5-HT3 receptor and transporter activity (uptake-1 and uptake-2) were investigated using a variety of in vitro assays and the in vivo mouse tail suspension test (TST). RESULTS: Although A7CDQ binds at 5-HT3 receptors with low affinity (Ki = 1975 nM) compared to A6CDQ (Ki = 80 nM), it retained 5-HT3 receptor antagonist action (IC50 = 5.77 and 0.26 µM, respectively). In the mouse TST A7CDQ produced antidepressant-like actions (ED50 = 0.09 mg/kg) comparable to that of A6CDQ. In addition, A6CDQ was found to be a 5-HT releasing agent (Km = 2.8 µM) at hSERT and a reuptake inhibitor (IC50 = 1.8 µM) at hNET, whereas A7CDQ was a weak reuptake inhibitor (Km = 43.6 µM) at SERT but a releasing agent (EC50 = 3.3 µM) at hNET. Moreover, A6CDQ and A7CDQ were potent inhibitors of uptake-2 (e.g.; OCT3 IC50 = 3.9 and 5.9 µM, respectively). CONCLUSIONS: A simple shift of a substituent in a common quinazoline scaffold from one position to another (i.e., a chloro group from the 6- to the 7-position) resulted in a common action in the TST but via a somewhat different mechanism. A6CDQ and A7CDQ might represent the first members of a new class of potential antidepressants with a unique multi-modal mechanism of action.

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.

Monoamine Transporters as Ionotropic Receptors.

It is well established that glutamate and GABA signal through both ionotropic and metabotropic receptors. Conversely, it is thought that, with one exception, monoamines (dopamine, serotonin, and norepinephrine) signal via metabotropic receptors. Given their capacity to generate fast-acting currents, I suggest that the monoamine transporters should be considered as ionotropic receptors.

A current view of serotonin transporters.

Serotonin transporters (SERTs) are largely recognized for one aspect of their function-to transport serotonin back into the presynaptic terminal after its release. Another aspect of their function, however, may be to generate currents large enough to have physiological consequences. The standard model for electrogenic transport is the alternating access model, in which serotonin is transported with a fixed ratio of co-transported ions resulting in net charge per cycle. The alternating access model, however, cannot account for all the observed currents through SERT or other monoamine transporters.  Furthermore, SERT agonists like ecstasy or antagonists like fluoxetine generate or suppress currents that the standard model cannot support.  Here we survey evidence for a channel mode of transport in which transmitters and ions move through a pore. Available structures for dopamine and serotonin transporters, however, provide no evidence for a pore conformation, raising questions of whether the proposed channel mode actually exists or whether the structural data are perhaps missing a transient open state.

Chloride requirement for monoamine transporters.

This review focuses on the Cl(-) requirement for dopamine, serotonin, and norepinephrine (DA, 5-HT, and NE) transport and induced current via the transporters for these transmitters, DAT, SERT, and NET. Indirect evidence exists for the passage of Cl(-) ions through monoamine transporters; however, direct evidence is sparse. An unanswered question is why in some preparations, notably native neurons, it appears that Cl(-) ions carry the current through DAT, whereas in heterologous expression systems Na(+) ions carry the current often referred to as the uncoupled current. It is suggested that different functional states in monoamine transporters represent conformational states that carry dominantly Cl(-) or Na(+). Structures of monoamine transporters contribute enormously to structure-function relationships; however, thus far no structural features support the functionally relevant ionic currents that are known to exist in monoamine transporters.

Amphetamine activates calcium channels through dopamine transporter-mediated depolarization.

Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca(2+) channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca(2+) channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca(2+) channels (CaV1.3 and CaV1.2) but not the N-type Ca(2+) channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca(2+) signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca(2+) channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca(2+) channel activation.

Structural analysis of dopamine- and amphetamine-induced depolarization currents in the human dopamine transporter.

Amphetamine (AMPH) induces depolarizing currents through the human dopamine transporter (hDAT). Recently we discovered that the S(+) enantiomer of AMPH induces a current through hDAT that persists long after its removal from the external milieu. The persistent current is less prominent for R(-)AMPH and essentially absent for dopamine (DA)-induced currents. Related agents such as methamphetamine also exhibit persistent currents, which are present in both frog oocyte and mammalian HEK expression systems. Here, we study hDAT-expressing Xenopus laevis oocytes voltage-clamped and exposed from outside to DA, S(+)AMPH, R(-)AMPH, and related synthesized compounds, including stereoisomers. The goal of the study was to determine how structural transitioning from dopamine to amphetamine influences hDAT potency and action. At saturating concentrations, S(+)AMPH or R(-)AMPH induce a sharply rising depolarizing current from -60 mV that is comparable in amplitude to DA-induced currents. The magnitude and duration of the currents and the presence or absence of persistent currents depend on the concentration, duration of exposure, and chemical structure and enantiomeric versions of the agents.

Electrical coupling between the human serotonin transporter and voltage-gated Ca(2+) channels.

Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca(2+) mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca(2+) permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca(2+) channels (CaV). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in CaV1.1-null muscle cells, thus implicating Ca(2+) channels. CaV1.3 and CaV2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type CaV1.3 channel show Ca(2+) transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and CaV1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca(2+) channels such as CaV1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca(2+)-driven signals in excitable cells.

Synthetic cathinones: chemical phylogeny, physiology, and neuropharmacology.

This mini-review summarizes the history of cathinone and its synthesized derivatives from early records to the present day, including the appearance of synthetic cathinones in the drug combination known as bath salts. Bath salts may consist of one compound (MDPV) or combinations of MDPV and one or more other synthetic cathinones, which may also appear alone without MDPV. We briefly review recent in vitro studies of bath salts components alone or in combination, focusing on pharmacological and biophysical studies. Finally we summarize new data from in vivo procedures that characterize the abuse-related neurochemical and behavioral effects of synthetic cathinones in rats.

"Deconstruction" of the abused synthetic cathinone methylenedioxypyrovalerone (MDPV) and an examination of effects at the human dopamine transporter.

Synthetic cathinones, ß-keto analogues of amphetamine (or, more correctly, of phenylalkylamines), represent a new and growing class of abused substances. Several such analogues have been demonstrated to act as dopamine (DA) releasing agents. Methylenedioxypyrovalerone (MDPV) was the first synthetic cathinone shown to act as a cocaine-like DA reuptake inhibitor. MDPV and seven deconstructed analogues were examined to determine which of MDPV's structural features account(s) for uptake inhibition. In voltage-clamped (-60 mV) Xenopus oocytes transfected with the human DA transporter (hDAT), all analogues elicited inhibitor-like behavior shown as hDAT-mediated outward currents. Using hDAT-expressing mammalian cells we determined the affinities of MDPV and its analogues to inhibit uptake of [3H]DA by hDAT that varied over a broad range (IC50 values ca. 135 to >25,000 nM). The methylenedioxy group of MDPV made a minimal contribution to affinity, the carbonyl group and a tertiary amine are more important, and the extended α-alkyl group seems most important. Either a tertiary amine, or the extended α-alkyl group (but not both), are required for the potent nature of MDPV as an hDAT inhibitor.

Mephedrone and methylenedioxypyrovalerone (MDPV), major constituents of "bath salts," produce opposite effects at the human dopamine transporter.

RATIONALE: Psychoactive "bath salts" represent a relatively new drug of abuse combination that was placed in Schedule I in October 2011. Two common ingredients of bath salts include the cathinone analogs: mephedrone and methylenedioxypyrovalerone (MDPV). The mechanism of action of these synthetic cathinone analogs has not been well investigated. MATERIALS AND METHODS: Because cathinone and methcathinone are known to act as releasing agents at the human dopamine transporter (hDAT), mephedrone and MDPV were investigated at hDAT expressed in Xenopus oocytes. RESULTS: Whereas mephedrone was found to have the signature of a dopamine-releasing agent similar to methamphetamine or methcathinone, MDPV behaved as a cocaine-like reuptake inhibitor of dopamine. CONCLUSIONS: Mephedrone and MDPV produce opposite electrophysiological signatures through hDAT expressed in oocytes. Implications are that the combination (as found in bath salts) might produce effects similar to a combination of methamphetamine and cocaine.

Bath salts components mephedrone and methylenedioxypyrovalerone (MDPV) act synergistically at the human dopamine transporter.

BACKGROUND AND PURPOSE: Bath salts is the street name for drug combinations that contain synthetic cathinone analogues, among them possibly mephedrone (MEPH) and certainly methylenedioxypyrovalerone (MDPV). In animal studies, cathinone and certain cathinone analogues release dopamine (DA), similar to the action of amphetamine (AMPH) and methamphetamine (METH). AMPH and METH act on the human DA transporter (hDAT); thus, we investigated MEPH and MDPV acting at hDAT. EXPERIMENTAL APPROACH: We recorded electrical currents mediated by hDAT expressed in Xenopus laevis oocytes and exposed to: DA, METH, a known hDAT stimulant and DA releaser, MEPH, MDPV, MEPH + MDPV, or cocaine, a known hDAT inhibitor. KEY RESULTS: DA, METH and MEPH induce an inward current (depolarizing) when the oocyte is held near the resting potential (-60 mV), therefore acting as excitatory hDAT substrates. Structurally analogous MDPV induces an outward (hyperpolarizing) current similar to cocaine, therefore acting as an inhibitory non-substrate blocker. CONCLUSIONS AND IMPLICATIONS: Two components of bath salts, MEPH and MDPV, produce opposite effects at hDAT that are comparable with METH and cocaine, respectively. In our assay, MEPH is nearly as potent as METH; however, MDPV is much more potent than cocaine and its effect is longer lasting. When applied in combination, MEPH exhibits faster kinetics than MDPV, viz., the MEPH depolarizing current occurs seconds before the slower MDPV hyperpolarizing current. Bath salts containing MEPH (or a similar drug) and MDPV might then be expected initially to release DA and subsequently prevent its reuptake via hDAT. Such combined action possibly underlies some of the reported effects of bath salts abuse.

4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter.

Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now, their use has mostly been successful to study the catecholamine transporters but not the serotonin (5HT) transporter. Here, we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP(+)) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP(+) analogs tested. APP(+) uptake is Na(+)- and Cl(-)-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP(+) specifically monitors hSERT activity. ASP(+), which was previously used to study catecholamine transporters, is 10 times less potent than APP(+) at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to -60 mV, APP(+) induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP(+) is a substrate, whereas ASP(+) induced hSERT-mediated outward currents and counteracted 5HT-induced hSERT currents, indicating ASP(+) possesses activity as an inhibitor. Extra-precise ligand receptor docking of APP(+) and ASP(+) in an hSERT homology model showed both ASP(+) and APP(+) docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP(+) is better suited than ASP(+) to study hSERT transport fluorometrically.

S(+)amphetamine induces a persistent leak in the human dopamine transporter: molecular stent hypothesis.

BACKGROUND AND PURPOSE: Wherever they are located, dopamine transporters (DATs) clear dopamine (DA) from the extracellular milieu to help regulate dopaminergic signalling. Exposure to amphetamine (AMPH) increases extracellular DA in the synaptic cleft, which has been ascribed to DAT reverse transport. Increased extracellular DA prolongs postsynaptic activity and reinforces abuse and hedonic behaviour. EXPERIMENTAL APPROACH: Xenopus laevis oocytes expressing human (h) DAT were voltage-clamped and exposed to DA, R(-)AMPH, or S(+)AMPH. KEY RESULTS: At -60mV, near neuronal resting potentials, S(+)AMPH induced a depolarizing current through hDAT, which after removing the drug, persisted for more than 30 min. This persistent leak in the absence of S(+)AMPH was in contrast to the currents induced by R(-)AMPH and DA, which returned to baseline immediately after their removal. Our data suggest that S(+)AMPH and Na(+) carry the initial S(+)AMPH-induced current, whereas Na+ and Cl(-) carry the persistent leak current. We propose that the persistent current results from the internal action of S(+)AMPH on hDAT because the temporal effect was consistent with S(+)AMPH influx, and intracellular S(+)AMPH activated the effect. The persistent current was dependent on Na(+) and was blocked by cocaine. Intracellular injection of S(+)AMPH also activated a DA-induced persistent leak current. CONCLUSIONS AND IMPLICATIONS: We report a hitherto unknown action of S(+)AMPH on hDAT that potentially affects AMPH-induced DA release. We propose that internal S(+)AMPH acts as a molecular stent that holds the transporter open even after external S(+)AMPH is removed. Amphetamine-induced persistent leak currents are likely to influence dopaminergic signalling, DA release mechanisms, and amphetamine abuse.

Amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in vivo.

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca(2+)](i) from internal stores compared with AMPH. [Ca(2+)](i) responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca(2+)](i) and for the enhanced effects of METH on [Ca(2+)](i) elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca(2+) responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.

Targeting the human serotonin transporter (hSERT) with quantum dots.

In this paper we report our work on the development of a human serotonin transporter (hSERT) antagonist that can be conjugated to quantum dots. This approach has been used to target and visualize the human serotonin transporter protein (hSERT). We demonstrate that labeling is blocked by the addition of high affinity hSERT antagonists such as paroxetine. This approach may be useful for the development of fluorescent assays to study the location and temporal dynamics of biogenic amine transporters and also holds promise for the development of plate-based high throughput assays used to identify novel transporter antagonists.

Na+, Cl-, and pH dependence of the human choline transporter (hCHT) in Xenopus oocytes: the proton inactivation hypothesis of hCHT in synaptic vesicles.

The recent cloning of the human choline transporter (hCHT) has allowed its expression in Xenopus laevis oocytes and the simultaneous measurement of choline transport and choline-induced current under voltage clamp. hCHT currents and choline transport are evident in cRNA-injected oocytes and significantly enhanced by the hCHT trafficking mutant L530A/V531A. The charge/choline ratio of hCHT varies from 10e/choline at -80 mV to 3e/choline at -20 mV, in contrast with the reported fixed stoichiometry of the Na+-coupled glucose transporter in the same gene family. Ion substitution shows that the choline uptake and choline-induced current are Na+ and Cl- dependent; however, the reversal potential of the induced current suggests a Na+-selective mechanism, consigning Cl- to a regulatory role rather than a coupled, cotransported-ion role. The hCHT-specific inhibitor hemicholinium-3 (HC-3) blocks choline uptake and choline-induced current; in addition, HC-3 alone reveals a constitutive, depolarizing leak current through hCHT. We show that external protons reduce hCHT current, transport, and binding with a similar pKa of 7.4, suggesting proton titration of residue(s) that support choline binding and transport. Given the localization of the choline transporter to synaptic vesicles, we propose that proton inactivation of hCHT prevents acetylcholine and proton leakage from the acidic interior of cholinergic synaptic vesicles. This mechanism would allow cholinergic, activity-triggered delivery of silent choline transporters to the plasma membrane, in which normal pH would reactivate the transporters for choline uptake and subsequent acetylcholine synthesis.