The main purpose of the present investigation was to examine the effects of lengthening the amine substituent of 4-MA on drug interactions at monoamine transporters and the resulting behavioral consequences. We observed that increasing the N-alkyl chain length of 4-MA from N-methyl to N-butyl decreased the potency to inhibit uptake at DAT, NET, and SERT in rat brain synaptosomes. Similarly, the potency of substrate activity at monoamine transporters decreased as the N-alkyl group was elongated, but the efficacy to promote release in synaptosomes differed markedly across the three transporters. In general, increasing the N-alkyl chain tended to convert DAT and NET substrates to inhibitors. For instance, when the amine substituent was lengthened from N-methyl to N-ethyl, release efficacy at DAT dropped from ~100% to ~60% of maximal, respectively. N-propyl and N-butyl analogs displayed <30% releasing efficacy at DAT consistent with the effects of DAT inhibitors. In contrast, N-methyl, N-ethyl, and N-propyl 4-MA displayed maximal or near-maximal release efficacy (80–100% of maximal) at SERT consistent with the profile of substrates. N-butyl 4-MA appeared to display no releasing activity in synaptosomes, but this lack of effect was most likely because of its extremely low potency. The structure–activity findings shown here are consistent with prior data demonstrating that increasing the steric bulk of amine substituents on amphetamine analogs converts substrates at DAT and NET to nontransported inhibitors (Rothman et al, 2012; Sandtner et al, 2016).

Previous investigations in synaptosomes and transporter-expressing cells have shown that potent uptake inhibitors can induce low-efficacy efflux of preloaded [3H]MPP+ in release assays, in the range of 20–30% of maximal releasing response (Baumann et al, 2013b; Scholze et al, 2000). Low-efficacy efflux of this magnitude, or ‘pseudoefflux’, is due to the diffusion of [3H]MPP+ out of synaptosomes that is unmasked in the presence of transporter blockade. Under these circumstances, the effects of transporter inhibitors in release assays could be erroneously interpreted as bona fide transporter-mediated reverse transport. Other studies in rat brain synaptosomes have identified transporter ligands exhibiting more substantial ‘partial releasing’ effects, in the range of 50–75% of maximal releasing response, similar to the effects of N-ethyl 4-MA at DAT observed here (Rothman et al, 2012; Sandtner et al, 2016). As a specific example, we demonstrated previously that 3,4-methylenedioxyethamphetamine (MDEA), the N-ethyl analog of 3,4-methylenedioxyamphetamine (MDA), induces partial release at DAT with ∼60% efficacy (Rothman et al, 2012). The precise molecular underpinnings of transporter-mediated partial release are not fully understood, but our prior data provide compelling evidence that the blunted releasing efficacy of MDEA at DAT is because of a much slower rate of [3H]MPP+ efflux (ie, a slower rate of reverse transport) when compared with full-efficacy releasers like MDA. The present findings with N-ethyl 4-MA reveal an important caveat of the synaptosome assays: methods using rat brain tissue cannot definitively discriminate transporter inhibitors from substrates when drugs display partial releasing actions. To further explore the mechanism of action of 4-MA analogs at transporters, we employed additional methods that measure different aspects of transporter activity.

By using the TEVC technique in X. laevis oocytes overexpressing monoamine transporters, we previously showed that transportable substrates produce large Na+-dependent inward currents, whereas nontransported inhibitors do not. In fact, transporter inhibitors evoke an apparent outward current because of blockade of the endogenous transporter ‘leak’ current (Cameron et al, 2013; Rodriguez-Menchaca et al, 2012; Solis, 2017; Solis et al, 2012). As expected, compounds eliciting maximal release efficacy in synaptosomes (eg, N-methyl 4-MA at DAT) also produced large inward currents through hDAT. In contrast, N-ethyl 4-MA displayed outward currents at DAT consistent with a nontransported inhibitor, and elongating to N-propyl or N-butyl 4-MA induced sequentially smaller inhibitor-like currents at DAT. The inhibitor-like outward currents produced by N-ethyl 4-MA in DAT-expressing oocytes might seem at odds with the partial substrate actions of this compound at DAT in synaptosomes. As noted above, partial releasing properties of N-ethyl analogs like MDEA in synaptosomes are characterized by a substantially slower rate of DAT-mediated efflux when compared with fully efficacious releasers. Thus, one possible explanation for the discordant findings with N-ethyl 4-MA in oocytes vs synaptosomes could be related to the different time courses for each assay. More specifically, the oocyte assays measure transporter-mediated currents on the timescale of seconds, whereas the synaptosome assays measure [3H]substrate efflux on the timescale of minutes. Because of the abbreviated time window of electrophysiological measurements, it seems feasible that only the inhibitor activity of 4-ethyl 4-MA at DAT is observed under the oocyte assay conditions. Other explanations for discrepancies in the data between oocytes and synaptosomes could be the existence of species differences in responsiveness to specific transporter ligands, or intermediate conformational states in transporter proteins that allow some reverse transport in the absence of an inward current. Further investigations are required to address these intriguing possibilities.

In contrast to the divergent effects of 4-MA analogs on DAT-mediated currents, all of the 4-MA analogs produced inward currents consistent with transporter substrates in hSERT-expressing oocytes. Although N-butyl 4-MA displayed no discernable activity at SERT in the synaptosome release assay, it elicited a small inward current through hSERT in oocytes indicative of substrate activity. Although the TEVC technique provides detailed mechanistic information about the action of compounds on transporters, there are significant drawbacks. For instance, hSERT is efficiently expressed in oocytes, whereas hDAT shows significant expression variability among oocyte batches. Moreover, hNET activity is undetectable in oocytes because it does not properly incorporate into oocyte plasma membranes. Thus, the oocyte TEVC system cannot be used for comparison of drug effects across DAT, NET, and SERT.

To overcome limitations of the synaptosome assays and TEVC methods, we employed a fluorometric assay that takes advantage of the electrogenic nature of transporter proteins (Cameron et al, 2015; Ruchala et al, 2014). This assay consists of HEK cell lines that coexpress either hDAT, hNET, or hSERT along with voltage-gated Ca2+ channels (VGCC) and uses the Fura2 dye to measure changes in Ca2+ influx into individual cells. In recent work, we demonstrated that transporter-mediated currents induced by substrates are strong enough to evoke opening of L-type Ca2+ channels in expression systems where transporters and Ca2+ channels are coexpressed (Cameron et al, 2015; Ruchala et al, 2014). Because monoamine transporter currents are electrically coupled to the opening of voltage-gated Ca2+ channels, the magnitude of Ca2+ influx is proportional to the concentration of substrate drug applied, allowing for the generation of reliable dose–response curves and potencies of different drugs to be determined (Cameron et al, 2015). Transporter inhibitors do not induce depolarizing currents and do not produce a Ca2+ signal, but they are able to block Ca2+ signals induced by substrates (Cameron et al, 2015; Ruchala et al, 2014). Using this assay, we first characterized the molecular mechanism of action for N-substituted 4-MA analogs at monoamine transporters, and used these results to classify the compounds as either transporter substrates or inhibitors. We then performed dose–response experiments in which we determined EC 50 values for substrate-type drugs and IC 50 values for inhibitor-type drugs. Importantly, the compounds that elicited maximal releasing efficacy in synaptosomes and inward currents in oocytes also elicited depolarization-induced Ca2+ responses in the fluorescence assay compounds. In contrast, the compounds that behaved as transporter inhibitors or partial releasers in synaptosomes, and behaved as blockers in the electrophysiological assay, did not elicit Ca2+ signals in the fluorescence assay. N-butyl 4-MA, which lacked measurable potency as a SERT substrate in synaptosomes, induced an inward current in oocytes and a positive response in the fluorometric Ca2+ assay. Importantly, the small outward hDAT current induced by N-butyl 4-MA in the TEVC experiments was substantiated by inhibition of dopamine-induced Ca2+ increases. These results highlight the sensitivity of the Ca2+ fluorescence assay to determine the molecular mechanism of action for compounds at monoamine transporters, even those with weak potency that cannot be effectively characterized in the synaptosome-based assays. It is worth mentioning that the differences in potency across the rat synaptosome and the transporter/Ca2+ channel fluorescence assays are likely because of technical differences in methods employed. For example, the IC 50 value for a compound blocking a Ca2+ response elicited by a transmitter depends on the concentration of the transmitter being inhibited (ie, if a lower concentration of transmitter is competed off by a compound, the IC 50 value would be more potent).

When we used the results from the transporter/Ca2+ channel assays to classify compounds as either transporter inhibitors or substrates at DAT, NET, and SERT, we observed remarkable correlations between the potencies obtained from the Ca2+ assays and rat synaptosome methods. More specifically, the IC 50 values for transporter inhibitors in the Ca2+ fluorescence assays were positively correlated with the IC 50 values for transporter inhibitors and partial substrates in the synaptosome uptake inhibition assays. Similarly, the EC 50 values for transporter substrates in the Ca2+ fluorescence assays were positively correlated with fully efficacious substrates in the synaptosome assays. Such correlations validate the Ca2+ fluorescence assay as a suitable and complementary technique to study monoamine transporter pharmacology as compared with the established [3H]transmitter uptake inhibition and release assays. In addition, the fluorometric transporter-Ca2+ channel assay has the added advantage to accurately discern the mechanism of action of a compound, possesses high signal-to-noise ratio, and is amenable to studying dose–response effects for many compounds. From the correlation results, we can make the case that monoamine transporters from distinct mammalian species (rats vs humans) interact similarly with substrates and blockers that differs from drug interactions with nonmammalian monoamine transporters. For example, cocaine has different actions at drosophila DAT and hDAT (Porzgen et al, 2001), and chicken SERT is much less sensitive to reuptake inhibitors than hSERT (Larsen et al, 2004).

Previous studies have identified DAT vs SERT selectivity as one important determinant of abuse-related effects of monoamine transporter ligands in ICSS procedures (Bauer et al, 2013; Bonano et al, 2014, 2015; Hutsell et al, 2016; Miller et al, 2015; Rosenberg et al, 2013), as well as in other behavioral procedures, such as drug self-administration (Czoty et al, 2002; Negus et al, 2007; Rothman et al, 2008; Wang and Woolverton, 2007). In general, transporter ligands with greater DAT selectivity are associated with abuse-related effects, whereas those with greater SERT selectivity tend to produce abuse-limiting effects. The behavioral effects of N-substituted 4-MA analogs in the ICSS experiments were generally consistent with their function at DAT and SERT, and selectivity for DAT vs SERT. In particular, N-methyl 4-MA produced the most potent and robust abuse-related facilitation of ICSS rates, whereas higher doses engendered rate-decreasing effects. This behavioral profile is consistent with the function of N-methyl 4-MA as a nonselective transporter substrate with somewhat greater potency at DAT. The expression of abuse-related effects and overall drug potency declined with increasing N-alkyl chain length that was associated with: (1) loss of selectivity for DAT vs SERT, (2) overall loss of potency at all transporters, and (3) change in function from DAT substrate to inhibitor. Thus, in comparison with N-methyl 4-MA, N-ethyl 4-MA produced weaker and less potent facilitation of ICSS rates, but displayed similar potency for depression of ICSS rates. This profile is consistent with the function of N-ethyl 4-MA as a DAT blocker with lower DAT potency than N-methyl 4-MA and its inverted selectivity for DAT<SERT. N-propyl 4-MA produced only low-potency depression of ICSS rates, agreeing with its low DAT potency and inverted DAT<SERT selectivity. Finally, N-butyl 4-MA also produced only depression of ICSS rates, consistent with its inactivity at DAT; however, N-butyl 4-MA also displayed very low potency at SERT. The finding that N-butyl 4-MA was more potent at depressing ICSS than N-propyl 4-MA, despite having weaker SERT potency, suggests that N-butyl 4-MA induced depression of ICSS rates independent of SERT.

In summary, the converging lines of evidence presented here demonstrate that lengthening the N-alkyl chain of 4-MA decreases potency to inhibit monoamine transporters. Perhaps more importantly, elongating the N-alkyl chain greater than a methyl group converts DAT substrates to inhibitors, whereas elongating the chain greater than an ethyl group converts NET substrates to inhibitors. Thus, the N-ethyl, N-propyl, and N-butyl analogs of 4-MA display hybrid transporter activity characterized by differential mechanisms at DAT, NET, and SERT for the same drug molecule (Blough et al, 2014; Saha et al, 2015). It is important to note that elucidating the complex pharmacology of 4-MA compounds could not have been accomplished without the multipronged approach utilized in our study. Although synaptosome assays provide physiologically relevant data from native tissue, and can discriminate fully efficacious transporter releasers from transporter inhibitors, these methods cannot discern the precise mechanism of action for drugs acting as partial releasers. The electrophysiological and Ca2+ fluorescence assays employed here demonstrate that partial releasers in the synaptosome assays can function as pure transporter inhibitors in cells expressing human transporters. Determining the molecular mechanisms responsible for the apparent disparities between the effects of partial releasers in native tissue preparations vs their effects in cell-based systems warrants further study. The in vivo ICSS data corresponded well with the in vitro data as increasing alkyl chain length reduced abuse-related effects of drugs in parallel with decreased DAT potency. Overall, our findings indicate that the N-alkylated analogs of 4-MA display less abuse liability than the parent compound. Given the continued appearance of stimulant-like NPS in the street drug marketplace, future investigations are warranted to evaluate the pharmacology and toxicology of these substances using an experimental approach that employs complementary in vitro and in vivo methods.