Preference for Distinct Functional Conformations of the Dopamine Transporter Alters the Relationship between Subjective Effects of Cocaine and Stimulation of Mesolimbic Dopamine

Abstract

Background Subjective effects related to cocaine abuse are primarily mediated by blockade of the dopamine (DA) transporter (DAT). The present study assessed the hypothesis that different conformational equilibria of the DAT regulate differences in extracellular DA induced by structurally diverse DA uptake inhibitors (DUI) and their cocaine-like subjective effects. Methods The relationship between cocaine-like subjective effects and stimulation of mesolimbic-DA levels by standard-DUIs (cocaine, methylphenidate, WIN35,428), and atypical-DUIs (benztropine analogs: AHN1-055, AHN2-005, JHW-007) was investigated using cocaine-discrimination and DA-microdialysis procedures in rats. Results All drugs stimulated DA-levels showing different time-courses and maximal effects. Standard-DUIs, which preferentially bind to the outward-facing DAT-conformation, fully substituted for cocaine, consistently producing those subjective effects at DA levels of 100-125% over basal values, regardless of dose or pretreatment time. The atypical-DUIs, with DAT binding minimally affected by DAT conformation, produced inconsistent cocaine-like subjective effects. Full effects were obtained, if at all, only at a few doses and pretreatment times, and at DA-levels 600-700% greater than basal values. Importantly, the linear, time-independent, relationship between cocaine-like subjective effects and stimulation of DA-levels, obtained with standard DUIs was not obtained with the atypical-DUIs. Conclusions These results suggest a time-related desensitization process underlying the reduced cocaine subjective effects of atypical-DUIs that may be differentially induced by the binding modalities identified using molecular approaches. Since the DAT is the target of several drugs for treating neuropsychiatric disorders, such as ADHD, these results help to identify safe and effective medications with minimal cocaine-like subjective effects that contribute to abuse liability.

Substrate and Inhibitor–Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry

“…This is of interest because seemingly subtle differences in the binding mechanism of SLC6 inhibitors have been demonstrated to have a profound influence on their in vivo effects. For example, the stimulant dopamine transporter inhibitor cocaine has been suggested to stabilize the outward-facing open conformation, whereas another class of dopamine transporter inhibitors, the benztropines, which lack stimulant effects, stabilize the outward-facing occluded conformation (Beuming et al, 2008;Loland et al, 2008;Kohut et al, 2014). Thus, determination of protein movements upon ligand binding may be an important aspect for understanding the molecular basis for such differences.…”

“…Current insight into mammalian SLC6 transporter conformations stabilized by drugs is mainly based on biochemical approaches, such as cysteine accessibility or protease and alkylation protection analyses (Zhang and Rudnick, 2006;Jacobs et al, 2007;Tavoulari et al, 2009;Koldso et al, 2013;Gaffaney et al, 2014) that do not offer direct experimental information on specific intraprotein motions occurring during drug binding. Studies of protein dynamics in purified bacterial SLC6 homologs using Förster resonance energy transfer and electron paramagnetic resonance spectroscopy techniques have identified specific motions associated with alternating access (Kazmier et al, 2014;Kohut et al, 2014); however, these techniques have not been available to study mammalian transporters. Voltage-clamp fluorometry (VCF) is a technique that provides real-time correlation between conformational and functional states of electrogenic membrane proteins in a native membrane environment (Gandhi and Olcese, 2008).…”

Behavioral, biological, and chemical perspectives on atypical agents targeting the dopamine transporter

“…An initial hypothesis derived from studies of the analogs of BZT focused on their relatively slow rate of association compared to that of cocaine. Recently, Kohut et al (2014) suggested that slower DAT association may create conditions favorable to desensitization of postsynaptic DA receptors that, in turn, would be less effective in transmitting DA signals. Such a desensitization process agrees with the low efficacy of atypical DAT inhibitors with regard to many of the often observed stimulant effects.…”

“…Coupled with pharmacokinetic studies indicating that many of the BZT analogs appear in brain within minutes of injection (Raje et al, 2003) at concentrations exceeding their in vitro K i values, the studies suggest a slow association rate for BZT analogs that was confirmed in vitro (Kopajtic et al, 2010). One hypothesis is that a slow association rate allows for compensatory mechanisms that blunt the effects of cocaine (Kohut et al 2014). A DAT inhibitor with a faster association with the DAT would not allow for that compensatory change.…”

Cocaine Antagonists; Studies on Cocaine Self-Administration

“…Studies on in vivo binding to DAT demonstrated slower apparent rates of occupancy with the DAT by several cocaine antagonists AHN 2-005, JHW 007, and RTI-371 relative to the standard dopamine uptake inhibitors cocaine, GBR 12909 or RTI-336 [3,[14][15][16]. Thus the slower association rates with DAT might result in a cocaine-antagonist action.…”