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A major theme of addiction research has focused on the neural substrates of individual differences in the risk for addiction; however, little is known about how vulnerable populations differ from those that are relatively protected. Here, we prospectively measured dopamine (DA) neurotransmission prior to cocaine exposure to predict the onset and course of cocaine use. Using in vivo voltammetry, we first generated baseline profiles of DA release and uptake in the dorsomedial striatum (DMS) and nucleus accumbens of drug-naive male rats prior to exposing them to cocaine using conditioned place preference (CPP) or operant self-administration. We found that the innate rate of DA uptake in the DMS strongly predicted motivation for cocaine and drug-primed reinstatement, but not CPP, responding when “price” was low, or extinction. We then assessed the impact of baseline variations in DA uptake on cocaine potency in the DMS using ex vivo voltammetry in naive rats and in rats with DA transporter (DAT) knockdown. DA uptake in the DMS of naive rats predicted the neurochemical response to cocaine, such that rats with innately faster rates of DA uptake demonstrated higher cocaine potency at the DAT and rats with DAT knockdown displayed reduced potency compared to controls. Together, these data demonstrate that inherent variability in DA uptake in the DMS predicts the behavioral response to cocaine, potentially by altering the apparent potency of cocaine.

The dopamine transporter (DAT) serves a critical role in controlling dopamine (DA)-mediated neurotransmission by regulating the clearance of DA from the synapse and extrasynaptic regions and thereby modulating DA action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DATs to alter their actions resulting in an enhancement in extracellular DA concentrations. We previously identified a novel allosteric site in the DAT and the related human serotonin transporter that lies outside the central orthosteric substrate- and cocaine-binding pocket. Here, we demonstrate that the dopaminergic psychostimulant sydnocarb is a ligand of this novel allosteric site. We identified the molecular determinants of the interaction between sydnocarb and DAT at the allosteric site using molecular dynamics simulations. Biochemical-substituted cysteine scanning accessibility experiments have supported the computational predictions by demonstrating the occurrence of specific interactions between sydnocarb and amino acids within the allosteric site. Functional dopamine uptake studies have further shown that sydnocarb is a noncompetitive inhibitor of DAT in accord with the involvement of a site different from the orthosteric site in binding this psychostimulant. Finally, DA uptake studies also demonstrate that sydnocarb affects the interaction of DAT with both cocaine and amphetamine. In summary, these studies further strengthen the prospect that allosteric modulation of DAT activity could have therapeutic potential.

Augmented vasoconstriction is a hallmark of hypertension and is mediated partly by hyper-stimulation of G protein couple receptors (GPCRs) and downstream signaling components. Although GPCR blockade is a key component of current anti-hypertensive strategies, whether hypertension is better managed by directly targeting G proteins has not been thoroughly investigated. Here, we tested whether inhibiting G(q/11) proteins in vivo and ex vivo using natural cyclic depsipeptide, FR900359 (FR) from the ornamental plant, Ardisia crenata, and YM-254890 (YM) from Chromobacterium sp. QS3666, or it’s synthetic analog, WU-07047 (WU), was sufficient to reverse hypertension in mice. All three inhibitors blocked G protein-dependent vasoconstriction, but to our surprise YM and WU and not FR inhibited K(+)-induced Ca(2+) transients and vasoconstriction of intact vessels. However, each inhibitor blocked whole-cell L-type Ca(2+) channel current in vascular smooth muscle cells. Subcutaneous injection of FR or YM (0.3 mg/kg, s.c.) in normotensive and hypertensive mice elicited bradycardia and marked blood pressure decrease, which was more severe and long lasting after the injection of FR relative to YM (FR(t1/2) ≅ 12 hr vs. YM(t1/2) ≅ 4 hr). In deoxycorticosterone acetate (DOCA)-salt hypertension mice, chronic injection of FR (0.3 mg/kg, s.c., daily for seven days) reversed hypertension (vehicle SBP: 149 ± 5 vs. FR SBP: 117 ± 7 mmHg), without any effect on heart rate. Our results together support the hypothesis that increased LTCC and G(q/11) activity is involved in the pathogenesis of hypertension, and that dual targeting of both proteins can reverse hypertension and associated cardiovascular disorders.

This Unit describes procedures for in vitro uptake assays for the functional characterization of DAT. All assays are performed using commonly available cell lines that transiently or stably express a particular transporter under investigation. The three main assays described here are: kinetic functional assay to calculate apparent affinity (KM) and maximal velocity (Vmax) of radiolabeled DA uptake into cells; dose-response assays to measure potencies (IC50/Ki values) of test compounds for inhibition of transporter function; and efflux assay to determine the ability and potency (EC50) of a ligand to elicit reverse transport of accumulated labeled DA in the cells. These procedures can be reproduced in any pharmacological research laboratory with minimal expertise and resources. Although the methods are described using DAT and its ligands, the same protocol can be directly applied to SERT and NET using their respective ligands.

The three human monoamine transporters (MATs) i.e. the dopamine transporter (DAT), the serotonin transporter (SERT) and the norepinephrine transporter (NET) are primarily responsible for controlling the neurotransmission mediated by their respective monoamine substrates, dopamine (DA), serotonin (5-HT) and norepinephrine (NE). MATs terminate the action of the neurotransmitters by translocating them from the synaptic space back into the presynaptic neurons. These neurotransmitters are responsible for controlling a number of physiological, emotional and behavioral functions and their transporters are a target of many marketed drugs prescribed for treatment and relief of disorders such as depression, anxiety, ADHD, schizophrenia, and psychostimulant abuse. This unit provides an overview of the location and regulation of the MATs and the structural aspects that mediate the translocation process. A brief description of the evolution of ligands that target these transporters is provided as well as more current insights regarding pharmacological effects, mechanisms of synthetic substrates, inhibitors and allosteric modulators. This unit also summarizes known data and evidence that provide structural details on the existence and significance of allosteric binding sites other than the natural substrate binding site. The purpose of this unit is to introduce researchers new to this field to basic background information on MATs, and the significance and application of unique effects elicited by ligands that interact with these transporters.

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), βLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.

Towards addressing the knowledge gap of how bupropion interacts with the dopamine transporter (DAT) and nicotinic acetylcholine receptors (nAChRs), a ligand was synthesized in which the chlorine of bupropion was isosterically replaced with an iodine and a photoreactive azide was added to the 4'-position of the aromatic ring. Analog (±)-3 (SADU-3-72) demonstrated modest DAT and α4β2 nAChR affinity. A radioiodinated version was shown to bind covalently to hDAT expressed in cultured cells and affinity-purified, lipid-reincorporated human α4β2 neuronal nAChRs. Co-incubation of (±)-[(125)I]-3 with non-radioactive (±)-bupropion or (-)-cocaine blocked labeling of these proteins. Compound (±)-[(125)I]-3 represents the first successful example of a DAT and nAChR photoaffinity ligand based on the bupropion scaffold. Such ligands are expected to assist in mapping bupropion-binding pockets within plasma membrane monoamine transporters and ligand-gated nAChR ion channels.

Non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored in contrast to tropane-based compounds such as cocaine. In order to fill this knowledge gap, a ligand was synthesized in which the aromatic ring of pyrovalerone was substituted with a photoreactive azido group. The analog 1-(4-azido-3-iodophenyl)-2-pyrrolidin-1-yl-pentan-1-one demonstrated appreciable binding affinity for the DAT (Ki = 78 ± 18 nM), suggesting the potential utility of a radioiodinated version in structure-function studies of this protein.