Amphetamine induces dopamine efflux through a dopamine transporter channel

Abstract

Drugs of abuse, including cocaine, amphetamine (AMPH), and heroin, elevate extracellular dopamine (DA) levels in the brain, thereby altering the activity͞plasticity of reward circuits and precipitating addiction. The physiological release of DA occurs through the calcium-dependent fusion of a synaptic vesicle with the plasma membrane. Extracellular DA is cleared by uptake through the Na ؉ ͞Cl ؊ -dependent DA transporter (DAT). In contrast, the substrate AMPH induces nonvesicular release of DA mediated by DAT. Extracellular AMPH is generally believed to trigger DA efflux through DAT by facilitating exchange for cytosolic DA. Here, in outside-out patches from heterologous cells stably expressing DAT or from dopaminergic neurons, by using ionic conditions in the patch pipette that mimic those produced by AMPH stimulation, we report that AMPH causes DAT-mediated DA efflux by two independent mechanisms: (i) a slow process consistent with an exchange mechanism and (ii) a process that results in rapid (millisecond) bursts of DA efflux through a channel-like mode of DAT. Because channel-like release of DA induced by AMPH is rapid and contains a large number of DA molecules, with a single burst of DA on par with a quantum of DA from exocytotic release of a vesicle, this burst mode of release may play a role in the synaptic actions and psychostimulant properties of AMPH and related compounds. Unlike AMPH, the endogenous substrate DA, when present on both sides of the plasma membrane, inhibits this channel-like activity, thereby suggesting that the DAT channel-like mode cannot accumulate DA against a concentration gradient.patch clamp ͉ amperometry D opamine (DA) transporter (DAT) is a member of a gene family that includes norepinephrine transporter (NET), serotonin transporter (SERT), and ␥-aminobutyric acid transporter 1 (GAT-1) (1-3). These neurotransmitter transporters are thought to play an important role in the reuptake (3, 4) and release (5, 6) of neurotransmitters. Their mechanism of transport is often described by using an alternating access model (7). In this model, the binding of cargo (DA, Na ϩ , and Cl Ϫ ) to an extracellularly oriented transporter induces a conformational rearrangement to an intracellularly oriented transporter from which the cargo is released into the cytosol, thereby completing the transport process. Consistent with this model, the recent crystal structure of the nonhomologous secondary transporter lactose permease (8) showed the transporter in what was inferred to be the ''inward-facing state'' with substrate bound within a cytoplasmic vestibule and no access route to the ''extracellular'' space.In the case of DAT, DA transport generates an electrical current because of the net movement of positive charge into the cell (9). DA efflux induced by the psychostimulant amphetamine (AMPH) is believed to result from the ability of AMPH to reverse this inward transport process, in that the inward transport of AMPH by DAT increases the number of ''inward-facing'' transporter binding sites and there...

Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson disease—resemblance to the effect of amphetamine drugs of abuse

“…This leads to an increase in the intracellular binding sites of DAT for dopamine, resulting in the exchange of extracellular amphetamine by intracellular dopamine, and leading to an increase in extracellular dopamine [289]. When present in higher extracellular concentrations, amphetamine may diffuse into the cell, due to its lipophilicity [290,291]. Amphetamine also interferes with VMATͲ2 function, impairing the active transport of the monoamines into synaptic vesicles, where they are stored.…”

Cellular and molecular mechanisms involved in the neurotoxicity of opioid and psychostimulant drugs

“…At low concentrations, amphetamine is transported by the DAT to the cytosol and increases the intracellular binding sites of the DAT for dopamine, resulting in the exchange of extracellular amphetamine by intracellular dopamine, and leading to an increase in extracellular dopamine (Jones et al, 1999). When present at higher extracellular concentrations, amphetamine may diffuse into the cell, due to its lipophilicity (Sulzer et al, 1995;Kahlig et al, 2005).…”

Dopamine Transporter Activity Is Modulated by α-Synuclein

“…Data were adapted from previous methods by Saha et al (31); midbrain tissues were dissected from C57Bl/6 mice on postnatal days 0 -3 (P0 -3) (The Jackson Laboratory) and were isolated and grown on a monolayer of glial cells on poly-D-lysinetreated glass-bottom dishes in neuronal media 10 containing 25 mg/ml insulin, 100 mg/2 ml of GBSS transferring, 45% glucose, and minimum Eagle's medium (Gibco) as we have described previously (9,30).…”