Ethanol Alters Endosomal Recycling of Human Dopamine Transporters

Abstract

Dynamic membrane trafficking of the monoamine dopamine transporter (DAT) regulates dopaminergic signaling. Various intrinsic and pharmacological modulators can alter this trafficking. Previously we have shown ethanol potentiates in vitro DAT function and increases surface expression. However, the mechanism underlying these changes is unclear. In the present study, we found ethanol directly regulates DAT function by altering endosomal recycling of the transporter. We defined ethanol action on transporter regulation by [ 3 H]DA uptake functional analysis combined with biochemical and immunological assays in stably expressing DAT HEK-293 cells. Shortterm ethanol exposure potentiated DAT function in a concentration-, but not time-dependent manner. This potentiation was accompanied by a parallel increase in DAT surface expression. Ethanol had no effect on function or surface localization of the ethanol-insensitive mutant (G130T DAT), suggesting a trafficking-dependent mechanism in mediating the ethanol sensitivity of the transporter. The ethanol-induced increase in DAT surface expression occurred without altering the overall size of DAT endosomal recycling pools. We found ethanol increased the DAT membrane insertion rate while having no effect on internalization of the transporter. Ethanol had no effect on the surface expression or trafficking of the endogenously expressing transferrin receptor, suggesting ethanol does not have a nonspecific effect on endosomal recycling. These results define a novel trafficking mechanism by which ethanol regulates DAT function. Dopamine (DA),2 a major central nervous system neurotransmitter, is involved in reward and reinforcing behaviors. DA signaling relies on a critical balance between release and removal of the neurotransmitter within synaptic clefts. Drugs of abuse, including psychostimulants and ethanol, cause maladaptive changes in DA signaling in mesolimbic areas of the brain, leading to addictive behaviors. Localized on pre-synaptic dopaminergic terminals, the dopamine transporter (DAT) is responsible for terminating DA signaling by rapidly removing the transmitter from the synaptic cleft region (1). DAT and other monoamine transporters, including norepinephrine, ␥-amino butyric acid, and serotonin (NET, GAT1, and SERT, respectively) clear extracellular transmitter via a reuptake mechanism (2).Regulation of DAT function is mediated by recycling of the transporters between intracellular compartments and the plasma membrane (3). This dynamic trafficking occurs in both a constitutive and regulated manner to increase or decrease the number of transporters on the cell surface that are available for transmitter reuptake. Trafficking modulators, such as activated protein kinase C (PKC), have been shown to alter basal transporter trafficking rates. PKC-mediated regulation causes an intracellular accumulation of DAT by increasing internalization and decreasing insertion of the transporter on the cell surface (4). In addition to intrinsic transporter modulators, various drugs of abus...

Chronic exposure to ethanol of male mice before mating produces attention deficit hyperactivity disorder‐like phenotype along with epigenetic dysregulation of dopamine transporter expression in mouse offspring

“…The effects of EtOH on DAT were investigated by several researchers. Methner and Mayfield () found that EtOH directly regulates DAT function by altering endosomal recycling of the transporter. For adult rats, it was shown that EtOH increases the number of DAT binding sites in numerous brain regions, including olfactory tubercle, striatum, hippocampus, ventral tegmental area, and substantia nigra (Jiao et al, ), although different results were obtained in brain regions of prenatally EtOH‐exposed rats (Druse et al, ).…”

Prenatal Alcohol Exposure in Rodents As a Promising Model for the Study of ADHD Molecular Basis

“…On the other hand, DAergic systems, which have neurocircuitries that overlap the HPA axis, are altered by PAE (Uban et al, 2013). PAE negatively affects DA biosynthesis and transport in midbrain neurons (Szot et al, 1999) and direct regulates DAT function by altering endosomal recycling of the transporter (Methner and Mayfield, 2010); also, PAE alters postnatal development of the spontaneous electrical activity of dopamine neurons in the ventral tegmental area (Choong and Shen, 2004). Taken together, all of these evidences lead us to suggest a physiological parallelism, at least in these three neurotransmitter systems (cholinergic, glutamatergic, and DAergic) between PAE and ADHD.…”

Cathepsin Inhibition Prevents Autophagic Protein Turnover and Downregulates Insulin Growth Factor-1 Receptor-Mediated Signaling in Neuroblastoma

“…Primary cathepsin B antibody was obtained from D. J. Buttle, University of Sheffield, UK (Buttle et al, 1988). Antibodies against the following proteins were purchased from manufacturers as listed below: actin (Sigma-Aldrich, St. Louis, MO) (Zhou et al, 2005), calnexin (Millipore, Carrigtwohill, Ireland) (Methner and Mayfield, 2010), EGF receptor (Cell Signaling Technologies, Danvers, MA) (Kawahara et al, 2010), Grb-2 (Cell Signaling Technologies) (Benitez et al, 2011), IGF-1R (Cell Signaling) (Michels et al, 2013), IRS-2 (Cell Signaling) (Gao et al, 2014), Lamp-1 (Cell Signaling), LC-3B (Cell Signaling) (Tong et al, 2012), MAPK (Cell Signaling) (Michels et al, 2013), Akt (Cell Signaling), p-IGF-1Rb (Cell Signaling), p-MAPK (Cell Signaling), p-Akt (Cell Signaling) (Michels et al, 2013), p-Shc A (Cell Signaling) (Krall et al, 2011), RAS (Cell Signaling), Shc A (Cell Signaling), N-Shc (BD Biosciences, San Jose, CA) (Tarr et al, 2002). The secondary goat fluor-568 anti-rabbit antibody was from Invitrogen.…”