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Schmitt & Reith DAT Regulation DAT-lacking animals. In particular, cocaine’s interaction with the serotonin transporter (SERT) has been hypothesized to contribute to continued self-administration in DAT-knockout animals. 7,8 Nevertheless, convincing preclinical evidence implicating specific activity at the DAT in cocaine dependence comes from the observation that the reinforcing effect of cocaine is lost in transgenic mice expressing a triple point–mutated DAT with preserved (albeit somewhat reduced) substrate translocation but little appreciable affinity for cocaine. 9 It is conceivable that elevated basal dopaminergic tone in these mice (a result of their reducedfunction DAT isoform) causes adaptive changes, altering the response to cocaine; however, knockdown mutant mice with a ∼90% reduction in DAT expression—but with functionally unmodified DATs and elevated basal dopamine—still exhibit robust, wild-type–like preference for cocaine, indicating that interaction with the DAT is necessary for the reinforcing effects of cocaine in animals that carry the DAT. 10 Moreover, a recent investigation by Thomsen et al. showed that a different strain of DAT –/– (DAT knockout) mice generally fail to acquire intravenous cocaine self-administration behavior. 11 Importantly, in the minority of mice that do initially show self-administration, this behavior is quickly extinguished as the response requirement (the “work” required to obtain a dose of cocaine) is increased. In contrast, both wild-type and SERT –/– mice maintain consistent self-administration, even when the response requirement for a single dose of intravenous cocaine is increased 50- to 100-fold. Unlike cocaine, other dopaminergic compounds (i.e., the dopamine D1-like receptor agonist SKF82958) and food rewards are comparably administered by DAT –/– and wild-type mice, indicating that elimination of the DAT does not globally hinder behavioral reinforcement. These data strongly suggest that cocaine is not a reliable reinforcer in the absence of the DAT, despite some intersubject differences in initial responses among DAT –/– mice. 11 The DAT governs both the duration and magnitude of dopaminergic neurotransmission by actively translocating dopamine from the extracellular space into presynaptic neurons. Extracellular dopamine is also subject to enzymatic catabolism—thus terminating its signaling potential—however, studies of DAT-knockout mice 6,12 have demonstrated that the DAT is the chief arbiter of dopaminergic signaling flux (for review, see Gainetdinov and Caron 3 ). Dopaminergic transmission is volume transmission with an extrasynaptic predominance of dopamine receptors and transporters; the function of the DAT is not to remove dopamine from the synapse but rather to regulate dopamine’s action at extrasynaptic dopamine receptors (see Rice and Cragg 13 for review and highly recommended, updated cartoon of a dopamine synapse). The overall rate of de novo DAT protein synthesis is a relatively slow process— the half-life of striatal transporter proteins, for example, is approximately 2–3 days. 14,15 Hence, it is not surprising that cells use an assortment of posttranslational regulatory strategies to rapidly alter DAT function—without requiring de novo protein synthesis or transcriptional-level modifications—to dynamically shift the tone of dopaminergic activity in response to various endogenous and exogenous stimuli. There are two conceivable manners in which a cell could modulate DAT function “on the fly” (on a time scale of seconds to minutes) in response to changing environmental conditions— either via direct modification of single-transporter parameters, such as intrinsic substrate permeation efficacy, or by affecting the trafficking of the transporter, redistributing DATs between the plasma membrane and intracellular endosomal compartments. 4 Experimental evidence has demonstrated that both processes may play some role in acute DAT regulation, but that redistribution of transporters to and from the cell surface is the predominant mechanism. 16,17 This regulatory method—rapid insertion or removal of plasmalemmal transporter proteins—may seem like a cumbersome way of acutely controlling neurotransmitter signaling; however, it makes logistical sense when we consider the kinetics of the substrate translocation cycle. That is, transporters must undergo a conformational shift between the discrete phases of extracellular substrate binding (outward facing) and cytosolic substrate release (inward facing), limiting the potential maximum uptake velocity to around one substrate molecule per second per transporter. 4 Ion channels, by contrast, can achieve flux velocities on the order of millions of ions per second per channel. 18 Because they offer a far wider dynamic range in “substrate” flux rate, ion channels are more amenable to regulation strategies involving posttranslational modification at the individual protein level than transporters, such as the Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences. 317
DAT Regulation Schmitt & Reith Figure 1. The many parallel factors influencing DAT trafficking and membrane distribution. (A) The DAT undergoes continuous constitutive recycling between the plasma membrane and early endosomal compartments. (B) and (C) Substrates, such as amphetamine trigger internalization of plasmalemmal DATs, whereas cocaine-like inhibitors upregulate surface DAT expression. (D) and (E) Alterations in the phosphorylation state of DAT and associated scaffolding proteins by intracellular signaling cascades modulates transporter trafficking—although direct phosphorylation of DAT proteins is probably not caused by PKC, this kinase is an integral participant in DAT dynamics. (F) Physical coupling of the DAT to dopamine D2 receptors encourages recruitment of DAT to the plasma membrane. (G) PKC is also involved in ubiquitin-mediated DAT regulation, with monoubiquitination hypothesized to target internalized DAT to the lysosomal degradation pathway. (In color in Annals online.) DAT. Moreover, as Deken et al. have proposed, the ability to rapidly alter transporter trafficking may allow cells to regulate the exocytic release of neurotransmitters and the insertion of surface transporters in parallel. 19 This hypothesis was prompted by the finding that the GABA transporter (GAT1) is trafficked to and from the plasma membrane in recycling vesicles similar in size and morphology to synaptic vesicles (but lacking synaptophysin and the vesicular GABA transporter) and that the rate of recycling depends on intracellular Ca 2+ concentration, indicating that transporter trafficking may indeed be coupled to transmitter release. 19 RegulationofDATactivitycanbetriggeredby a myriad of exogenous factors and cellular events, such as (1) small-molecule ligands targeting either the DAT itself (i.e., substrates and inhibitors) or presynaptic G protein-coupled receptors (GPCRs) that affect DAT, (2) enzymatic modification (e.g., phosphorylation) involving intracellular secondmessenger cascades, and (3) protein–protein interactions between the DAT and other transmembrane or cytoskeletal scaffolding proteins. A graphical overview of the putative factors influencing DAT membrane trafficking is given in Figure 1. Our primary focus in this review will be on short-term DAT regulation in response to acute exposure to psychostimulants, such as cocaine- and amphetaminelike drugs. Although the complete signaling mechanisms underlying substrate- and inhibitor-mediated 318 Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences.
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