InteractiveFly: GeneBrief

Dopamine transporter: Biological Overview | References

BIOLOGICAL OVERVIEW

Sleep and arousal are known to be regulated by both homeostatic and circadian processes, but the underlying molecular mechanisms are not well understood. It has been reported that the Drosophila rest/activity cycle has features in common with the mammalian sleep/wake cycle, and it is expected that use of the fly genetic model will facilitate a molecular understanding of sleep and arousal. This study reports the phenotypic characterization of a Drosophila rest/activity mutant known as fumin (fmn). fmn mutants have abnormally high levels of activity and reduced rest (sleep); genetic mapping, molecular analyses, and phenotypic rescue experiments demonstrate that these phenotypes result from mutation of the Drosophila Dopamine transporter gene. Consistent with the rest phenotype, fmn mutants show enhanced sensitivity to mechanical stimuli and a prolonged arousal once active, indicating a decreased arousal threshold. Strikingly, fmn mutants do not show significant rebound in response to rest deprivation as is typical for wild-type flies, nor do they show decreased life span. These results provide direct evidence that dopaminergic signaling has a critical function in the regulation of insect arousal (Kume, 2005).

Although fmn flies exhibit such rest/activity phenotypes, there is apparently no effect of the mutation on development or longevity. This contrasts markedly with the results observed for mutations of two other genes that have been implicated in the regulation of Drosophila rest: cyc and Shaker (Sh). Mutations in cyc or Sh reduce life span, relative to genetic background controls, although complete life-span curves were not reported for Sh and there appears to be only a small effect on longevity for the Sh¹⁰² allele. The different effects of these mutations on longevity may reflect the relatively selective effect of fmn on arousal. Alternatively, the effects of Sh and cyc mutations on life span might reflect requirements for these genes in developmental or physiological processes other than rest. Cyc protein is a broadly expressed basic helix-loop-helix transcription factor, whereas Sh is a voltage-activated potassium channel with a broad localization in the nervous system. In contrast, DAT deficits only affect dopaminergic neuromodulation and therefore might have a less general impact on development and physiological processes (Kume, 2005).

Fmn flies carry a mutation in the Drosophila dopamine transporter gene, indicating that alterations of dopamine signaling are responsible for the observed phenotypes. dDAT functions in the dopaminergic pathway, as shown by (1) dDAT protein has significant sequence similarity to comparable mammalian and invertebrate transporters, (2) dDAT gene expression is restricted to dopaminergic neurons (as expected for a presynaptic transporter), (3) this transporter has a substrate specificity paralleling that of the mammalian DATs, with dopamine and tyramine being the preferred substrates, and (4) the dDAT transporter mediates uptake of dopamine in cell-based assays and responds to dopamine when expressed in Xenopus oocytes (Kume, 2005).

Dopamine is cleared from the synaptic cleft via presynaptic DAT, and DAT mutant mice exhibit altered presynaptic autoreceptor function, dopamine clearance, and biosynthetic rate in addition to behavioral alterations including spontaneous hyperlocomotion and hyperactivity. These phenotypes are presumably caused by the elevated persistence of released dopamine in these mice. Similarly, it seems likely that increased dopamine signaling in fmn is responsible for the observed hyperactivity and shortening of the rest phase, regarded as sleep in Drosophila (Kume, 2005).

Previous studies in Drosophila implicate biogenic amines in the modulation of activity. In larval Drosophila, 5-HT, OA, TA, and DA regulate locomotion or the sensory-motor circuitry on which such behavior depends. In adult flies, evidence suggests that DA and 5-HT function to regulate locomotor activity and flight, respectively. The study by Lima (2005) is particularly informative in that it demonstrates state-dependent effects of dopamine neuronal stimulation in behaving flies. In flies with low basal activity, the response to targeted (and transient) stimulation of dopamine neurons is an increased probability of locomotor bouts, whereas a similar stimulation of flies showing high basal activity leads to an inhibition. These results are most consistent with a biphasic role for modulation of locomotor activity by released dopamine, with the highest levels of dopamine release leading to locomotor inhibition. This is in agreement with studies that have examined the responses of flies to the psycho-stimulant cocaine, an inhibitor of aminergic transporters. Cocaine stimulation of flies results in transient stereotypies and hyperactivity that are strikingly similar to those seen after cocaine exposure to vertebrate animals. However, the most severely affected flies become akinesic, consistent with a biphasic effect of high extracellular dopamine (Kume, 2005).

Based on the decreased arousal threshold and the prolonged responses of fmn flies to mechanical stimulation, it is suggested that this mutant is characterized by an arousal state with enhanced alertness associated with the expected increase in extracellular dopamine. The absence of significant rest rebound in fmn supports this conclusion, along with the finding that activity level during each arousal period is normal. This is the first direct evidence that altered arousal threshold and decreased rebound can result from perturbations of dopaminergic signaling. Previous results have indirectly implicated DA in arousal. Those studies examined animals with lesions of dopaminergic neuronal populations, changes in firing rates of dopaminergic neurons as a function of sleep states and arousal, or the actions of wake-promoting drugs, some of which modulate DAT or DA receptor activity. The analysis of fmn shows directly that a selective lesion of DAT, presumably with accompanying increased DA levels, results in an alteration of arousal threshold (Kume, 2005).

It is of interest that mouse DAT mutants, like fly fmn, have abnormal sleep, although arousal sensitivity has not been explicitly examined. DAT mutant mice show enhanced spontaneous locomotor hyperactivity that is greatly enhanced by the stimulating effects of novel environment. More importantly, mouse DAT mutants have significantly increased wake bout duration and moderately increased activity levels during the latter half of the active (night) phase of the diurnal cycle. The extension of the active phase in these mice mimics the phenotype of fmn flies that have a lengthened active period and shortened rest phase during the diurnal cycle. DAT knock-out mice also exhibit altered responses to wake-promoting drugs such as GBR12909, modafinil, and caffeine. Such mice show increased sensitivity to caffeine and decreased sensitivity to GBR12909 and modafinil, suggesting that the latter two drugs act on DAT to promote wakefulness. Modafinil has wake-promoting properties in Drosophila, and it will be of interest to determine whether modafinil action is altered in fmn as it is in DAT mutant mice. A role for DAT and dopaminergic signaling in regulating wakefulness in flies and mice provides additional evidence for a similarity between mammalian sleep and insect rest (Kume, 2005).

Dopamine is probably also important for the regulation of arousal in humans. Parkinson's disease patients, who have reduced dopamine levels, often complain of sleepiness. When treated with L-3,4-dihydroxyphenylalanine (L-DOPA), which increases dopamine levels, they recover from sleepiness, but with excessive L-DOPA, they exhibit insomnia. Moreover, patients and animals with narcolepsy, which show increased wakefulness and excessive sleepiness, show a compensational increase in brain D₂-like receptors, suggesting a reduced level of dopamine (Kume, 2005).

Although fmn mutants have higher basal levels of activity, they nonetheless exhibit diurnal and circadian rhythms in locomotor activity. However, rhythmicity is less evident compared with that observed in wild-type flies, presumably because there is a higher baseline of activity at all times of day and therefore a corresponding decrease in the amplitude of rhythmicity (Kume, 2005).

Part of the evidence that fmn is a mutation in dDAT comes from transgenic rescue using pan-neurally driven expression of dDAT. It is notable that this rescue is only partial. One obvious explanation for this partial rescue is that expression of ELAV-GAL4 in the dopamine neurons, the sole site of dDAT localization, is weak. However, even stronger dopamine neuron expression of dDAT, under the control of TH-GAL4, yields even weaker rescue. This counterintuitive result may indicate that the precise level of dDAT in dopamine neurons is critical for normal dopamine homeostasis. It is possible that homeostatic mechanisms potentially overcompensate for high DAT levels and reduced synaptic DA by hyperactivating postsynaptic receptors, as has been seen after inhibition of dopamine and serotonin neurons. Thus, dDAT may be a component of a precisely regulated dopamine homeostatic mechanism that controls arousal threshold and overall activity levels (Kume, 2005).

This study of a Drosophila DAT mutant indicates another striking parallel between Drosophila and vertebrates with regard to the functions of biogenic amine systems. It demonstrates that the regulated reuptake of dopamine by DAT is important for setting arousal threshold. Equally important, the identification of a mutation in the pharmacologically important dopamine transporter opens new avenues for use of this genetically tractable model in pharmacological and behavioral studies (Kume, 2005).

Direct evidence that two cysteines in the dopamine transporter form a disulfide bond

A fully functional dopamine transporter (DAT) mutant (dmDATx7) has been generated with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces the disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. These results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface (Chen, 2007).

X-ray structure of dopamine transporter elucidates antidepressant mechanism

Antidepressants targeting Na⁺/Cl^--coupled neurotransmitter uptake define a key therapeutic strategy to treat clinical depression and neuropathic pain. However, identifying the molecular interactions that underlie the pharmacological activity of these transport inhibitors, and thus the mechanism by which the inhibitors lead to increased synaptic neurotransmitter levels, has proven elusive. This study presents the crystal structure of the Drosophila melanogaster Dopamine transporter at 3.0 Å resolution bound to the tricyclic antidepressant nortriptyline. The transporter is locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure of the dopamine transporter is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7. Taken together, the dopamine transporter structure reveals the molecular basis for antidepressant action on sodium-coupled neurotransmitter symporters and elucidates critical elements of eukaryotic transporter structure and modulation by lipids, thus expanding understanding of the mechanism and regulation of neurotransmitter uptake at chemical synapses (Penmatsa, 2013).

The structure of DAT_cryst captures the transporter in an inhibitor-bound, outward-open conformation. The TCA nortriptyline targets the primary substrate site and stabilizes the open conformation by sterically preventing closure of the extracellular gate. One chloride and two sodium ions are located adjacent to the ligand, suggesting that the binding of ions and inhibitor are directly coupled. A cholesterol molecule bound to a crevice flanking TM1a probably stabilizes the outward-open, inhibitor-bound conformation. The structure reveals a C-terminal latch that makes extensive interactions with the cytoplasmic face of the transporter, proximal to the cytoplasmic gate, and thus in a position to modulate transport activity. Taken together, the structure of a eukaryotic DAT reveals novel insights into antidepressant recognition and structural elements implicated in the regulation of neurotransmitter transport, providing a foundation for drug design strategies (Penmatsa, 2013).

Neurotransmitter and psychostimulant recognition by the dopamine transporter

Na(+)/Cl(-)-coupled biogenic amine transporters are the primary targets of therapeutic and abused drugs, ranging from antidepressants to the psychostimulants cocaine and amphetamines, and to their cognate substrates. This study determine X-ray crystal structures of the Drosophila melanogaster dopamine transporter (dDAT) bound to its substrate dopamine, a substrate analogue 3,4-dichlorophenethylamine, the psychostimulants d-amphetamine and methamphetamine, or to cocaine and cocaine analogues. All ligands bind to the central binding site, located approximately halfway across the membrane bilayer, in close proximity to bound sodium and chloride ions. The central binding site recognizes three chemically distinct classes of ligands via conformational changes that accommodate varying sizes and shapes, thus illustrating molecular principles that distinguish substrates from inhibitors in biogenic amine transporters (Wang, 2015).

Substrate-induced conformational dynamics of the dopamine transporter

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na(+)-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. This study probed the global Na(+)- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. Na⁺- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, ligand-dependent slow cooperative fluctuations of helical stretches were detected in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. These results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter (Nielsen, 2019).

A network of phosphatidylinositol (4,5)-bisphosphate (PIP2) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila melanogaster

Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. This study developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). Previous work has shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP2), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. This study demonstrates that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. This study showed that PIP2 also interacts with intracellular loop 4 at R443. Further, R443 was shown to electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, it was determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. This inhibition is associated with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, this study showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. This study presents the first evidence linking PIP2, DAT, DA efflux, and phosphorylation processes with AMPH reward (Belovich, 2019).

Structural determinants of the dopamine transporter regulation mediated by G proteins

Dopamine clearance in the brain is controlled by the dopamine transporter (DAT), a protein residing in the plasma membrane, which drives reuptake of extracellular dopamine into presynaptic neurons. Studies have revealed that the βγ subunits of heterotrimeric G proteins modulate DAT function through a physical association with the C-terminal region of the transporter. This study refined the crystal structure of the Drosophila melanogaster DAT (dDAT), modeling de novo the N- and C-terminal domains; subsequently, the full-length dDAT structure was used to generate a comparative model of human DAT (hDAT). Both proteins were assembled with Gβ1γ2 subunits employing protein-protein docking, and subsequent molecular dynamics simulations were run to identify the specific interactions governing the formation of the hDAT:Gβγ and dDAT:Gβγ complexes. A [L/F]R[Q/E]R sequence motif containing the residues R588 in hDAT and R587 in dDAT was found as key to bind the Gβγ subunits through electrostatic interactions with a cluster of negatively charged residues located at the top face of the Gβ subunit. Alterations of DAT function have been associated with multiple devastating neuropathological conditions; therefore, this work represents a step toward better understanding DAT regulation by signaling proteins, allowing therapeutic target regions to be predicted (Rojas, 2020).

Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters

Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. This study employed the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. This study also reports structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, it was observed that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. These experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters (Pidathala, 2021).

The Role of the Dopamine Transporter in the Effects of Amphetamine on Sleep and Sleep Architecture in Drosophila

The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake, and thereby plays an important homeostatic role in dopaminergic neurotransmission. Amphetamines exert their stimulant effects by targeting DAT and inducing the reverse transport of DA, leading to a dramatic increase of extracellular DA. Animal models have proven critical to investigating the molecular and cellular mechanisms underlying transporter function and its modulation by psychostimulants such as amphetamine. This study established a behavioral model for amphetamine action using adult Drosophila melanogaster. This model was used to characterize the effects of amphetamine on sleep and sleep architecture. The data show that amphetamine induces hyperactivity and disrupts sleep in a DA-dependent manner. Flies that do not express a functional DAT (dDAT null mutants) have been shown to be hyperactive and to exhibit significantly reduced sleep at baseline. The data show that, in contrast to its action in control flies, amphetamine decreases the locomotor activity of dDAT null mutants and restores their sleep by modulating distinct aspects of sleep structure. To begin to explore the circuitry involved in the actions of amphetamine on sleep, the localization of dDAT throughout the fly brain is described, particularly in neuropils known to regulate sleep. Together, these data establish Drosophila as a robust model for studying the regulatory mechanisms that govern DAT function and psychostimulant action (Karam, 2021).

Ring Finger Protein 11 (RNF11) Modulates Dopamine Release in Drosophila

Recent work indicates a role for RING finger protein 11 (RNF11) in Parkinson disease (PD) pathology, which involves the loss of dopaminergic neurons. However, the role of RNF11 in regulating dopamine neurotransmission has not been studied. This work tested the effect of RNF11 RNAi knockdown or overexpression on stimulated dopamine release in the larval Drosophila central nervous system. Dopamine release was stimulated using optogenetics and monitored in real-time using fast-scan cyclic voltammetry at an electrode implanted in an isolated ventral nerve cord. RNF11 knockdown doubled dopamine release, but there was no decrease in dopamine from RNF11 overexpression. RNF11 knockdown did not significantly increase stimulated serotonin or octopamine release, indicating the effect is dopamine specific. Dopamine clearance was also changed, as RNF11 RNAi flies had a higher V(max) and RNF11 overexpressing flies had a lower V(max) than control flies. RNF11 RNAi flies had increased mRNA levels of dopamine transporter (DAT) in RNF11, confirming changes in DAT. In RNF11 RNAi flies, release was maintained better for stimulations repeated at short intervals, indicating increases in the recycled releasable pool of dopamine. Nisoxetine, a DAT inhibitor, and flupenthixol, a D2 antagonist, did not affect RNF11 RNAi or overexpressing flies differently than control. Thus, RNF11 knockdown causes early changes in dopamine neurotransmission, and this is the first work to demonstrate that RNF11 affects both dopamine release and uptake. RNF11 expression decreases in human dopaminergic neurons during PD, and that decrease may be protective by increasing dopamine neurotransmission in the surviving dopaminergic neurons (Champaloux, 2022).

Search PubMed for articles about Drosophila Dopamine transporter

Belovich, A. N., Aguilar, J. I., Mabry, S. J., Cheng, M. H., Zanella, D., Hamilton, P. J., Stanislowski, D. J., Shekar, A., Foster, J. D., Bahar, I., Matthies, H. J. G. and Galli, A. (2019). A network of phosphatidylinositol (4,5)-bisphosphate (PIP2) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila melanogaster. Mol Psychiatry. PubMed ID: 31796894

Champaloux, E. P., Donelson, N., Pyakurel, P., Wolin, D., Ostendorf, L., Denno, M., Borman, R., Burke, C., Short-Miller, J. C., Yoder, M. R., Copeland, J. M., Sanyal, S. and Jill Venton, B. (2020). Ring Finger Protein 11 (RNF11) Modulates Dopamine Release in Drosophila. Neuroscience. PubMed ID: 33176188

Chen, R., Wei, H., Hill, E. R., Chen, L., Jiang, L., Han, D. D. and Gu, H. H. (2007). Direct evidence that two cysteines in the dopamine transporter form a disulfide bond. Mol. Cell. Biochem. 298(1-2): 41-8. PubMed ID: 17131045

Karam, C. S., Williams, B. L., Jones, S. K. and Javitch, J. A. (2021). The Role of the Dopamine Transporter in the Effects of Amphetamine on Sleep and Sleep Architecture in Drosophila. Neurochem Res. PubMed ID: 33630236

Kume, K., Kume, S., Park, S. K., Hirsh, J. and Jackson, F. R. (2005). Dopamine is a regulator of arousal in the fruit fly. J. Neurosci. 25(32): 7377-84. PubMed ID: 16093388

Nielsen, A. K., Moller, I. R., Wang, Y., Rasmussen, S. G. F., Lindorff-Larsen, K., Rand, K. D. and Loland, C. J. (2019). Substrate-induced conformational dynamics of the dopamine transporter. Nat Commun 10(1): 2714. PubMed ID: 31221956

Penmatsa, A., Wang, K. H. and Gouaux, E. (2013). X-ray structure of dopamine transporter elucidates antidepressant mechanism. Nature 503: 85-90. PubMed ID: 24037379

Pidathala, S., Mallela, A. K., Joseph, D. and Penmatsa, A. (2021). Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters. Nat Commun 12(1): 2199. PubMed ID: 33850134

Rojas, G., Orellana, I., Rosales-Rojas, R., Garcia-Olivares, J., Comer, J. and Vergara-Jaque, A. (2020). Structural determinants of the dopamine transporter regulation mediated by G proteins. J Chem Inf Model. PubMed ID: 32525311

Wang, K. H., Penmatsa, A. and Gouaux, E. (2015). Neurotransmitter and psychostimulant recognition by the dopamine transporter. Nature 521: 322-327. PubMed ID: 25970245

date revised: 30 May 2023

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