Dopamine release mediated by the dopamine transporter, facts and consequences

Spontaneous and/or stimulated neural activity of the nigrostriatal dopamine (DA) pathway makes amines run out from the neurons. This DA dynamic follows a rather complex path, running in or out the terminals, and flushing or diffusing into the extracellular space. The location of this leakage is not limited to the axon terminals; it also occurs from the cell bodies and dendrites. This molecular release mechanism was, for a long time, considered as being produced, in part, by the exocytosis of previously stored vesicles. The DA carrier protein (DAT, DA transporter) embedded in the DA cell membrane is known to clear previously released amines through an inward DA influx. The DAT also appears to be an active vector of amine release. Particular local conditions and the presence of numerous psychostimulant substances are able to trigger an outward efflux of DA through the DAT. This process, delivering slowly large amounts of amine could play a major regulatory role in extracellular DA homeostasis.     

An inverse correlation between the apparent rate of dopamine clearance and tonic autoinhibition in subdomains of the rat striatum: a possible role of transporter-mediated dopamine efflux

The dopaminergic terminal field in the rat striatum is compartmentalized into sub-domains that exhibit distinct dynamics of electrically evoked dopamine release. The fast striatal domains, where dopamine release is predominantly vesicular, exhibit conventional dopaminergic activity. However, vesicular dopamine release is tonically autoinhibited in the slow domains, which suggests that dopamine reaches the autoreceptors via a non-vesicular route. Hence, it appears that the domains use distinct mechanisms to regulate the basal dopamine concentration available to activate, or not, pre-synaptic autoinhibitory receptors. However, direct detection of local variations in tonic extracellular dopamine concentrations is not yet possible. So, the present study employed voltammetry to test the hypothesis that the apparent rate of dopamine clearance from the extracellular space should be domain-dependent. The apparent rate of dopamine clearance is equal to the difference in the rates of dopamine release and uptake that determine extracellular dopamine concentrations. This study confirms that the apparent rate of dopamine clearance is slower in the slow striatal domains where vesicular dopamine release is tonically autoinhibited. These findings support the view that the basal concentration in slow domains is maintained by a non-vesicular release process, possibly transporter-mediated efflux.     

Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.     

Dual Action of Zn2+ on the Transport Cycle of the Dopamine Transporter

The dopamine transporter shapes dopaminergic neurotransmission by clearing extracellular dopamine and by replenishing vesicular stores. The dopamine transporter carries an endogenous binding site for Zn²⁺, but the nature of the Zn²⁺-dependent modulation has remained elusive: both, inhibition and stimulation of DAT have been reported. Here, we exploited the high time resolution of patch-clamp recordings to examine the effects of Zn²⁺ on the transport cycle of DAT: we recorded peak currents associated with substrate translocation and steady-state currents reflecting the forward transport mode of DAT. Zn²⁺ depressed the peak current but enhanced the steady-state current through DAT. The parsimonious explanation is preferential binding of Zn²⁺ to the outward facing conformation of DAT, which allows for an allosteric activation of DAT, in both, the forward transport mode and substrate exchange mode. We directly confirmed that Zn²⁺ dissociated more rapidly from the inward- than from the outward-facing state of DAT. Finally, we formulated a kinetic model for the action of Zn²⁺ on DAT that emulated all current experimental observations and accounted for all previous (in part contradictory) findings. Importantly, the model predicts that the intracellular Na⁺ concentration determines whether substrate uptake by DAT is stimulated or inhibited by Zn²⁺. This prediction was directly verified. The mechanistic framework provided by the current model is of relevance for the rational design of allosteric activators of DAT. These are of interest for treating de novo loss-of-function mutations of DAT associated with neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD).     

多巴胺受体的cDNA克隆

通过多巴胺受体的5个cDNA克隆,综述和分析了5个多巴胺受体(D₁R-D₅R)的基因结构,在染色体上的定位及其mRNA在中枢脑区的分布;比较了这5个受体cDNA克隆的结构特征和药理学性质.     

Polypyrimidine tract-binding protein 1 regulates the alternative splicing of dopamine receptor D(2)

Dopamine receptor D(2) (DRD2) has two splicing isoforms, a long form (D2L) and short form (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is unknown. In this study, we examined which splicing factors regulate the expression of D2L and D2S by over-expressing several RNA-binding proteins in HEK293 cells. In a cellular splicing assay, the over-expression of polypyrimidine tract-binding protein 1 (PTBP1) reduced the expression of D2S, whereas the knockdown of PTBP1 increased the expression of D2S. We also identified the regions of DRD2 that are responsive to PTBP1 using heterologous minigenes and deletion mutants. Our results indicate that PTBP1 regulates the alternative splicing of DRD2. Considering that DRD2 inhibits cAMP-dependent protein kinase A, which modulates the intracellular localization of PTBP1, PTBP1 may contribute to the autoregulation of DRD2 by regulating the expression of its isoforms.     

Prenatal cocaine exposure disrupts the dopaminergic system and its postnatal responses to cocaine

Impaired attention is the hallmark consequence of prenatal cocaine exposure (PCE), affecting brain development, learning, memory and social adaptation starting at an early age. To date, little is known about the brain structures and neurochemical processes involved in this effect. Through focusing on the visual system and employing zebrafish as a model, we show that PCE reduces expression of dopamine receptor Drd1, with levels reduced in the optic tectum and other brain regions, but not the telencephalon. Organism‐wide, PCE results in a 1.7‐fold reduction in the expression of the dopamine transporter (dat), at baseline. Acute cocaine administration leads to a 2‐fold reduction in dat in drug‐naive larvae but not PCE fish. PCE sensitizes animals to an anxiogenic‐like behavioral effect of acute cocaine, bottom‐dwelling, while loss of DAT due to genetic knockout (DATKO) leads to bottom‐dwelling behavior at baseline. Neuronal calcium responses to visual stimuli in both PCE and DATKO fish show tolerance to acute cocaine in the principal regions of visual attention, the telencephalon and optic tectum. The zebrafish model can provide a sensitive assay by which to elucidate the molecular mechanisms and brain region‐specific consequences of PCE, and facilitate the search for effective therapeutic solutions.     

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.     

GZ‐793A,a lobelane analog,interacts with the vesicular monoamine transporter‐2 to inhibit the effect of methamphetamine

(R)‐3‐[2,6‐cis‐Di(4‐methoxyphenethyl)piperidin‐1‐yl]propane‐1,2‐diol (GZ‐793A) inhibits methamphetamine‐evoked dopamine release from striatal slices and methamphetamine self‐administration in rats. GZ‐793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter‐2 (VMAT2). This study determined GZ‐793A's ability to evoke [³H]dopamine release and inhibit methamphetamine‐evoked [³H]dopamine release from isolated striatal synaptic vesicles. Results show GZ‐793A concentration‐dependent [³H]dopamine release; nonlinear regression revealed a two‐site model of interaction with VMAT2 (High‐ and Low‐EC₅₀ = 15.5 nM and 29.3 μM, respectively). Tetrabenazine and reserpine completely inhibited GZ‐793A‐evoked [³H]dopamine release, however, only at the High‐affinity site. Low concentrations of GZ‐793A that interact with the extravesicular dopamine uptake site and the High‐affinity intravesicular DA release site also inhibited methamphetamine‐evoked [³H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration‐response was evident with increasing concentrations of GZ‐793A, and the Schild regression slope was 0.49 ± 0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ‐793A interaction at more than one site on the VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High‐affinity tetrabenazine‐ and reserpine‐sensitive site, dopamine release via a Low‐affinity tetrabenazine‐ and reserpine‐insensitive site, and a low‐affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ‐793A inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse.

     

Nomifensine amplifies subsecond dopamine signals in the ventral striatum of freely-moving rats

The present experiment examined the effect of the dopamine transporter blocker nomifensine on subsecond fluctuations in dopamine concentrations, or dopamine transients, in the nucleus accumbens and olfactory tubercle. Extracellular dopamine was measured in real time using fast-scan cyclic voltammetry at micron-dimension carbon fibers in freely-moving rats. Dopamine transients occurred spontaneously throughout the ventral striatum in the absence of apparent sensory input or change in behavioral response. The frequency of dopamine transients increased at the presentation of salient stimuli to the rat (food, novel odors and unexpected noises). Administration of 7 mg/kg nomifensine amplified spontaneous dopamine transients by increasing both amplitude and duration, consistent with its known action at the dopamine transporter and emphasizing the dopaminergic origin of the signals. Moreover, nomifensine increased the frequency of detected dopamine transients, both during baseline conditions and at the presentation of stimuli, but more profoundly in the nucleus accumbens than in the olfactory tubercle. This difference was not explained by nomifensine effects on the kinetics of dopamine release and uptake, as its effects on electrically-evoked dopamine signals were similar in both regions. These findings demonstrate the heterogeneity of dopamine transients in the ventral striatum and establish that nomifensine elevates the tone of rapid dopamine signals in the brain.     

Repeated alcohol administration during adolescence causes changes in the mesolimbic dopaminergic and glutamatergic systems and promotes alcohol intake in the adult rat

Adolescence is a developmental period which the risk of drug and alcohol abuse increases. Since mesolimbic dopaminergic system undergoes developmental changes during adolescence, and this system is involved in rewarding effects of drugs of abuse, we addressed the hypothesis that ethanol exposure during juvenile/adolescent period over-activates mesolimbic dopaminergic system inducing adaptations which can trigger long-term enduring behavioural effects of alcohol abuse. We treated juvenile/adolescent or adult rats with ethanol (3 g/kg) for two-consecutive days at 48-h intervals over 14-day period. Here we show that intermittent ethanol treatment during the juvenile/adolescence period alters subsequent ethanol intake. In vivo microdialysis demonstrates that ethanol elicits a similar prolonged dopamine response in the nucleus accumbens of both adolescent and adult animals pre-treated with multiple doses of ethanol, although the basal dopamine levels were higher in ethanol-treated adolescents than in adult-treated animals. Repeated ethanol administration also down-regulates the expression of DRD2 and NMDAR2B phosphorylation in prefrontal cortex of adolescent animals, but not of adult rats. Finally, ethanol treatment during adolescence changes the acetylation of histones H3 and H4 in frontal cortex, nucleus accumbens and striatum, suggesting chromatin remodelling changes. In summary, our findings demonstrate the sensitivity of adolescent brain to ethanol effects on dopaminergic and glutamatergic neurotransmission, and suggest that abnormal plasticity in reward-related processes and epigenetic mechanisms could contribute to the vulnerability of adolescents to alcohol addiction.     

DARPP—32的结构,功能及其调节机制

DARPP-32是一种多巴胺（DA）和cAMP调节的磷蛋白，存在于所有接受DA能投射的神经元中，在中枢神经系统的分布与DAD1受体的分布非常一致。DA通过D1受体使DARPP-32第34位苏氨酸磷酸化，磷酸化DARPP-32成为蛋白磷酸酶1（PP-1）的强效抑制剂，在两个不同位点与PP-1相互作用，从而抑制PP-1活性。DARPP-32／PP-1级联反应在调节，如钙通道、电压依赖性钠通道、Na＋,K＋-ATPase和NMDANR1受体的功能等神经元兴奋性过程中起重要作用。DA对DARPP—32的磷酸化状态有双向调节作用，其他许多神经递质亦可调节其磷酸化状态。     

Dopamine transmission in the tail striatum: Regional variation and contribution of dopamine clearance mechanisms

The striatum can be divided into four anatomically and functionally distinct domains: the dorsolateral, dorsomedial, ventral and the more recently identified caudolateral (tail) striatum. Dopamine transmission in these striatal domains underlies many important behaviours, yet little is known about this phenomenon in the tail striatum. Furthermore, the tail is divided anatomically into four divisions (dorsal, medial, intermediate and lateral) based on the profile of D₁ and D₂ dopamine receptor-expressing medium spiny neurons, something that is not seen elsewhere in the striatum. Considering this organisation, how dopamine transmission occurs in the tail striatum is of great interest. We recorded evoked dopamine release in the four tail divisions, with comparison to the dorsolateral striatum, using fast-scan cyclic voltammetry in rat brain slices. Contributions of clearance mechanisms were investigated using dopamine transporter knockout (DAT-KO) rats, pharmacological transporter inhibitors and dextran. Evoked dopamine release in all tail divisions was smaller in amplitude than in the dorsolateral striatum and, importantly, regional variation was observed: dorsolateral ≈ lateral > medial > dorsal ≈ intermediate. Release amplitudes in the lateral division were 300% of that in the intermediate division, which also exhibited uniquely slow peak dopamine clearance velocity. Dopamine clearance in the intermediate division was most dependent on DAT, and no alternative dopamine transporters investigated (organic cation transporter-3, norepinephrine transporter and serotonin transporter) contributed significantly to dopamine clearance in any tail division. Our findings confirm that the tail striatum is not only a distinct dopamine domain but also that each tail division has unique dopamine transmission characteristics. This supports that the divisions are not only anatomically but also functionally distinct. How this segregation relates to the overall function of the tail striatum, particularly the processing of multisensory information, is yet to be determined.

     

多巴胺重摄取的分子机制

多巴胺转运体(Dopamine transporter,DAT)位于多巴胺能神经元表面,主要负责将细胞外的多巴胺重摄取至多巴胺能神经元内,控制细胞外多巴胺的浓度,进而影响多巴胺的信号强度和时长。多巴胺转运体与注意力缺陷多动症、抑郁、成瘾等中枢神经系统的功能异常相关。多巴胺转运体的重摄取功能受多种因素的调节,包括底物的浓度、自身位点的翻译后修饰、细胞内蛋白激酶的活性、细胞外的调节信号等。本文就近年来DAT的分子调节机制以及在脑疾病发病机制中作用的研究进展做一综述。     

Gap Junction Coupling Shapes the Encoding of Light in the Developing Retina

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