The dopamine transporter (DAT) plays a key role in the regulation of dopaminergic signaling wherein it controls both the spatial and temporal actions of dopamine. Here we evaluated the behavioral and neurochemical consequences of increased DAT function by generating DAT transgenic mice (DAT-tg) that overexpress the transporter. These mice were generated by pronuclear injection of a bacterial artificial chromosome containing the mouse DAT locus, yielding an anatomical expression pattern of DAT-tg identical to WT. In DAT-tg mice there is a 3-fold increase in the levels of total and membrane-expressed DAT, but synaptic plasma membrane fractions of DAT-tg mice show only a 30% increase in transporter levels. Functional studies reveal that in the DAT-tg animals there is a 50% increase in the rate of dopamine (DA) uptake resulting in extracellular levels of DA that are decreased by approximately 40%. Behaviorally, DAT-tg animals display similar locomotor stimulation when treated with DAT blockers such as GBR12909, methylphenidate, and cocaine. However, these mice demonstrate markedly increased locomotor responses to amphetamine compared with WT animals. Furthermore, compared with controls, there is a 3-fold greater increase in the amount of DA released by amphetamine in DAT-tg mice that correlates with the 3-fold increase in protein expression. Finally, DAT-tg animals show reduced operant responding for natural reward while displaying preference for amphetamine at much lower doses (0.2 and 0.5 mg/kg) than WT mice (2 mg/kg). These results suggest that overexpression of DAT leads to a marked increase in sensitivity to psychomotor and rewarding properties of amphetamine.
Serotonin is involved in a variety of physiological processes in the central nervous system and the periphery. As the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase plays an important role in modulating these processes. Of the two variants of tryptophan hydroxylase, tryptophan hydroxylase 2 (TPH2) is expressed predominantly in the central nervous system, whereas tryptophan hydroxylase 1 (TPH1) is expressed mostly in peripheral tissues. Although the two enzymes share considerable sequence homology, the regulatory domain of TPH2 contains an additional 41 amino acids at the N terminus that TPH1 lacks. Here we show that the extended TPH2 N-terminal domain contains a unique sequence involved in the regulation of enzyme expression. When expressed in cultured mammalian cells, TPH2 is synthesized less efficiently and is also less stable than TPH1. Removal of the unique portion of the N terminus of TPH2 results in expression of the enzyme at a level similar to that of TPH1, whereas protein chimeras containing this fragment are expressed at lower levels than their wild-type counterparts. We identify a region centered on amino acids 10-20 that mediates the bulk of this effect. We also demonstrate that phosphorylation of serine 19, a protein kinase A consensus site located in this N-terminal domain, results in increased TPH2 stability and consequent increases in enzyme output in cell culture systems. Because this domain is unique to TPH2, these data provide evidence for selective regulation of brain serotonin synthesis. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
Pharmacological evaluation of nicotine-stimulated dopamine release from striatum has yielded data consistent with activation of a single population of nicotinic acetylcholine receptors (nAChR). However, discovery that α-conotoxin MII (α-CtxMII) partially inhibits the response indicates that two classes of presynaptic nAChRs mediate dopamine release. We have investigated the pharmacology and subunit composition of these two classes of nAChR. Inhibition of nicotine-stimulated dopamine release from mouse striatal synaptosomes by α-CtxMII occurs within minutes; recovery is slow. The IC50 is 1 to 3 nM. α-CtxMII-sensitive and -resistant components have significant differences in pharmacology. The five agonists tested were more potent at activating the α-CtxMII-sensitive nAChRs; indeed, this receptor is the highest affinity functional nAChR found, so far, in mouse, brain. In addition, cytisine was more efficacious at the α-CtxMII-sensitive sites. Methyllycaconitine was 9-fold more potent at inhibiting the α-CtxMII-sensitive sites, whereas dihydro-β -erythroidine was a 7-fold more potent inhibitor of the α -CtxMII-resistant response. Both the transient and persistent phases of nicotine-stimulated dopamine release were partially inhibited by α-CtxMII with equal potency. The subunit composition of functional nAChRs, was assessed in mice with null mutations for individual nAChR subunits. The β2 subunit is an absolute requirement for both classes. In contrast, deletion of β4 or α7 subunits had no effect. The α-CtxMII-sensitive response requires β3 and is partially dependent upon α4 subunits, probably α6β3β2 and α4α6β3β2, whereas the α-CtxMII-resistant release requires α4 and is partially dependent upon α5 subunits, probably α4β2 and α4α5β2.