Maarten E.A. Reith

Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6

The SLC6 family is a diverse set of transporters that mediate solute translocation across cell plasma membranes by coupling solute transport to the cotransport of sodium and chloride down their electrochemical gradients. These transporters probably have 12 transmembrane domains, with cytoplasmic N- and C-terminal tails, and at least some may function as homo-oligomers. Family members include the transporters for the inhibitory neurotransmitters GABA and glycine, the aminergic transmitters norepinephrine, serotonin, and dopamine, the osmolytes betaine and taurine, the amino acid proline, and the metabolic compound creatine. In addition, this family includes a system B0+ cationic and neutral amino acid transporter, and two transporters for which the solutes are unknown. In general, SLC6 transporters act to regulate the level of extracellular solute concentrations. In the central and the peripheral nervous system, these transporters can regulate signaling among neurons, are the sites of action of various drugs of abuse, and naturally occurring mutations in several of these proteins are associated with a variety of neurological disorders. For example, transgenic animals lacking specific aminergic transporters show profoundly disturbed behavioral phenotypes and probably represent excellent systems for investigating psychiatric disease. SLC6 transporters are also found in many non-neural tissues, including kidney, intestine, and testis, consistent with their diverse physiological roles. Transporters in this family represent attractive therapeutic targets because they are subject to multiple forms of regulation by many different signaling cascades, and because a number of pharmacological agents have been identified that act specifically on these proteins.

Extracellular dopamine, norepinephrine, and serotonin in the ventral tegmental area and nucleus accumbens of freely moving rats during intracerebral dialysis following systemic administration of cocaine and other uptake blockers

Abstract Extracellular levels of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) were measured by microdialysis in conscious rats equipped with dual probes, one in the ventral tegmental area (VTA) and another one in the contralateral nucleus accumbens (NACC). Dialysate content of all amines in both regions was essentially abolished by local infusion of tetrodotoxin (1 μM) or Ca2+-free buffer. Injection of the selective DA uptake blocker GBR 12935 (15 mg/kg IP) increased DA, as well as NE and, to a lesser extent, 5-HT in the VTA; it increased DA more than 5-HT in the NACC. The selective NE uptake blocker desipramine (10 mg/kg IP) increased NE but also 5-HT in the VTA and NACC; the DA level was persistently enhanced in the VTA, whereas in the NACC it initially rose and then fell below baseline value. The selective 5-HT uptake blocker citalopram (15 mg/kg IP) was generally more effective in elevating dialysate level of 5-HT than that of other amines in both regions. Cocaine (20 mg/kg IP) was non-selective in enhancing all three amines in both regions. There is considerable crosstalk between monoamine systems occurring upon systemic administration of uptake blockers, and the VTA and NACC are notably different in the time course of the DA effect (long-lasting versus transient).

Determination of release and uptake parameters from electrically evoked dopamine dynamics measured by real-time voltammetry

Quantifying mechanisms underlying extracellular signaling by the neurotransmitter dopamine (DA) is a difficult task, particularly in the complex extracellular microenvironment of the intact brain. In this study, two methods for evaluating release and uptake from DA dynamics monitored by real-time voltammetry are described. Both are based on a neurochemical model characterizing electrically evoked levels of DA as a balance between these opposing mechanisms. The theoretical basis of what is called here nonlinear regression and single curve analyses is given. Fitting simulated data tests the reliability of the methods. The two analyses are also compared with an experimental data set describing the effects of pharmacologically inhibiting the DA transporter in the caudate-putamen (CP) and nucleus accumbens (NAc). The results indicate that nonlinear regression and single curve analyses are suitable for quantifying release and uptake mechanisms underlying DA neurotransmission. Additionally, the most important experimental finding of this technical study was the independent confirmation of high affinity (approximately 0.2 microM) DA uptake in the intact striatum.

Structure and function of the dopamine transporter.

The dopamine transporter mediates uptake of dopamine into neurons and is a major target for various pharmacologically active drugs and environmental toxins. Since its cloning, much information has been obtained regarding its structure and function. Binding domains for dopamine and various blocking drugs including cocaine are likely formed by interactions with multiple amino acid residues, some of which are separate in the primary structure but lie close together in the still unknown tertiary structure. Chimera and site-directed mutagenesis studies suggest the involvement of both overlapping and separate domains in the interaction with substrates and blockers, whereas recent findings with sulfhydryl reagents selectively targeting cysteine residues support a role for conformational changes in the binding of blockers such as cocaine. The dopamine transporter can also operate in reverse, i.e. in an efflux mode, and recent mutagenesis experiments show different structural requirements for inward and outward transport. Strong evidence for dopamine transporter domains selectively influencing binding of dopamine or cocaine analogs has not yet emerged, although the development of a cocaine antagonist at the level of the transporter remains a possibility.

Pharmacology and regulation of the neuronal dopamine transporter

The dopamine transporter, a member of the family of Na+,Cl(-)-dependent transporters, mediates uptake of dopamine into dopaminergic neurons by an electrogenic, Na(+)- and Cl(-)-transport-coupled mechanism. Dopamine and blockers of uptake such as cocaine probably bind to both shared and separate domains on the transporter, which can be influenced dramatically by the presence of cations. Regulation of the dopamine transporter occurs both by chronic occupancy with blocker and by acute effects of D2 dopamine receptors or second messengers such as diacylglycerol (protein kinase C) and arachidonic acid. The dopamine transporter is involved in the uptake of toxins generating Parkinsons disease; it is also an important target for psychostimulant drugs, ligands for in vivo imaging and medications used for neurologic diseases involving changes in the dopamine system.

Dizocilpine (MK-801) increases not only dopamine but also serotonin and norepinephrine transmissions in the nucleus accumbens as measured by microdialysis in freely moving rats

The extracellular concentrations of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) in the nucleus accumbens (NACC) of freely moving rats were monitored simultaneously via intracerebral microdialysis. Local infusion of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine) (5-250 microM) produced significant increases in extracellular levels of DA, NE and 5-HT in a concentration-dependent fashion. Perfusion with tetrodotoxin (TTX, 1 microM) blocked the ability of focal MK-801 (50 microM) to increase DA, NE and 5-HT in the dialysate. Systemic administration of MK-801 (0.3 mg/kg, i.p.) also produced small, but statistically significant, increases in extracellular concentrations of DA, NE and 5-HT in the NACC. Our microdialysis results are consistent with the hypothesis that, in addition to dopaminergic, serotonergic and noradrenergic neurotransmissions in the NACC are involved in the mechanism by which MK-801 alters behavior in rats. Also, the present study gives further support to the concept that NMDA receptors within the NACC do not regulate DA release through direct excitatory control.

Effects of locally applied cocaine, lidocaine, and various uptake blockers on monoamine transmission in the ventral tegmental area of freely moving rats: a microdialysis study on monoamine interrelationships.

Abstract: Microdialysis was used to compare the effect of local perfusion of cocaine with that of functionally similar compounds on extracellular norepinephrine, dopamine, and serotonin (measured simultaneously) in the ventral tegmental area of freely moving rats. Tetrodotoxin (1 µM) potently inhibited both basal and cocaine‐induced dialysate monoamine outputs. The local anesthetic lidocaine produced little or no effect on the monoamine output, whereas all uptake blockers tested (at 0.1–1,000 µM) increased the monoamine output in a dose‐dependent manner. The selective norepinephrine‐uptake blockers desipramine and nisoxetine did not show any selectivity for norepinephrine, whereas the selective serotonin‐uptake blockers fluoxetine and citalopram, as well as the selective dopamine‐uptake blocker GBR 12935, preferentially (but not exclusively) increased their target amine. Cocaine at low concentrations (1–10 µM) increased the three amines similarly, but at higher concentrations (100–1,000 µM) caused a relatively higher dopamine output. A positive relationship between blocker‐induced dialysate norepinephrine and dopamine outputs suggests significant interactions between monoamine systems. The present results indicate that cocaines action in the ventral tegmental area involves not only a dopamine‐, but also a norepinephrine‐ and a serotonin‐related component, and that cocaine‐induced monoamine increase is independent of its local anesthetic property.

The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site-directed mutagenesis

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na+/Cl−‐dependent transporters. We have explored the roles of these residues using non‐conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10–24‐fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [3H]DA uptake, but caused undetectable [3H]CFT binding. Upon anti‐C‐terminal‐hDAT immunoblotting, D345N appeared as broad bands of 66–97 kDa, but this band could not be photoaffinity labeled with cocaine analog [125I]‐3β‐(p‐chlorophenyl)tropane‐2β‐carboxylic acid ([125I]RTI‐82). Unexpectedly, in this mutant, cocaine‐like drugs remained potent inhibitors of [3H]DA uptake. CFT solely raised the Km of [3H]DA uptake in wild‐type hDAT, but increased Km and decreased Vmax in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.

Monoamine Interactions Measured by Microdialysis in the Ventral Tegmental Area of Rats Treated Systemically with (±)-8-Hydroxy-2-(Di-n-Propylamino)tetralin

Abstract: The effect of (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), a selective serotonin 5‐HT1A agonist, on levels of extracellular norepinephrine (NE), dopamine (DA), and 5‐HT (measured simultaneously) was investigated by microdialysis in the ventral tegmental area (VTA) of freely moving rats, and their behavioral activity was monitored. At 50 µg/kg s.c., 8‐OH‐DPAT reduced 5‐HT levels but enhanced NE and DA levels in VTA dialysate. These effects were not altered by pretreatment with systemic idazoxan (5 mg/kg i.p.), a selective α2 antagonist, or local sulpiride (10 µM), a selective D2/D3 antagonist. At 500 µg/kg s.c., 8‐OH‐DPAT further enhanced or more persistently reduced dialysate NE or 5‐HT content but had little effect on dialysate DA content. Its DA level‐increasing effect could be seen dramatically with local infusion of cocaine (30 µM) and, to a lesser extent, sulpiride (10 µM). Depletion of endogenous 5‐HT with p‐chlorophenylalanine attenuated both the 5‐HT level‐reducing and DA level‐enhancing effects of 8‐OH‐DPAT without affecting its maximal NE effect and the locomotor‐stimulatory effect. Partial depletion of endogenous NE with N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine failed to change the monoamine response but diminished the locomotion induced by 8‐OH‐DPAT. These results suggested that (a) the low dose of 8‐OH‐DPAT may act at presynaptic 5‐HT1A receptors to modulate 5‐HT and DA release, while acting at postsynaptic 5‐HT1A receptors to modulate NE release; (b) the high dose of 8‐OH‐DPAT may activate D2 receptors to offset its DA level‐increasing effect; and (c) the locomotor‐stimulatory effect of 8‐OH‐DPAT might be mediated primarily by postsynaptic 5‐HT1A receptors and the NE system.

Extracellular Dopamine, Norepinephrine, and Serotonin in the Nucleus Accumbens of Freely Moving Rats During Intracerebral Dialysis with Cocaine and Other Monoamine Uptake Blockers

Abstract: Monoamine‐uptake blockers were applied focally (0.1–1,000 µM) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine‐uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin‐uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine‐uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.