Nianhang Chen

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.

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.

Aspartate 345 of the Dopamine Transporter Is Critical for Conformational Changes in Substrate Translocation and Cocaine Binding

The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na+/Cl--dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low Vmax and Km values for DA uptake, converted the inhibitory effect of Zn2+ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2β-carbomethoxy-3β-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn2+ but not by Na+. These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K+ substitution for Na+. Thus, cocaine binding to D345N likely induces a different and Na+-dependent conformational change, which may contribute to its Na+-dependent uptake inhibitory activity.

Amygdala Kindling of Forebrain Seizures and the Occurrence of Brainstem Seizures in Genetically Epilepsy‐Prone Rats

Forebrain seizures were kindled in rats by daily electrical stimulation of the amygdala. Genetically epilepsy‐prone rats scoring 9 (GEPR‐9s) on the seizure severity scale during audiogenic seizure (AGS) screening (“brainstem seizure‐experienced”) required fewer stimulations to achieve fully kindled seizures (forelimb clonus with rearing and falling) than control rats. AGS‐naive GEPR‐9s required an intermediate number of stimulations, indicating a role for both genetic predisposition and previous acoustically evoked brainstem seizure experience. Other forebrain kindling indices such as afterdischarge thresholdlduration and seizure latencylduration also involved genetic as well as phenotypic (previous seizure experience) factors. In most GEPR‐9s in both groups, severe brainstem seizures occurred after forebrain stimulation. The occurrence of brainstem seizures had a random nature and was not related to the sequence of kindling‐dependent forebrain seizure progression. The lack of a difference in the occurrence of brainstem seizures between seizure‐experienced and AGS‐naïve GEPR‐9s suggest that genetic predisposition is the major factor in forebrain seizure‐induced activation of brainstem seizure circuitry. This brainstem seizure activity appears to model pertinent aspects of secondary generalization observed in human partial seizures.

GBR 12909 and 12935 block dopamine uptake into brain synaptic vesicles as well as nerve endings

GBR 12909 and 12935, commonly used as potent neuronal dopamine uptake blockers, also inhibit dopamine uptake into brain synaptic vesicles. The concentrations required for the latter activity (34-45 nM) are one order of magnitude higher than those required for inhibiting neuronal uptake of dopamine (1-6 nM). In contrast, the two activities differ by three orders of magnitude for cocaine (137 microM versus 0.35 microM). We propose that the vesicular effect of GBR-type dopamine uptake blockers should be taken into account when interpreting in vivo experiments.

Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2α-carbomethoxy-3α-(4-fluorophenyl)tropane

In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+‐free medium. Na+ markedly increased the transporters affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+] from 20 to 155 mm reduced Li+‐induced increase in DA Ki, but not CFT Kd. The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+‐independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+‐insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+s inhibitory effect, though they caused Na+ protection of DA Ki against H+s inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+‐bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding.

Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations

Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4°C than 25°C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell‐free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA–DAT interaction and the integration of Na+ modulation in this interaction.

Facilitation of Amygdala Kindling Development and Kindled Seizures by Metaphit

Summary: The effect of metaphit (a phencyclidine analogue with an acylating isothiocyanate) on kindling development and kindled seizures from amygdala was investigated in rats pretreated once with metaphit. Administration of a single dose of metaphit (10 or 20 mg/kg intraperitoneally i.p.) 4 h before the first electrical stimulation of the amygdala did not in itself induce seizures, but greatly facilitated development of behavioral seizures during kindling. This effect persisted throughout the whole process of electrical amygdala kindling without further dosing. In contrast, metaphit only transiently and modestly increased the growth of afterdischarge (AD) duration. In kindled rats, pretreatment with a single dose of metaphit (20 mg/kg) 8 h before the test stimulation reduced the threshold current required to elicit a stage 5 seizure and shortened the latency for bilateral forelimb clonus (BFC) without changing AD duration or BFC duration. The facilitation of kindling development and kindled seizures may be due to an excessive excitatory transmission by metaphit in the limbic seizure circuitry.