Rescue by 4-phenylbutyrate of several misfolded creatine transporter-1 variants linked to the creatine transporter deficiency syndrome.

Diseases arising from misfolding of SLC6 transporters have been reported over recent years, e.g. folding-deficient mutants of the dopamine transporter and of the glycine transporter-2 cause infantile/juvenile Parkinsonism dystonia and hyperekplexia, respectively. Mutations in the coding sequence of the human creatine transporter-1 (hCRT-1/SLC6A8) gene result in a creatine transporter deficiency syndrome, which varies in its clinical manifestation from epilepsy, mental retardation, autism, development delay and motor dysfunction to gastrointestinal symptoms. Some of the mutations in hCRT-1 occur at residues, which are highly conserved across the SLC6 family. Here, we examined 16 clinically relevant hCRT-1 variants to verify the conjecture that they were misfolded and that this folding defect was amenable to correction. Confocal microscopy imaging revealed that the heterologously expressed YFP-tagged mutant CRTs were trapped in the endoplasmic reticulum (ER), co-localised with the ER-resident chaperone calnexin. In contrast, the wild type hCRT-1 reached the plasma membrane. Preincubation of transiently transfected HEK293 cells with the chemical chaperone 4-phenylbutyrate (4-PBA) restored ER export and surface expression of as well as substrate uptake by several folding-deficient CRT-1 mutants. A representative mutant (hCRT-1-P544L) was expressed in rat primary hippocampal neurons to verify pharmacochaperoning in a target cell: 4-PBA promoted the delivery of hCRT-1-P544L to the neurite extensions. These observations show that several folding-deficient hCRT-1 mutants can be rescued. This proof-of-principle justifies the search for additional pharmacochaperones to restore folding of 4PBA-unresponsive hCRT-1 mutants. Finally, 4-PBA is an approved drug in paediatric use: this provides a rationale for translating the current insights into clinical trials. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Rapid, transient effects of the protein kinase C activator phorbol 12-myristate 13-acetate on activity and trafficking of the rat high-affinity choline transporter.

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V(max) of choline uptake, but had no effect on K(m) for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.

MDMA (Ecstasy) and human dopamine, norepinephrine, and serotonin transporters: implications for MDMA-induced neurotoxicity and treatment.

RATIONALE: 3,4-Methylenedioxymethamphetamine (MDMA, designated as "Ecstasy" if illicitly marketed in tablet form) induces significant decrements in neuronal serotonin (5-HT) markers in humans, nonhuman primates, and rats as a function of dosing and dosing regimen. In rats, MDMA-mediated effects are attributed, in part, to selective high-affinity transport of MDMA into 5-HT neurons by the 5-HT transporter (SERT), followed by extensive 5-HT release. OBJECTIVES: To clarify whether SERT-selective effects of MDMA at human monoamine transporters can account for the reported MDMA-induced selective toxicity of serotonin neurons in primate brain. METHODS: We investigated the interaction of [(3)H](+/-, RS)- (+, S)- and (-, R)-MDMA with the human SERT, dopamine (DA) transporter (DAT), and norepinephrine (NE) transporter (NET) in stably transfected human embryo kidney (HEK)-293 cells. RESULTS: The human DAT, NET, and SERT actively transported [(3)H]RS(+/-)-MDMA saturably, stereoselectively, and in a temperature-, concentration-, and transporter-dependent manner. MDMA exhibited the highest affinity for the NET>>SERT>or=DAT, the same rank order for MDMA inhibition of [(3)H]DA, [(3)H]NE, and [(3)H]5-HT transport and stimulated release of the [(3)H]monoamines, which differed from reports derived from rodent monoamine transporters. The extent of MDMA-induced release of 5-HT was higher compared with release of DA or NE. CONCLUSIONS: The affinity of MDMA for the human SERT in transfected cells does not clarify the apparent selective toxicity of MDMA for serotonin neurons, although conceivably, its higher efficacy for stimulating 5-HT release may be a distinguishing factor. The findings highlight the need to investigate MDMA effects in DAT-, SERT-, and NET-expressing neurons in the primate brain and the therapeutic potential of NET or DAT inhibitors, in addition to SERT-selective inhibitors, for alleviating the pharmacological effects of MDMA.