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J Biol Chem. 2016 Sep 30;291(40):20876-20890. doi: 10.1074/jbc.M116.737551. Epub 2016 Aug 1.

Functional Rescue of a Misfolded Drosophila melanogaster Dopamine Transporter Mutant Associated with a Sleepless Phenotype by Pharmacological Chaperones.

Author information

1
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria.
2
the Department of Pharmacology, Faculty of Veterinary Medicine, Mansoura University, 35516 Mansoura, Egypt, and.
3
the Department of Neurobiology, University of Vienna, A-1090 Vienna, Austria.
4
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria, michael.freissmuth@meduniwien.ac.at.

Abstract

Folding-defective mutants of the human dopamine transporter (DAT) cause a syndrome of infantile dystonia/parkinsonism. Here, we provide a proof-of-principle that the folding deficit is amenable to correction in vivo by two means, the cognate DAT ligand noribogaine and the HSP70 inhibitor, pifithrin-μ. We examined the Drosophila melanogaster (d) mutant dDAT-G108Q, which leads to a sleepless phenotype in flies harboring this mutation. Molecular dynamics simulations suggested an unstable structure of dDAT-G108Q consistent with a folding defect. This conjecture was verified; heterologously expressed dDAT-G108Q and the human (h) equivalent hDAT-G140Q were retained in the endoplasmic reticulum in a complex with endogenous folding sensors (calnexin and HSP70-1A). Incubation of the cells with noribogaine (a DAT ligand selective for the inward-facing state) and/or pifithrin-μ (an HSP70 inhibitor) restored folding of, and hence dopamine transport by, dDAT-G108Q and hDAT-G140Q. The mutated versions of DAT were confined to the cell bodies of the dopaminergic neurons in the fly brain and failed to reach the axonal compartments. Axonal delivery was restored, and sleep time was increased to normal length (from 300 to 1000 min/day) if the dDAT-G108Q-expressing flies were treated with noribogaine and/or pifithrin-μ. Rescuing misfolded versions of DAT by pharmacochaperoning is of therapeutic interest; it may provide opportunities to remedy disorders arising from folding-defective mutants of human DAT and of other related SLC6 transporters.

KEYWORDS:

Drosophila; chaperone; dopamine; dopamine transporter; drug action; endoplasmic reticulum (ER)

PMID:
27481941
PMCID:
PMC5076501
DOI:
10.1074/jbc.M116.737551
[Indexed for MEDLINE]
Free PMC Article

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