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

Ameya Kasture,Ali El-Kasaby,Daniel Szöllősi,H M Mazhar Asjad,Alexandra Grimm,Thomas Stockner,Thomas Hummel,Michael Freissmuth,Sonja Sucic

doi:10.1074/jbc.m116.737551

Ameya Kasture, Ali El-Kasaby + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m116.737551

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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.

Highlights

Folding of proteins can be assisted by small molecules
We examined whether the combination of noribogaine and pifithrin-␮ exerted an additive action; human embryonic kidney 293 cells (HEK293) cells stably expressing Drosophila dopamine transporter (DAT) (dDAT)-G108Q were treated with increasing concentrations of noribogaine (0 –30 ␮M) in the presence or absence of pifithrin-␮ (5 ␮M)
We capitalized on a serendipitous mutation, which had been introduced into the gene encoding DAT of D. melanogaster, to provide a proof-of-principle for pharmacochaperoning in a living organism

Summary

Pharmacochaperoning of a Misfolded DAT Mutant

A phenotype of reduced sleep was identified in fruit flies (Drosophila melanogaster), which had been subjected to random mutagenesis, and linked to a point mutation in the gene encoding Drosophila DAT (dDAT) [13]. The mutation substitutes glycine 108 by glutamine and phenocopies the effect caused by deletion of DAT in flies, which are referred to as fumin (ϭ sleepless) flies [14]. This suggests that dDAT-G108Q is a loss-of-function mutation. In SERT, IL-1 interacts with the C terminus via a salt bridge to stabilize the folded structure [10]. An analogous mechanism may operate in dDAT, because the crystal structure of dDAT visualizes a cation-␲ interaction between a Trp in the C terminus and an Arg in IL-1 [15]. It appears justified to surmise that dDAT-G108Q has a folding defect. The magnitude of the effect sufficed to restore sleep in flies expressing dDAT-G108Q

Results

Discussion

Experimental Procedures