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.
Pubmed ID: 27481941 RIS Download
Publication data is provided by the National Library of Medicine ® and PubMed ®. Data is retrieved from PubMed ® on a weekly schedule. For terms and conditions see the National Library of Medicine Terms and Conditions.
Software package of molecular simulation programs. It is distributed into AmberTools15 and Amber14. AmberTools15 is a software package which can carry out complete molecular dynamics simulations with either explicit water or generalized Born solvent models. It is distributed in source code format and must be compiled in order to be used. Amber14 builds on AmberTools15 by adding the pmemd program, which provides better performance on multiple CPUs and dramatic speed improvements on GPUs compared to sander (molecular dynamics). GPU info, manuals, and tutorials are available on the website.
View all literature mentionsSoftware tool as Program for Comparative Protein Structure Modelling by Satisfaction of Spatial Restraints. Used for homology or comparative modeling of protein three dimensional structures. User provides alignment of sequence to be modeled with known related structures and MODELLER automatically calculates model containing all non hydrogen atoms.
View all literature mentionsServes Drosophila research community by collecting and distributing DNA clones and vectors; collecting and distributing Drosophila cell lines; developing and testing genomics technologies for use in Drosophila and assisting members of the research community in their use.
View all literature mentionsCell line HEK293 is a Transformed cell line with a species of origin Homo sapiens (Human)
View all literature mentions