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

Neuropharmacology. 2019 Dec 15:161:107572. doi: 10.1016/j.neuropharm.2019.03.015. Epub 2019 Mar 15.


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'.

Keywords: 4-Phenylbutyrate; Creatine deficiency syndrome; Creatine transporter-1; Pharmacochaperoning; Protein misfolding; Severe mental retardation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain Diseases, Metabolic, Inborn / drug therapy*
  • Brain Diseases, Metabolic, Inborn / genetics
  • Calnexin / metabolism
  • Cell Membrane / metabolism
  • Creatine / deficiency*
  • Creatine / genetics
  • Endoplasmic Reticulum / metabolism
  • Endoplasmic Reticulum / ultrastructure
  • HEK293 Cells
  • Humans
  • Mental Retardation, X-Linked / drug therapy*
  • Mental Retardation, X-Linked / genetics
  • Mutation / genetics
  • Nerve Tissue Proteins / drug effects*
  • Nerve Tissue Proteins / genetics
  • Neurites / metabolism
  • Neurons / metabolism
  • Phenylbutyrates / pharmacology*
  • Plasma Membrane Neurotransmitter Transport Proteins / deficiency*
  • Plasma Membrane Neurotransmitter Transport Proteins / drug effects
  • Plasma Membrane Neurotransmitter Transport Proteins / genetics
  • Primary Cell Culture
  • Proteostasis Deficiencies / drug therapy*
  • Proteostasis Deficiencies / genetics
  • Rats


  • CANX protein, human
  • Nerve Tissue Proteins
  • Phenylbutyrates
  • Plasma Membrane Neurotransmitter Transport Proteins
  • SLC6A8 protein, human
  • Calnexin
  • 4-phenylbutyric acid
  • Creatine

Supplementary concepts

  • Creatine deficiency, X-linked