A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate

J Neurochem. 2005 Jul;94(2):352-9. doi: 10.1111/j.1471-4159.2005.03199.x.

Abstract

Previously, we reported that Phe105 in transmembrane domain 2 of the mouse dopamine transporter (DAT) is crucial for high-affinity cocaine binding. In the current study, we investigated whether other residues surrounding Phe105 also affect the potency of cocaine inhibition. After three rounds of sequential random mutagenesis at these residues, we found a triple mutant (L104V, F105C and A109V) of mouse DAT that retained over 50% uptake activity and was 69-fold less sensitive to cocaine inhibition when compared with the wild-type mouse DAT. The triple mutation also resulted in a 47-fold decrease in sensitivity to methylphenidate inhibition, suggesting that the binding sites for cocaine and methylphenidate may overlap. In contrast, the inhibition of dopamine uptake by amphetamine or methamphetamine was not significantly changed by the mutations, suggesting that the binding sites for the amphetamines differ from those for cocaine and methylphenidate. Such functional but cocaine-insensitive DAT mutants can be used to generate a knock-in mouse line to study the role of DAT in cocaine addiction.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution / physiology
  • Amphetamine / pharmacology
  • Animals
  • Biological Transport / drug effects
  • Biological Transport / physiology
  • Blotting, Western / methods
  • Cell Line
  • Cocaine / pharmacology*
  • Dopamine / pharmacokinetics*
  • Dopamine Plasma Membrane Transport Proteins
  • Dopamine Uptake Inhibitors / pharmacology*
  • Dose-Response Relationship, Drug
  • Haplorhini
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Membrane Transport Proteins / chemistry
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Methylphenidate / pharmacology*
  • Mice
  • Mice, Knockout
  • Mutagenesis / physiology
  • Mutation*
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Phenylalanine / metabolism
  • Protein Structure, Tertiary / physiology
  • Radioligand Assay
  • Transfection / methods
  • Tritium / pharmacokinetics

Substances

  • Dopamine Plasma Membrane Transport Proteins
  • Dopamine Uptake Inhibitors
  • Membrane Glycoproteins
  • Membrane Transport Proteins
  • Nerve Tissue Proteins
  • Slc6a3 protein, mouse
  • Tritium
  • Methylphenidate
  • Phenylalanine
  • Amphetamine
  • Cocaine
  • Dopamine