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Related Experiment Videos

Dopamine transporter: expression in Xenopus oocytes.

G R Uhl1, B O'Hara, S Shimada

  • 1Laboratory of Molecular Neurobiology, ARC/NIDA, Baltimore, MD.

Brain Research. Molecular Brain Research
|January 1, 1991
PubMed
Summary

Xenopus oocytes expressing mRNA from rat midbrain exhibit specific dopamine uptake. This functional assay aids in purifying the dopamine transporter cDNA.

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Pharmacology

Background:

  • Xenopus oocytes are a versatile system for expressing foreign proteins.
  • Dopamine transporter (DAT) function is crucial for neurotransmission and targeted by drugs.
  • Previous methods for studying DAT were limited in their ability to purify the transporter.

Purpose of the Study:

  • To establish a functional assay in Xenopus oocytes for dopamine uptake.
  • To identify and purify the complementary DNA (cDNA) encoding the dopamine transporter.
  • To characterize the properties of the expressed dopamine transporter.

Main Methods:

  • Injection of Xenopus oocytes with messenger RNA (mRNA) from rat ventral midbrain and PC12 cells.
  • Incubation of mRNA-injected oocytes with radiolabeled dopamine ([3H]dopamine).

Related Experiment Videos

  • Pharmacological characterization of dopamine uptake using inhibitors like cocaine and mazindol.
  • Construction of a size-selected rat midbrain cDNA library in a plasmid vector for mRNA transcription.

    • Main Results:

    • mRNA-injected oocytes displayed pharmacologically specific dopamine uptake.
    • Uptake was dependent on time, sodium, and temperature, and blocked by cocaine and mazindol.
    • Water-injected oocytes showed minimal dopamine uptake.
    • mRNA from a size-selected rat midbrain library induced cocaine-blockable [3H]dopamine uptake.

    Conclusions:

    • Xenopus oocytes provide a functional expression system for the dopamine transporter.
    • This system serves as an effective assay for the purification of dopamine transporter cDNA.
    • The findings facilitate molecular cloning and further study of DAT.