Electrochemical monitoring of transport by a vesicular monoamine transporter expressed in Xenopus oocytes
| Autor: | Yongjian Liu, Joseph A. Near, Jeffrey Freeman, Janet Whitley, Robert H. Edwards, Jessica Parsons |
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| Rok vydání: | 2004 |
| Předmět: |
Microinjections
Dopamine Tetrabenazine Fast-scan cyclic voltammetry Xenopus Gene Expression Endocytosis Synaptic vesicle Dihydrotetrabenazine Xenopus laevis chemistry.chemical_compound Vesicular Biogenic Amine Transport Proteins Electrochemistry medicine Animals RNA Messenger Enzyme Inhibitors Binding Sites Membrane Glycoproteins biology General Neuroscience Neuropeptides Membrane Transport Proteins Substrate (chemistry) Biological Transport Hydrogen-Ion Concentration biology.organism_classification Oocyte Rats Vesicular monoamine transporter medicine.anatomical_structure chemistry Biochemistry Vesicular Monoamine Transport Proteins Mutation Oocytes Biophysics Environmental Monitoring |
| Zdroj: | Journal of Neuroscience Methods. 133:191-199 |
| ISSN: | 0165-0270 |
| Popis: | Xenopus laevis oocytes were injected with synthetic mRNA coding for a rat VMAT2 mutant (rVMAT2-I483A/L484A) shown previously to be retained on the plasma membrane as a result of a presumed reduction of endocytosis. Binding of the specific VMAT inhibitor [3H]dihydrotetrabenazine indicated that expression did occur at a level of approximately 3 fmol per oocyte. To determine if rVMAT2-I483A/L484A expressed in oocytes was capable of substrate transport, oocytes were placed in buffer at pH 6.0, dopamine substrate was injected into the cell, and egress of substrate was monitored by fast scan cyclic voltammetry using a carbon fiber microelectrode. Under these conditions, transport by oocytes injected with RNA coding for rVMAT2-I483A/L484A ranged from approximately 0.5 to more than 2.5 pmol/min. Water-injected and uninjected control oocytes did not exhibit appreciable transport activity. Transport by rVMAT2-I483A/L484A-injected oocytes was reduced to control levels by tetrabenazine, a known inhibitor of VMAT transport activity. Comparison of subtracted voltammograms obtained from transport assays with those for calibration experiments confirmed that the transported species was dopamine. These results suggest that expression of VMATs in oocytes may provide a useful model system for mechanistic and regulatory studies that would not be feasible using traditional methods. |
| Databáze: | OpenAIRE |
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