Electrochemical monitoring of transport by a vesicular monoamine transporter expressed in Xenopus oocytes.
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.
Whitley
Janet
J
Medical Science, Indiana University School of Medicine, Jordan Hall 104, Bloomington, IN 47405-6401, USA.
Parsons
Jessica
J
Freeman
Jeffrey
J
Liu
Yongjian
Y
Edwards
Robert H
RH
Near
Joseph A
JA
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D004298:Dopamine / Q000378:metabolism; D004563:Electrochemistry / Q000379:methods*; D004784:Environmental Monitoring / Q000379:methods; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics; D015870:Gene Expression / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008845:Microinjections; D009154:Mutation; D009479:Neuropeptides; D009865:Oocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013747:Tetrabenazine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D014982:Xenopus laevis
D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D012333:RNA, Messenger; C493454:Slc18a2 protein, rat; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; C032811:dihydrotetrabenazine; D004298:Dopamine; D013747:Tetrabenazine