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In Vitro Assessment of Dopamine Uptake and Methamphetamine‐Induced Dopamine Efflux at the Vesicular Monoamine Transporter‐2
Author(s) -
Volz Trent J,
Hanson Glen R,
Fleckenstein Annette E
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a684-a
Subject(s) - tetrabenazine , chemistry , vesicular monoamine transporter 2 , vesicular monoamine transporter , efflux , monoamine neurotransmitter , methamphetamine , vesicular transport protein , kinetics , dopamine , biophysics , vesicle , dopamine transporter , transporter , pharmacology , biochemistry , biology , membrane , neuroscience , receptor , serotonin , gene , physics , quantum mechanics
The vesicular monoamine transporter‐2 (VMAT‐2) is a vesicular transporter protein that utilizes proton electrochemical gradients to sequester cytoplasmic dopamine (DA) into vesicles for storage and subsequent release. Herein, we report the first use of rotating disk electrode (RDE) voltammetry to measure DA uptake and efflux by the VMAT‐2 in small synaptic vesicles. DA uptake profiles followed mixed order kinetics with apparent zero order kinetics for the first 25 s and apparent first order kinetics thereafter. Vesicular DA uptake was temperature‐ and ATP‐dependent and was blocked by the VMAT‐2 inhibitor tetrabenazine. Initial velocities of DA uptake followed Michaelis‐Menten kinetics with a K m and V max of 289 ± 59 nM and 1.9 ± 0.2 fmol / (s * μg protein), respectively. The VMAT‐2 substrate analog, methamphetamine, caused DA efflux while the VMAT‐2 inhibitor, tetrabenazine, did not. Methamphetamine‐induced DA efflux had an initial velocity of 0.54 ± 0.08 fmol / (s * μg protein) and was blocked by tetrabenazine. These results suggest that RDE voltammetric measurements are selective for VMAT‐2 uptake activity in preparations of small synaptic vesicles and will allow the design of experiments that could reveal important information about the kinetics of VMAT‐2 activity and its inhibition. Additionally, methamphetamine‐induced DA efflux in these preparations is not diffusive but rather is mediated by the VMAT‐2. This work was supported by grants DA 00689, DA 04222, DA 13367, DA 11389, DA 019447, and DA 00378 from the National Institute on Drug Abuse.