Measurement of kinetically resolved vesicular dopamine uptake and efflux using rotating disk electrode voltammetry.

The vesicular monoamine transporter-2 (VMAT-2) sequesters cytoplasmic dopamine (DA) into vesicles for storage and subsequent release. VMAT-2 activity has traditionally been measured in small synaptic vesicles isolated from rat striatum by monitoring [3H] DA uptake and in cellular expression systems using fast scan cyclic voltammetry. This is the first report using rotating disk electrode (RDE) voltammetry to measure VMAT-2 DA uptake and efflux 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 were kinetically resolved and displayed Michaelis-Menten kinetics with a Km and Vmax of 289 +/- 59 nM and 1.9 +/- 0.2 fmol/(s microg protein), respectively. Methamphetamine-induced DA efflux was blocked by tetrabenazine and kinetically resolved with an initial velocity of 0.54 +/- 0.08 fmol/(s microg protein). These results suggest that RDE voltammetry can be used to make kinetically resolved measurements of vesicular DA uptake and efflux and will allow the design of experiments that could reveal important information about the kinetics of VMAT-2 activity and its inhibition.