Abstract:To elucidate the regulation of the rat dopamine transporter (rDAT), we established several PC12 variants overexpressing the rDAT. Treating these cells with a nicotinic agonist (1,1-dimethyl-4-phenylpiperazinium iodide, 30 M) depolarized the plasma membrane potential from Ϫ31 Ϯ 2 to 43 Ϯ 5 mV and inhibited rDAT activity significantly in a calcium-and protein kinase C-independent manner. Membrane depolarization by a high external K ϩ concentration or two K ϩ channel blockers (tetraethylammonium hydroxide and BaCl 2 ) also resulted in a marked inhibition of rDAT activity. Such inhibition of dopamine uptake is due to a reduction in V max , with no marked effect on the K m for dopamine. The potency of cocaine in inhibiting dopamine uptake was not significantly altered, whereas that of amphetamine was slightly enhanced by membrane depolarization. Removing extracellular Ca 2ϩ or blocking the voltage-sensitive L-type calcium channels using nifedipine did not exert any significant effect on the inhibition of rDAT activity by depolarization. These data confirm that calcium influx on depolarization is not required for inhibition of the rDAT. Collectively, our data suggest that rDAT activity can be altered by a neurotransmitter that modulates the membrane potential, thus suggesting an exquisite mechanism for the fine-tuning of dopamine levels in the synapse. Key Words: Dopamine transporter-Nicotinic acetylcholine receptor-Depolarization-Protein kinase C-Calcium.