ATP regulation of dopamine transport into presynaptic cells

Abstract

The objective of my Ph.D. thesis was to develop a better understanding of how ATP affects and regulates re-uptake of dopamine (DA) back into presynaptic cells, using rat pheochromocytoma (PC12) cells as my model system. In PC12 cells, extracellular ATP significantly increases DA uptake with maximal stimulation (200% over basal) occurring by 0.7 mM ATP. Kinetically, this ATP stimulation translates into a 3 fold increase in Vmax, without a significant change in Km. The ATP-regulated DA transport pathway is distinguished from basal transport because of differences in its ionic requirements and pharmacological profile. This thesis research has led to the identification of the major transport pathway for the uptake of the potent parkinsonism-producing neurotoxin, MPP{dollar}\sp+.{dollar} Extracellular ATP (2 mM) stimulates the transport of MPP{dollar}\sp+{dollar} into the PC12 cell line by 271% over basal levels. Kinetically, in the absence of extracellular ATP, the V{dollar}\sb{lcub}\rm max{rcub}{dollar} of MPP{dollar}\sp+{dollar} transport represents only 15% of the transport observed when 2 mM ATP is present. The kinetic parameters also show that extracellular ATP causes the PC12 monoamine transporter to have less affinity for MPP{dollar}\sp+.{dollar} These results support the hypothesis that the route of transport for the bulk of MPP{dollar}\sp+{dollar} in vivo is through the ATP-regulated uptake component. Lithium has become one of the most important agents utilized for the treatment of manic depression. LiCl (1 mM) shows significant stimulation (49%) of the ATP-regulated moiety of DA uptake. These results offer an explanation for the direct effect of LiCl on DA transport which is consistent with its therapeutic modality in the treatment of mania. A COS cell line transiently transfected with the rat dopamine (DA) transporter (DAT) was utilized to study the effect of extracellular ATP on DA transport. Extracellular ATP (0.3-1.0 mM) enhances DA uptake, with a maximal stimulation of 71.5% over basal uptake. The removal of an intracellular serine residue at amino acid position 261 effectively abolishes this stimulation. These results have revealed that ATP most likely transmits its signal through direct interaction with the DA transporter, and that this mechanism has an absolute requirement for Ser261.