Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis.
Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 microM treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K(m), in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three-fivefold in both cell types exposed to 50 microM DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.
Wiesinger
Jason A
JA
Department of Nutritional Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Buwen
James P
JP
Cifelli
Christopher J
CJ
Unger
Erica L
EL
Jones
Byron C
BC
Beard
John L
JL
D000818:Animals; D019921:Biotinylation / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D003676:Deferoxamine / Q000494:pharmacology*; D004298:Dopamine / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007930:Leucine / Q000378:metabolism; D009447:Neuroblastoma; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017262:Siderophores / Q000494:pharmacology*; D019311:Staurosporine / Q000494:pharmacology; D013347:Subcellular Fractions / Q000187:drug effects; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D017262:Siderophores; D014316:Tritium; D011493:Protein Kinase C; D007930:Leucine; D019311:Staurosporine; D003676:Deferoxamine; D004298:Dopamine