Species differences in dopamine transporters: postmortem changes and glycosylation differences.
The apparent molecular masses of photoaffinity-labeled dopamine transporters (DATs) from rat, human, dog, and primate kidney COS cells expressing the rat DAT1 cDNA differ. Sequences predicted from cDNA cloning reveal only one amino acid difference between the length of the rat and human DAT but one less site for potential N-linked glycosylation in the human DAT. Possible posttranslational and postmortem bases for species differences in DAT molecular mass were explored. Rat DAT proteins from striata subjected to approximately 5 h of postmortem delay modeled after the human postmortem delay process revealed small but consistent losses in apparent molecular mass and in cocaine analogue binding; the DAT molecular mass displayed no further losses for up to 30 h of model postmortem treatment. Degradative postmortem changes could thus contribute to molecular mass differences between rat and human DATs. Neuraminidase treatment reduced the apparent molecular mass of native rat DAT but not that of the rat DAT expressed in COS cells, suggesting that the sugars added to the DAT expressed in COS cells were different than those added to the rat brain striatal transporter. These differences could account for the somewhat higher Km values for expressed DAT cDNA in COS cells when compared with the wild-type striatal transporter. These results are in accord with the differences in number of predicted N-linked glycosylation sites between rat and human DATs and with cell-type specificity in transporter posttranslational processing.
Patel
A
A
Molecular Pharmacology Section, NIDA Addiction Research Center, Baltimore, Maryland 21224.
Uhl
G
G
Kuhar
M J
MJ
D000345:Affinity Labels; D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D004285:Dogs; D050483:Dopamine Plasma Membrane Transport Proteins; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D009439:Neuraminidase / Q000494:pharmacology; D010777:Photochemistry; D011180:Postmortem Changes; D011499:Protein Processing, Post-Translational; D051381:Rats; D013045:Species Specificity
D000345:Affinity Labels; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; D009439:Neuraminidase