Chimeric dopamine-norepinephrine transporters delineate structural domains influencing selectivity for catecholamines and 1-methyl-4-phenylpyridinium.
The dopamine (DA) and norephinephrine (NE) transporters demonstrate important differences in their selectivity for catecholamines and the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+), yet their primary sequences and predicted topology are strikingly similar. To delineate discrete structural domains contributing to pharmacologic and kinetic differences between the DA and NE transporters, a series of recombinant chimeras was generated by a restriction site-independent method and expressed in mammalian cells. Functional analyses of the chimeras delineate two discrete regions spanning the first through the third transmembrane domains (TM1-3) and TM10-11 that contribute to differences in their apparent affinities for DA, NE, and MPP+. These studies also suggest that TM2-3 of the DA transporter have a role in selectively increasing the rate of DA uptake as compared with NE. TM4-8 of the DA transporter may influence the relative rate with which MPP+ is taken up into cells and could contribute to its selective toxicity in neurons expressing the DA transporter. These structure-function studies using chimeras of members of the superfamily of Na(+)- and Cl(-)-dependent transporters provide a framework for identifying the specific structural or regulatory determinants contributing to substrate recognition and translocation by the DA and NE transporters.
Buck
K J
KJ
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Amara
S G
SG
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry*; D002395:Catecholamines / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000737:chemistry; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D027981:Symporters; D014162:Transfection
D002352:Carrier Proteins; D002395:Catecholamines; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C493227:SLC6A2 protein, human; D027981:Symporters; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine