Delineation of discrete domains for substrate, cocaine, and tricyclic antidepressant interactions using chimeric dopamine-norepinephrine transporters.
Neurotransmitter transporters determine the intensity and duration of signal transduction by controlling the rapid removal of transmitter molecules from the synaptic cleft. The importance of their function is further reflected by the medical and social implications of compounds that inhibit their activity such as the antidepressants and cocaine. Molecular characterization of these transporters has revealed that they are members of a large family of membrane proteins with 12 putative transmembrane domains. However, little information exists as to whether discrete domains of these proteins mediate the various defined functions of these transporters. In this study, we constructed a series of chimeras between two structurally related but pharmacologically distinct transporters, the dopamine and norepinephrine transporters. The properties of these chimeric transporters suggest that distinct regions of these molecules determine these individual functions. Regions from the amino-terminal through the first five transmembrane domains are likely to be involved in the uptake mechanisms and ionic dependence. Regions within transmembrane domains 6-8 determine tricyclic antidepressant binding and cocaine interactions, whereas the carboxyl-terminal region encompassing transmembrane domain 9 through the COOH-terminal tail appears to be responsible for the stereoselectivity and high affinity for substrates. The dissociation of the substrate uptake and cocaine binding properties of these transporters further raises the possibility that antagonists of cocaine action devoid of uptake blockade activity might be developed for the clinical management of cocaine addiction.
Giros
B
B
Howard Hughes Medical Institute Laboratory, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Wang
Y M
YM
Suter
S
S
McLeskey
S B
SB
Pifl
C
C
Caron
M G
MG
D000929:Antidepressive Agents, Tricyclic / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003042:Cocaine / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D018492:Dopamine Antagonists; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009638:Norepinephrine / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D027981:Symporters
D000929:Antidepressive Agents, Tricyclic; D002352:Carrier Proteins; D018492:Dopamine Antagonists; 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; D003042:Cocaine; D004298:Dopamine; D009638:Norepinephrine