Transport-dependent accessibility of a cytoplasmic loop cysteine in the human dopamine transporter.
The effect of covalent sulfhydryl modification on dopamine uptake by the human dopamine transporter was determined by rotating disc electrode voltammetry. A transporter construct, X5C, with five mutated cysteines (C90A, C135A, C306A, C319F, and C342A) and the constructs into which the wild-type cysteines were substituted back into X5C, one at a time, all showed nearly normal binding affinity for [(3)H]CFT and for cocaine, but they displayed significant reductions in K(m) and V(max) for DA uptake. Reaction of Cys-90 or Cys-306 with impermeant methanethiosulfonate derivatives enhanced dopamine uptake to a similar extent as the previously observed enhancement of [(3)H]CFT binding caused by the same reaction, suggesting that cocaine may bind preferentially to a conformation in the transport cycle. m-Tyramine increased the rate of reaction of (2-aminoethyl)methanethiosulfonate (MTSEA) with X-A342C, the construct with a cytoplasmic loop residue Cys-342 restored. This m-tyramine-induced increase in reactivity appeared to require the inward transport rather than the outward transport or external binding of m-tyramine, and it was prevented by cocaine. Thus, inward translocation of substrates may involve structural rearrangement of hDAT, which likely exposes Cys-342 to reaction with MTSEA, and Cys-342 may be located on a part of the transporter associated with cytoplasmic gating.
Chen
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Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA. nhc@uic.edu
Ferrer
J V
JV
Javitch
J A
JA
Justice
J B
JB
D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003042:Cocaine / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D013997:Time Factors; D014439:Tyramine / Q000494:pharmacology
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate; D003042:Cocaine; C028169:3-tyramine; D003545:Cysteine; D014439:Tyramine