Transport-dependent Accessibility of a Cytoplasmic Loop Cysteine in the Human Dopamine Transporter

• Published in: The Journal of biological chemistry Vol. 275; no. 3; pp. 1608 - 1614
• Main Authors: Chen, Nianhang, Ferrer, Jasmine V., Javitch, Jonathan A., Justice, Joseph B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.01.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Biological Transport - drug effects; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Line; cocaine; Cocaine - pharmacology; Cysteine - metabolism; Cytoplasm - metabolism; Dopamine Plasma Membrane Transport Proteins; dopamine transporter; Dopamine Uptake Inhibitors - pharmacology; Ethyl Methanesulfonate - analogs & derivatives; Ethyl Methanesulfonate - metabolism; Humans; Indicators and Reagents - metabolism; Inhibitory Concentration 50; Kinetics; Membrane Glycoproteins; Membrane Transport Proteins; Mutagenesis, Site-Directed; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Protein Binding; Protein Conformation; Temperature; Time Factors; Tyramine - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.3.1608

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 [3H]CFT and for cocaine, but they displayed significant reductions in Km andVmax 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 [3H]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.