Transport-dependent accessibility of a cytoplasmic loop cysteine in the human dopamine transporter

The effect of covalent sulfhydryl modification on dopamine uptake by the hu man dopamine transporter was determined by rotating disc electrode voltamme try. A transporter construct, X5C, with five mutated cysteines (C90A, C135A , C306A, C319F, and C342A) and the constructs into which the wild-type cyst eines were substituted back into X5C, one at a time, all showed nearly norm al binding affinity for [H-3]CFT and for cocaine, but they displayed signif icant reductions in K-m and V-max for DA uptake. Reaction of Cys-90 or Cys- 306 with impermeant methanethiosulfonate derivatives enhanced dopamine upta ke to a similar extent as the previously observed enhancement of [H-3]CFT b inding caused by the same reaction, suggesting that cocaine may bind prefer entially to a conformation in the transport cycle. m-Tyramine increased the rate of reaction of (2-amino-ethyl)methanethiosulfonate (MTSEA) with X-A34 2C, the construct with a cytoplasmic loop residue Cys-342 restored. This m- tyramine-induced increase in reactivity appeared to require the inward tran sport rather than the outward transport or external binding of m-tyramine, and it was prevented by cocaine. Thus, inward translocation of substrates m ay involve structural rearrangement of hDAT, which likely exposes Cys-342 t o reaction with MTSEA, and Cys-342 may be located on a part of the transpor ter associated with cytoplasmic gating.